Efficient Conjugation of Chelating Agents to IgG and Accurate Colorimetric Determination of the Chelate-to-IgG Molar Ratio.

Efficient Conjugation of Chelating Agents to IgG and Accurate Colorimetric Determination of the Chelate-to-IgG Molar Ratio. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficient Conjugation of Chelating Agents to IgG and Accurate Colorimetric Determination of the Chelate-to-IgG Molar Ratio. Masayuki Yokoyama, Kouichi Shiraishi, Teppei Komatsu, Yasuyuki Iguchi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8683063/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Chelate conjugation is an essential step for metal-ion labelling of proteins, and the chelate-to-protein (chelate/protein) molar ratio of the resulting protein-chelate conjugate critically influences the conjugate’s functions. Therefore, both controlling and measuring the chelate/protein molar ratio are important technical considerations. Among available methods for chelate/protein determination, a colorimetric method using the Y(III)–Arsenazo III reagent is convenient because it requires neither large-scale facilities nor expensive instruments. We examined this assay method for three representative chelating agents (DOTA-NHS, DTPA-di, and CHX-A”-DTPA). Concerning the conjugation reactions of chelating agents, high and reproducible reaction yields are desired. However, the reported yields have varied widely, and no standardized, high-yield, and reproducible procedure has been established. Results We established new measurement methods to determine the chelate-to-protein molar ratio for DOTA-NHS and CHX-A”-DTPA using human IgG as the target protein for conjugation. We also established a conjugation procedure that affords high conjugation-reaction yields for these chelating agents. We achieved these outcomes both by preparing stock solutions of the chelating agents in dehydrated DMSO and by employing the “dry-handling technique” throughout the conjugation procedure. Through the combination of these two practices, we successfully obtained high reproducibility and high yields in the reactions. Conclusions The establishment of the two measurement methods for the chelate-to-protein molar ratio and the conjugation procedure delivering the above yields may substantially contribute to research and clinical applications of metal ion-labeled proteins in many fields of science and medicine, particularly in the field of nuclear medicine. chelates chelating agents conjugates metal ions DOTA-NHS DTPA-di CHX-A”-DTPA Arsenazo III Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Chelate compounds are essential chemicals that serve importantly as a metal-ion coordinator of proteins for medical diagnoses and therapies. For example, the chelating agent DTPA (diethylenetriaminepentaacetic acid) and its derivatives have been widely used in both research and clinical applications. Likewise, the chelating agent DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and its derivatives have also been extensively used in these same applications owing to their higher coordination affinities than DTPA’s. This higher coordination affinity exhibited by DOTA is attributable to its cyclic chemical structure. Representative metal ions used for imaging diagnoses include 67 Ga, 99m Tc, and 111 In, while 90 Y, 177 Lu, and 211 At are used for radionuclide-based therapies. Additionally, Gd is clinically employed as a positive MRI contrast agent in coordinated structures within low-molecular-weight chelates. To conduct medical diagnoses and therapies involving metal ions, medical practitioners use conjugates consisting of proteins, chelating agents, and metal ions. The first step in preparing these conjugates is the conjugation of chelate compounds to the given protein. For avoidance of protein denaturation, this conjugation reaction must be conducted in either pure aqueous buffers or aqueous buffers containing small amounts of water-miscible organic solvents such as ethanol and DMSO. Consequently, the reaction yields for this type of conjugation are typically much lower than those obtained in the standard organic chemistry carried out in organic solvents. These lower reaction yields occur because active chemical species used for the conjugation—such as N-hydroxysuccinimide esters, acid anhydrides, and phenyl isothiocyanates—react not only with the primary amino groups of proteins but also with hydroxide ions (OH − ) present in aqueous buffers. Moreover, these conjugation reactions are favored at higher pH because only the unprotonated form (–NH2) of the primary amino group can react with these active chemical species. In contrast, the protonated form (–NH3 + ) that is predominant at low pH cannot react with the active chemical species. This explains why reported reaction yields for chelate compounds were generally low, typically ranging from 2% to less than 15% (Abadi et al. 2021 ; Alirezapour et al. 2013 ; Arano et al. 1996 ; Hoppman et al. 2011; Sudo et al. 2023 ; Winter et al. 2023 ). Another potential problem in the conjugation of chelating agents to proteins is the poor reproducibility of the reaction yields. Most published papers report only a single value for the reaction yield, and to our knowledge, no paper has examined and reported the yield reproducibility. In previously published papers concerning the conjugation of chelating agents to proteins, an array of experimental procedures have been reported involving, for example, various kinds of buffers, pH levels, temperatures, reaction times, and concentrations of chelating agents and proteins. Therefore, many readers of these papers, including the authors of the present study, have had great difficulty ascertaining which reaction conditions are generally optimal. In the current paper, we aim to identify several experimental factors affecting the conjugation of chelating agents to proteins and we present a facile and reproducible procedure that achieves high reaction yields. It is well known that the coordination affinity for metal ions is higher in DOTA and its derivatives than in DTPA and its derivatives. This superior affinity constitutes an important advantage of DOTA-based chelates over the other types of chelates. However, the coordination reactions in which metal ions form complexes with DOTA and its derivatives proceed much more slowly than the corresponding coordination reactions involving other types of chelates, such as DTPA and its derivatives. The relative slowness of the kinetics for DOTA-based chelates not only hinders coordination reactions where metal ions serve as imaging markers and therapeutic agents, but also poses a technical problem for determining chelate concentrations via a Y³⁺-based colorimetric agent, the Y(III)–Arsenazo III complex reagent. We therefore examined both the reaction temperatures and reaction periods to optimize the colorimetric determination of the DOTA moieties conjugated to human IgG as a model protein. We encountered yet another technical problem when we measured the chelate-to-IgG (chelate/IgG) molar ratios for CHX-A”-DTPA-based IgG–chelate conjugates. Present in the chemical structure of this chelating agent is one phenyl (benzene) ring, which—because it can exhibit absorbance at 280 nm (A280)—may significantly interfere with the A280 measurement of IgG concentrations, [IgG]. To address this problem, we performed three steps: (1) we synthesized a model compound that mimicked the CHX-A”-DTPA moiety conjugated to IgG, (2) we estimated how much the model compound contributed to absorbance at 280 nm, and (3) we obtained the true chelate/IgG molar ratio values of the IgG–CHX-A”-DTPA conjugates. Methods Chemicals We obtained a chelate compound, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS, Supplementary Fig. S1 ) bearing an N-hydroxysuccinimide ester reactive group through an ordered synthesis made by Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). We purchased two commercially available chelate compounds; diethylenetriaminepentaacetic acid (DTPA, Fig. S1 ) and diethylenetriaminepentaacetic dianhydride (DTPA-di, Fig. S1 ) from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). We purchased a chelate compound, p-SCN-Bn-CHX-A”-DTPA (CHX-A”-DTPA, Fig. S1 ) from Macrocyclics, Inc. (Plano, USA). We purchased the Arsenazo III (2,2'-[1,8-Dihydroxy-3,6-disulfo-2,7-naphthalene] bis(azo)dibenzenearsonic acid) reagent from FUJIFILM Wako Pure Chemical Corp. (Tokyo, Japan) and YCl3·6H2O from Sigma-Aldrich Japan G.K. (Tokyo, Japan). We prepared a colorimetric agent, the Y(III)–Arsenazo III solution, according to a specific reference (Pippin et al. 1992 ). We created a 2-fold-concentrated Y(III)–Arsenazo III stock solution containing 3.2 µM Y(III) and 10 µM Arsenazo III in 0.15 M sodium acetate buffer at pH 4.0. We also made the following two purchases: dehydrated DMSO from FUJIFILM Wako Pure Chemical Corp. (Tokyo, Japan); and human IgG (Product No. I4506) from Sigma-Aldrich Japan G.K. (Tokyo, Japan). Other reagents were of reagent grade. A monoclonal IgG1 was kindly provided by the Laboratory of RIN Institute Inc. (Tokyo, Japan). For mass spectroscopy, we purchased sinapic acid (Product No. 198-13363) from FUJIFILM Wako Pure Chemical Corp. (Tokyo, Japan) and protein standard II (Part No. 8207234) from Bruker, Japan (Tokyo, Japan). The conjugation of chelating agents to immunoglobulin G (IgG): Preparation of IgG–chelate conjugates We added 100 µL of human IgG solution (10.0 mg/mL in serine) to a 1.5 mL Eppendorf tube. Then, we added 100 µL of either 0.10 M borate-KCl buffer or 0.20 M HEPES buffer. Most commonly, we used the former buffer at pH 8.5. Next, we added a chelating-agent solution (5 mM) in dehydrated DMSO. For DOTA-NHS, we stirred this reaction mixture at 4°C for 2.0 h, followed by reaction at room temperature (r.t.) for 1.5 h. For DTPA-di and CHX-A”-DTPA, the reaction took place at 25°C for 2.0 h. After the reactions, we purified the reaction mixtures by means of gel-filtration chromatography using a PD MidiTrap™ G-25 column (Cytiva, Tokyo, Japan). We collected a 0.5–1.5 mL fraction eluted with Dulbecco’s phosphate-buffered saline (D-PBS) to remove unreacted low-molecular-weight chelating reagents. Colorimetric determination of the chelate concentrations We performed colorimetric assays of chelate concentrations using the Y(III)–Arsenazo III reagent solution. To this end, we followed (with some modifications) the steps presented in the previously mentioned reference (Pippin et al. 1992 ). In the presence of chelate compounds, the Y 3+ ion transfers from the Arsenazo III moiety to the chelate, altering the visible (VIS) light spectrum; we used absorbance at 652 nm (A652) to determine the chelate concentration. We added 450 µL of the 2-fold concentrated Y(III)–Arsenazo III solution to a 1.5 mL Eppendorf tube. To it, we then added (450 – x) µL of 0.15 M sodium acetate buffer at pH 4.0, followed by x µL of the IgG–chelate conjugate solution in D-PBS. Consequently, the total volume of the reaction mixture was 900 µL. The added volume (x µL, typically 20–30 µL) of the IgG–chelate solution was adjusted on the basis of absorbance at 280 nm (A280). Our reason for taking this step was to maintain, throughout one series of experiments, a fixed [IgG] ranging from 0.03 to 0.12 µM in the 900-µL reaction mixture. In parallel, we prepared a standard curve of A652 values plotted against chelate concentrations. For this standard curve, we added 450 µL of the 2-fold concentrated Y(III)–Arsenazo III solution to a 1.5 mL Eppendorf tube. To it, we then added (450 – x – y) µL of 0.15 M sodium acetate buffer at pH 4.0, followed, first, by x µL of an intact IgG solution in saline at 2.7–5.4 µM and, thereafter, by y µL of a chelate solution in 0.15 M sodium acetate buffer at pH 4.0 (Added in this order). The total volume of the reaction mixture was 900 µL. The x µL of intact IgG solution was set for the same IgG concentration as that of the IgG–chelate conjugates, and the y µL of the chelate solution varied in a range extending from 0.2 to 1.2 µM in the 900 µL solution. Chelates used for this standard-curve preparation were DOTA for the IgG–DOTA-NHS conjugate and DTPA for both the IgG–DTPA-di and the IgG–CHX-A”-DTPA conjugates. The reaction-duration conditions for IgG–DTPA-di and IgG–CHX-A”-DTPA were r.t. and 10 min. In these cases, the VIS spectra of IgG–chelate conjugates were unchanged over the 10-min reaction period. Reaction conditions for IgG–DOTA-NHS were 37°C and 3 days. These conditions were optimized after pre-examination at various temperatures and time periods. Details of this pre-examination will be described in the Results section. Measurements We recorded UV-VIS spectra using a V-750 UV-Visible Spectrophotometer (JASCO International Co. Ltd., Tokyo, Japan). To determine IgG concentration ([IgG]), we measured absorbance at 280 nm (A280). In the colorimetric assay with the Y(III)–Arsenazo III solution, we measured absorbance at 652 nm (A652). In actual measurement procedures, we subtracted background absorbance at 600 nm for the IgG assay and at 800 nm for the colorimetric assay to compensate for minute changes in quartz-cell conditions in each measurement. Therefore, (A280 – A600) values and (A652 – A800) values were used in the IgG assays and the colorimetric chelate assays, respectively, although only A280 and A652 are described in the rest of this paper. We obtained mass spectra of intact IgG and IgG–DOTA-NHS conjugates by means of MALDI-TOF mass spectroscopy using an Autoflex Speed (Bruker Daltonics GmbH, Germany). After preparing protein solutions in Milli Q water containing 0.10 vol.% TFA at approximately 1 mg protein/mL, we mixed the protein solution with a saturated sinapic acid solution in absolute ethanol containing 0.10 vol.% of TA30 (TA30: 30 vol.% acetonitrile and 0.07 vol.% TFA in MilliQ water). The mixing volume ratio was 1:1. We deposited 0.5 µL of the saturated sinapic acid solution onto the appropriate spot on a ground-steel MALDI target plate to dry. Then, we deposited 1.0 µL of a protein sample solution onto the same spot, which was covered with the dried sinapic acid layer. After the sample solution dried, we measured three MALDI-TOF mass spectra from one spot on the target plate: three spots were measured for each sample. Consequently, nine measurements were conducted for one sample. We obtained the average ± standard deviation (S.D.) of the most intense molecular ion peak from a total of nine spectra. Results Optimization of reaction conditions for the colorimetric assay of DOTA. We first performed a colorimetric DOTA assay (the chemical structure of DOTA is shown in Fig. S1 ) with the Y(III)–Arsenazo III solution. In performing this assay, we carefully referred to the reaction conditions reported for a DTPA derivative in a previous study (Pippin et al. 1992 ). As shown in Fig. 1a, after a 10-min r.t. reaction, A652 was significantly lower for 1 µM DTPA (red line) than for the control (black line), which involved only the Y(III)–Arsenazo III solution. By contrast, A652 for DOTA (blue line) was nearly identical to A652 for the control under these same reaction conditions. The rigid tetraazacyclododecane structure of DOTA prevented the Y 3+ ion coordination reaction from proceeding at r.t.. It has been well documented that coordination rates of Gd 3+ metal ions to DOTA are very low (Sherry et al. 1989 ; Wang et al. 1992 ). Studies along these lines have also uncovered evidence of low coordination rates for 14 kinds of lanthanide metal ions, including Y 3+ (Kodama et al. 1991 ) and for 11 kinds of divalent metal ions, including Mg 2+ , Ca 2+ , and Cu 2+ (Kasprzyk et al. 1982). As a result of these low coordination rates, reaction yields of radionucleotides conjugated to DOTA were not quantitative (100%) for 90 Y 3+ (Li et al. 1994 ; Sugyo et al. 2015 ), 111 In 3+ (Suzuki et al. 2024 ), and 90 Y 3+ and 111 In 3+ (Li et al. 2023) under mild conditions (r.t. to 40°C), whereas a quantitative reaction yield is commonly obtained with DTPA under the mild conditions. Taking these results and reported facts into consideration, we examined the reaction of DOTA at 80°C for 20 min: as shown in Fig. 1b, the degree of reduction in A652 was almost the same for 1 µM of DOTA (blue line) as for 1 µM of DTPA (red line). Additionally, as shown in Fig. S2, DTPA and DOTA provided the same degree of reductions after a 20-min reaction at 80°C when the concentrations of these two chelates were identical at 0.5, 1.0, and 2.0 µM. We thus concluded that the reduction in A652 resulted from the elimination of the Y 3+ ion from the Arsenazo III moiety. We further concluded that chelate-coordinated Y 3+ ions exhibited no absorbance at 652 nm irrespective of the differences between the chemical structure of DTPA and that of DOTA. The high temperature (80°C) examined in the above-mentioned results is suitable for DOTA determination in the absence of proteins; however, it is not applicable to protein-chelate conjugates because proteins are easily denatured at such a high temperature. Therefore, we examined reactions at 37°C for 3 days. Figure 1c shows the spectrum changes with 1.0 µM of DTPA (red line) and 1.0 µM of DOTA (blue line). Both lines completely overlap at about 652 nm. This fact indicates that these reaction conditions (37°C, 3 days) seem appropriate for the DOTA systems. However, absorbance at 652 nm (A652) of a control (37°C, 3 days, black dotted line) was lower than A652 of the negative control (r.t., 10 min, black solid line). This comparison indicates that the reduction in A652 occurring over 3 days of incubation at 37°C was due possibly to the effects of ambient room light on the Y(III)–Arsenazo III reagent over this lengthy period of time. To test this possibility, we compared the behavior of the Y(III)–Arsenazo III solution in the dark and under room light for 3 days at 37°C. Fig. S3a shows that A652 for the lighted sample at 37°C for 3 days (blue line) was markedly lower than A652 for the control at r.t. for 10 min (black line), while A652 for the darkened sample at 37°C for 3 days (red line) was essentially identical to A652 for the previously mentioned negative control (indeed, the two lines almost completely overlapped). For the conditions involving a temperature of 37°C and a duration of 3 days, the reaction had to proceed in the dark as our aim was to avoid A652 values lower than the negative control. Thanks to this basic finding, we made sure that all the results presented in Fig. 2 and Fig. 3 and in Fig. S3b through Fig. S5 were obtained in the dark. Under these dark conditions, we successfully confirmed a well-correlated linear relationship between [DOTA] and A652 (see Fig. 2a). In the next step, we examined reaction periods over time (the period extended from 10 min to 7 days). For 1.0 µM of DOTA, absorbance at about 550 nm progressively increased and absorbance at 652 nm progressively decreased (Fig. S3b) during the period beginning at the 10-min marker, passing through the 1-day marker, and ending at the 3-day marker (with the isosbestic point observed at ~ 580 nm); however, during the period beginning at the 3-day marker, passing through the 5-day marker, and ending at the 7-day marker, absorbance progressively decreased at about 550 nm and at 652 nm (with no isosbestic point observed). We surmised that the substantial degradation of the Y(III)–Arsenazo III reagent was due to the lengthy incubation periods (i.e., periods over 3 days in length) at 37°C. After performing the above experiments, we concluded that the optimal conditions for [DOTA] determination were a 3-day duration, a 37°C temperature, and a darkened setting. Under these conditions, we successfully obtained a standard curve of A652 values plotted against [DOTA] with a high R 2 value (0.9788), as shown in Fig. 2a. Next, we set out to determine the optimal reaction conditions for IgG–DOTA-NHS conjugates. In the reaction conditions involving a temperature of 80°C and a duration of 10 min, we observed that peaks at 652 nm would markedly shift to longer wavelengths (red shift) in the presence of IgG. Fig. S4a presents this shift: the red solid line shows 0.5 µM [DOTA] in the presence of 0.20 µM IgG and the blue solid line shows 1.0 µM [DOTA] in the presence of 0.20 µM IgG. This peak red shift is an easily identifiable sign of IgG denaturation. As shown in Fig. S4b, the peak shift was also clearly observed at 56°C, which is a temperature commonly used for complement inactivation of serum. In contrast, the reaction at 37°C did not exhibit the peak shift, as demonstrated by the curve of the blue solid line in Fig. S4b. When the reaction was performed at 37°C for 3 days, the peak shift was not observed, as shown in Fig. 2b. Therefore, we have concluded that 37°C is an appropriate choice for the IgG–DOTA-NHS assays. We then attempted to establish a standard curve for [DOTA] in the presence of IgG. As shown in Fig. S5, we found that the presence of IgG was associated with relatively low A652 values even for a short period of incubation in the Y(III)–Arsenazo III solution. The sky-blue line (DOTA + IgG, 37°C, 1h) illustrates this finding. Absorbance around 550 nm was lower for the sky-blue line than for the negative control (black solid line). This finding indicates that the spectrum change was closely linked not to a transfer of Y(III) ions from the Arsenazo III moiety to the chelating agent, but to the Y(III)–Arsenazo III molecules’ adsorption to and interaction with the IgG molecules. The same pattern and degree of spectrum change were observed in an experiment we conducted with + IgG at r.t. for 10 min (see the green line in Fig. S5). This finding indicates that IgG’s adsorption to and interaction with IgG occurred much faster than the Y(III)’s transfer from the Arsenazo III moiety to DOTA. A652 for the sky-blue line (+ IgG, 37°C, 1 h) was lower than A652 for the green line (+ IgG, r.t., 10 min). However, A652 for the positive control (80°C, 10 min) was even lower than A652 for the 1-hour reaction at 37°C (sky-blue line). Based on all the above-mentioned results, we set the standard reaction conditions for [DOTA] determination in the presence of IgG at 37°C for 3 days in the dark. As shown in Fig. 2b, we observed that absorbances at 652 nm and absorbances at about 550 nm were markedly lower for all the colored lines ([DOTA] = 0.2, 0.4, 0.6, 0.8, 1.0 µM) than for the negative control (black solid line). More specifically, these differences in absorbance occurred because the Y(III)–Arsenazo III reagent interacted with and adsorbed to the IgG. We observed [DOTA]-dependent spectrum changes in A652 and in absorbance at about 550 nm for the five colored lines. A652 was lower and absorbance at about 550 nm was higher for the colored lines than those of the black dotted line ([DOTA] = 0 µM). From the A652 values presented in Fig. 2b, we successfully obtained a standard, strongly linear curve depicting the relationship between A652 and [DOTA]. (see Fig. 2c). The high R 2 value (0.984) of the standard curve proved the strong linearity. When we measured concentrations of the DOTA moiety conjugated to IgG, we set [IgG] at a fixed value both for the standard curve and for the IgG–DOTA-NHS conjugate samples. This fixed [IgG] value ranged from 0.06 µM to 0.12 µM. From this range, we applied a particular value to each experiment in consideration of the DOTA-NHS/IgG molar ratio estimated for each experiment: the smaller the estimated DOTA-NHS/IgG molar ratio was, the larger the [IgG] was. As a consequence of these experiments and measurements, we successfully obtained the DOTA-NHS/IgG molar ratios of the IgG–DOTA-NHS conjugates from the [DOTA-NHS] that we had obtained in the colorimetric assay and the [IgG] that we had fixed in all the measurements of the standard curve and the conjugates. DOTA-NHS conjugation to IgG In the current study, we prepared a stock solution of DOTA-NHS in dehydrated DMSO and allowed the DOTA-NHS to react with the primary amino group of the IgG molecule by using the “dry-handling technique” described in detail in the Supplementary Information. Table 1 summarizes the results of the DOTA conjugation reaction to IgG. Across all tested pH values and all DOTA-NHS/IgG feed molar ratios, we obtained reaction yields of 43% or more for the DOTA-NHS. These values for the DOTA-NHS conjugation to proteins are greater than previously reported values (Abadi et al. 2021 ; Alirezapour et al. 2013 ; D'Huyvetter et al. 2012 ; Hoppmann et al. 2011 ; Rossin et al. 2011 ; Sudo et al. 2023 ) for the DOTA-NHS conjugation reaction to proteins (Table S2 summarizes the previously reported DOTA-NHS reaction yields.). Comparisons of the reaction pH levels reveal that reaction yields did not change markedly for the pH range of 8.3–9.2 in a 0.05 M borate-KCl buffer, as shown in Runs 1–5 of Table 1. By fixing the reaction pH at 8.5, we examined conjugation reactions three times at a DOTA-NHS/IgG feed molar ratio of 4.0 and 12.0, as summarized in Runs 6–11. We obtained high reaction yields with small standard deviations. The conjugation procedure proved to have good reproducibility in terms of the DOTA-NHS reaction yields. Comparing two methods for DOTA-NHS/IgG molar-ratio determinations In addition to the colorimetric method, we determined DOTA-NHS/IgG molar ratios of IgG–DOTA-NHS conjugates by means of mass spectroscopy (Cassells et al. 2021 ; Rossin et al. 2011 ). For this study, we used a monoclonal IgG that was kindly supplied by the Laboratory of RIN Institute Inc. (Tokyo, Japan). For this monoclonal IgG, we used three DOTA-NHS/IgG feed ratios (4.0, 10.0, and 20.0) and obtained three IgG–DOTA-NHS conjugates, which we coded as A1, A2, and A3 (for a summary, see Table 2). Figure 4 presents mass spectra of intact IgG and three IgG–DOTA conjugates; A1, A2 and A3. By subtracting the mass-to-charge ratio of the intact IgG from the mass-to-charge ratios of the conjugates, we obtained the products’ DOTA-NHS/IgG molar ratios using a molecular weight (386.4) of one DOTA moiety conjugated to IgG. Table 3 compares these “mass spectroscopy” DOTA-NHS/IgG molar-ratio values with the “colorimetric assay” DOTA-NHS/IgG molar-ratio values. The two sets of ratio values are in good agreement with each other. Conjugation of DTPA-di to IgG and measurements of the DTPA-di/IgG molar ratios of the IgG–DTPA-di conjugates For conjugates obtained with the DTPA-di reagent, we prepared a standard curve of [DTPA]. In the DTPA experiment, the reaction readily yielded a reduction in A652 in the Y(III)–Arsenazo III solution at r.t. for a very short period, such as 15 min. As shown in Fig. 3, we obtained a standard, strongly linear curve depicting the relationship between A652 and [DTPA] in the presence of IgG. The large R 2 value (0.9996) proved the strong linearity. When we measured the chelate-to-IgG (chelate/IgG) molar ratios, we set [IgG] at a fixed value for both the standard curve of DTPA and the IgG–DTPA-di conjugate samples. These fixed [IgG] values were in a range of 0.06–0.12 µM. On the standard curve depicting the DTPA in the presence of a fixed concentration of IgG, the reduction in A652, which was due to the Y(III)–Arsenazo III reagent’s adsorption to and interaction with IgG, was much smaller than the corresponding reduction that is observable on the DOTA’s standard curve. We can observe this relative smallness by comparing the x-intercept presented in Fig. 2c (0.0605 for the DOTA standard curve) and the corresponding x-intercept presented in Fig. 3 (0.0986 for the DTPA standard curve). This smallness stems from the fact that the DTPA experiments were conducted at a lower reaction temperature, and for a much shorter reaction period than was the case for the DOTA experiments. We conjugated the DTPA moiety to IgG using the DTPA-di reagent at various pH levels in two buffer systems. Results are summarized in Runs 1–11 of Table 4. By using the [DTPA] standard curve, we measured the DTPA-di/IgG molar ratios of the IgG–DTPA-di conjugates. We prepared Runs 1–11 with a DTPA-di/IgG feed molar ratio of 10.0 and obtained large DTPA-di/IgG product molar ratios of 7.0 or more. When the DTPA-di/IgG feed molar ratio was reduced from 10, considerably high reaction yields were also obtained, as shown in Runs 12 and 13 at pH 8.5. These reaction yield values are much greater than previously reported values for the conjugation of DTPA-di to proteins (Arano et al. 1996 ; McLarty et al. 2009 ; Scollard et al. 2011 ; Tang et al. 2005 ) (see Table S2). Conjugation of CHX-A”-DTPA to IgG and measurements of the CHX-A”-DTPA /IgG molar ratios of the IgG–CHX-A”-DTPA conjugate For conjugates obtained with the CHX-A”-DTPA chelating reagent, we used a standard curve of [DTPA]. When we determined chelate/IgG molar ratios, we set two fixed values: a fixed value for [IgG] for the standard DTPA curve, and a fixed value for the volume of CHX-A"-DTPA conjugate samples. Below, we explain our reason for fixing the latter value. After conjugation of the CHX-A”-DTPA reagent to IgG, one phenyl ring is present for each CHX-A”-DTPA moiety conjugated to IgG. This phenyl ring may affect A280-based [IgG] measurement results by biasing them upward, leading to underestimation of the chelate/IgG ratio, if there is no correction of the estimated [IgG]. To correct for this potential bias with respect to the CHX-A"-DTPA/IgG molar ratios, we relied on a model compound that simulated the CHX-A"-DTPA moiety conjugated to IgG. A chemical structure of this model compound is shown in scheme (1) of Fig. S6. Details of this correction process are provided in the Supplementary Information (the text and Table S1 ). Table 5 summarizes the post-correction CHX-A"-DTPA/IgG molar ratios obtained at various pH values. Reaction yields of the CHX-A”-DTPA reagent ranged from 32% to 45%. These values, despite being smaller than those of the DOTA-NHS and DTPA-di reagents, are still reasonably high for reaction yields of chelating agents working in aqueous buffers. The reaction yields that we obtained for the CHX-A"-DTPA reagent were either much greater than previously reported values (Price et al. 2016 ; Winter et al. 2023 ) or approximately equal to them (Cassells et al. 2021 ; Strand et al. 2021 ). The “dry-handling technique” for chelating-agent solutions and its effect on reaction yields In following the handling procedures for chelating-agent solutions, we strictly maintained dry (dehydrated) and inert environments (see the Supplementary Information for details). Because we were unsure whether or not this dry-handling technique would sufficiently raise the reaction yields of the chelating agents, we carried out a set of experiments comparing the dry-handling technique with the normal-handling technique. In the normal-handling technique, we prepared 5-mM chelating-agent solutions by diluting the 50-mM chelating-agent solutions in dehydrated DMSO with reagent-grade (not dehydrated) DMSO. Therefore, the 5-mM chelate solutions prepared according to the normal-handling technique contained 90% reagent-grade DMSO. Additionally, before using them, we incubated (at r.t. for 1 day) three kinds of 5-mM chelate stock solutions containing 90% reagent-grade DMSO. If water contaminated in the reagent-grade DMSO reacts significantly with the chelating agents during this 1-day incubation, the reaction yields of these chelating agents should be lower than the reaction yields obtained in the dry-handling technique. Results are summarized in Table 6. For DOTA-NHS, the dehydrated environment that we created for the dry-handling technique provided markedly higher reaction yields than did the normal environment, even though the difference did not reach statistical significance (p = 0.09, n = 3) in an unpaired Student’s t-test. No marked differences between the reaction yields for the dehydrated environment and those for the normal environment were observed for the other reagents, DTPA-di and CHX-A"-DTPA. 1) Reactions were conducted at a chelating agent/IgG molar ratio of 10.0 in a 50 mM pH 8.5 borate-KCl buffer. The reaction conditions were: 0°C for 2.0 h and r.t. for 1.5 h for DOTA-NHS, and 25°C for 2.0 h for DTPA-di and CHX-A”-DTPA. For each reagent, we carried out three runs, and from them, we calculated the averages and standard deviations. Discussion Conjugation reactions involving chelating agents are an essential process for the metal-ion labeling of proteins. This labelling allows for protein-biodistribution analyses and pharmacokinetic analyses in animal experiments and human clinical practices. Additionally, metal-ion labeling can endow proteins with critical cancer-fighting cytotoxic functions. In these experimental and clinical applications, chemical conjugation reactions that involve facile procedures, high yields, and high repeatability are desirable. An assay method for determining the chelate-to-protein (chelate/protein) molar ratio is a critical factor in these conjugation reactions, as this ratio governs the capacity of the chelating agents to label metal ions and to preserve the proteins’ original biological functions. If the ratios are too small, the diagnostic and therapeutic functions of the metal ions will be deficient. If the ratios are too large, the desired biological functions of the proteins may be compromised or even entirely extinguished owing to excessive protein modification by the chelating moieties. In general, three assay methods are available for measurements of the chelate/protein molar ratios: (1) the radioisotope method, (2) the mass spectroscopic method, and (3) the colorimetric method . (1) The radioisotope method: Known amounts of a mixture of a nonradioactive (“cold”) metal ion and a radioactive metal ion of the same element are coordinated into a protein-chelate conjugate. In this feed of both the metal ions, the total ion mole number must be greater than the expected chelate’s mole number. After the coordination reaction of these ions into the conjugate is complete, the chelate/protein molar ratio is calculated from the molar ratio of the coordinated radioactive metal ion to the uncoordinated radioactive metal ion, as measured by means of thin-layer chromatography (Arano et al. 1996; McLarty et al. 2009; Meares et al. 1984; Oskar et al.2021; Scollard et al. 2011; Strand et al. 2021; Sudo et al. 2023; Tang et al. 2005; Timmermand et al. 2021), high-performance liquid chromatography, or other methods. The radioisotope method requires a facility where radioactive materials can be handled. (2) The mass spectroscopic method (Cassells et al. 2021; D'Huyvetter et al. 2012; Hoppmann et al. 2011; Price et al. 2016; Rossin et al. 2011;Sugyo et al. 2015;): Molecular weights of an intact protein and a protein–chelate conjugate are measured typically by means of MALDI-TOF mass spectroscopy. The chelate/protein molar ratio is calculated from the difference between the two molecular weights. The mass spectroscopic method requires an expensive analytical instrument: a mass spectrometer. (3) The colorimetric method (Abadi et al. 2021; Alirezapour et al. 2013; Pippin et al. 1992; Winter et al. 2023): The amount of chelate is measured by the use of a chemical colorimetric reagent. One well-known example of a colorimetric reagent is the reagent that we used in the current study: the Y(III)–Arsenazo III reagent. Y 3+ ions are coordinated within the Arsenazo III moieties. The coordination affinity of Y 3+ ions in the Arsenazo III moiety is much lower than the affinities in such representative chelate compounds as DOTA and DTPA. Therefore, in the presence of these chelates, the Y 3+ ion transfers from the Arsenazo III moiety to the chelate moieties. This transfer causes a change in the VIS light spectrum, and this change at 652 nm can be measured with a spectrometer. Consequently, one can calculate the amount of the chelate by calculating the precise reduction in absorbance at 652 nm. (A652). The colorimetric method can be performed if a relatively inexpensive instrument, a UV-VIS spectrometer, is available. However, reaction conditions for the transfer reaction must be optimized for specific chelate compounds and specific proteins. For the present study, we examined the colorimetric method in conjunction with the Y(III)–Arsenazo III reagent and then compared some of these results with our mass-spectroscopy results. For DTPA-di-derived IgG–DTPA conjugates, we easily performed the quantitative colorimetric analysis of the DTPA-moiety/IgG molar ratio by following the reaction conditions (r.t., 15 min.) described in a previously published paper (Pippin et al. 1992). In conducting the colorimetric assay for IgG–DOTA-NHS conjugates derived from DOTA-NHS, we had to identify and resolve several technical problems. Initially, we planned to conduct the assay on the basis of two papers published by the same research group (Abadi et al. 2021 and Alirezapour et al. 2013). However, neither of the papers provided any information whatsoever regarding the two most critical reaction conditions: temperature and duration. Therefore, to obtain a standard curve for [DOTA] determination, we conducted our first attempted assay at r.t. for 15 min. However, as shown in Fig. 1a, no change in the VIS light spectrum was observed under these reaction conditions for DOTA in the Y(III)–Arsenazo III solution. A well-known disadvantage of DOTA and its derivatives is the slowness with which they react to metal ions (Kasprzyk et al. 1982; Kodama et al. 1991; Li et al. 2023; Sherry et al. 1989; Sugyo et al. 2015; Suzuki et al. 2024; Wang et al. 1992). Therefore, for the reaction between DOTA and the Y(III)–Arsenazo III reagent, we tried high temperatures at 80°C and 56°C, and we found a [DOTA]-dependent colorimetric change of the Y(III)–Arsenazo III reagent at these high temperatures. (see Fig. 1b, Figs. S4a, and S4b) However, at these high temperatures, we observed an unfavorable 652 nm peak shift toward longer wavelengths (red shift). We speculated that the red shift was probably due to protein denaturing at these high temperatures. Consequently, we again sought to optimize the reaction conditions for the [DOTA] determination, this time by fixing the temperature at 37°C and the duration at 3 days. From this experiment, we successfully obtained the same degree of reduction in A652 values as we had obtained for the [DTPA] determination, but we avoided any red shift at 652 nm. This last experiment strongly indicates that the reduction in A652 was due to a transfer of Y 3+ ions from Arsenazo III moieties to any chelate possessing a Y 3+ -ion coordination affinity higher than the one associated with Arsenazo III moieties. Furthermore, this fact indicates that the standard curve obtained with a parent chelate compound (e.g., DTPA and DOTA) can be used to determine the chelate derivatives that are found in IgG–chelate conjugates. For example, an intact DOTA molecule possesses 8 functional groups for coordination, while the DOTA derivative moiety that was conjugated to IgG possesses only 7 functional groups for coordination, as one functional group is lost in the conjugation of the DOTA-NHS to IgG. We found that, when present, IgG reduced the levels of A652 in the Y(III)–Arsenazo III solution, as shown in Fig. 2bs and Fig. S5. This reduction in A652 occurred even in the DTPA-based assay systems involving relatively short reaction periods, such as 15 min. In order to deal with this reduction in A652, we maintained a fixed concentration of IgG ([IgG]) throughout all the colorimetric measurements for both the standard-curve samples and the conjugate samples. Concentrations of the chelate moieties, [chelate moiety], in the IgG–chelate conjugates were obtained in colorimetric measurements, while the concentration of IgG, [IgG], in these same conjugates was obtained from absorbance measurements at 280 nm. Accordingly, the chelate/IgG molar ratio can be obtained from these [chelate moiety] and [IgG] values. We encountered another technical problem in the colorimetric determination of the chelate/IgG molar ratios for IgG–CHX-A"-DTPA conjugates derived from CHX-A"-DTPA. Because the CHX-A"-DTPA reagent contains one phenyl (benzene) ring structure, and because this phenyl ring remains in the CHX-A"-DTPA derivative of the IgG–CHX-A"-DTPA conjugate, the UV-absorption property of the phenyl ring may interfere with A280 determination of [IgG] values. In order to solve this problem, we synthesized a model compound that, derived from the CHX-A"-DTPA reagent and ethylenediamine, could simulate the chemical structure of the CHX-A"-DTPA moiety conjugated to IgG. Then, we obtained a standard curve of the model compound’s A280 plotted against the [model compound], as shown in Figure S7b. From the reduction in A652, we obtained the [CHX-A"-DTPA moiety] of the IgG–CHX-A"-DTPA conjugates. We then calculated the contribution of the CHX-A"-DTPA moiety to the A280 values of the conjugates by using the standard curve of the model compound. Accordingly, by subtracting the calculated A280 value of the CHX-A"-DTPA moiety from the initially measured A280 value that contained both absorbance of IgG and absorbance of the CHX-A"-DTPA moiety, we could compensate for the true A280 value representing only [IgG]. Furthermore, we examined possible effects of impurities contaminating in DMSO and ethylenediamine reagents on the A280 measurements. As described closely in Supplemental Information and Fig. S8, these effects were turned out to be negligible. Yet another problem presented itself, this time regarding the IgG–CHX-A"-DTPA conjugate. The true [IgG] in the IgG–CHX-A"-DTPA conjugate solutions was unknown prior to the colorimetric assay owing to the presence of one phenyl ring from the conjugated CHX-A"-DTPA moiety, as described above. Therefore, we conducted the colorimetric assay by adding a fixed volume (24 mL) of the conjugate sample solutions into the Y(III)–Arsenazo III solution, irrespective of the samples’ A280 values. Because of this fixed volume, a potential problem arose: a difference between the [IgG] set for the standard curve and the true [IgG] of the conjugate in the colorimetric assay might cause an error in the estimation of the chelate/IgG molar ratios. We examined this potential problem (as described in the text of Supplemental Information and Figure S9) and determined that any errors stemming from this problem would be negligible. Thanks to the many carefully executed steps described above, we successfully established the colorimetric assay methods for three kinds of chelate-IgG conjugates with the Y(III)–Arsenazo III reagent. We compared the chelate/IgG molar ratios obtained from the colorimetric method with the chelate/IgG molar ratios obtained from the mass spectroscopic method. As summarized in Table 3, these molar-ratio values were in good agreement with each other. Therefore, researchers can safely choose either of these two methods, with the right choice dependent on, for example, the availability of instruments in a lab and the lab’s relative familiarity with the methods and their specific techniques. Using the colorimetric assays with the Y(III)–Arsenazo III reagent, we analyzed the chelate/IgG molar ratios for three kinds of reactive chelating agents: DOTA-NHS, DTPA-di, and CHX-A"-DTPA. We conducted a comparison between our study’s reaction yields for these three kinds of chelating agents (see Table 6) and previous studies’ reported reaction yields (see Table S2). The values of our study’s yields are much higher than the values reported in the previous studies, with only two exceptions (both of which were for CHX-A"-DTPA: Cassells et al. (2021) and Strand et al. (2021). These comparisons demonstrate that we have successfully established conjugation procedures achieving very high reaction yields for the three kinds of chelating agents studied herein. We believe the central reason for these high reaction yields was our use of chelate stock solutions in dehydrated DMSO. Each of the three chelating agents involved a unique reactive group: DOTA-NHS used N-hydroxysuccinimide ester, DTPA-di used acid anhydride, and CHX-A"-DTPA used phenyl thioisocyanate. Regarding conjugation to proteins in aqueous buffers, all these reactive groups favor high pH levels because only proteins’ free amino groups (–NH2), which are the favorable amino-group form at high pH levels, can react with these reactive groups, and because the protonated form of the amino group (–NH3 + ), which is the favorable form at low pH levels, cannot react with the reactive groups of the chelating agents. That is why we frequently carried out the conjugation reactions of the chelating agents at high pH levels, such as 8.5 and 9.2. When the conjugation reactions of these chelating agents are conducted in pure aqueous buffers that contain no DMSO, the first step in the multi-step synthesizing procedures must be to dissolve the chelating agents in the buffers. During this dissolution step, considerable amounts of the chelating agents may react with hydroxide ions, resulting in a substantial loss of these reagents before they react with the primary amino groups of the proteins. In particular, this side reaction occurs aggressively at high pH levels, where high concentrations of hydroxide ions exist. Furthermore, the time period required for complete dissolution can vary substantially depending on an experimenter’s technical skill. In sum, the longer the period, the larger the reagent loss. Therefore, significant variation in the dissolution period can lead to significant variation in the reaction yields of the chelating agents. We used a synthesizing procedure in which we added the chelating-agent solutions (in dehydrated DMSO) to the IgG solutions in aqueous buffers. In this procedure, the chelating agents started their reactions as soon as the addition of the chelating-agent solutions to the IgG solutions was complete. The rapid onset of the reactions enabled us to avoid any loss of the chelating agents that would have arisen from their side reactions with hydroxide ions during the dissolution step. The result was a series of high reaction yields. Additionally, we believe that, because we can evade technical differences among experimenters in our synthetic procedure, we obtained a narrow band of inconsistent variation in the reaction yields of the chelating agents. Therefore, in our conjugation of the three chelating agents to IgG, we achieved both high reaction yields and high reaction-yield repeatability. In some previously published papers (Arano et al. 1996; Cassells et al. 2021; Sugyo et al. 2015), chelating-agent solutions were used in the conjugation of either DMSO or dehydrated DMSO to proteins. However, these papers omit important details about the reaction procedures or fail to examine the reaction yields of the chelating agents relative to the optimum pH levels of the reaction buffers. Additionally, we elucidate the effects that dehydrated solvents and dry-handling techniques can have on the reaction yields. Therefore, we believe the results of the current paper constitute very valuable technical information for researchers conducting research on the conjugation of chelating agents to proteins, particularly in the field of nuclear medicine. Conclusions In the present study, we examined the conjugation of three chelating agents (DOTA-NHS, DTPA-di, and CHX-A"-DTPA) to human IgG and measured the chelate/IgG molar ratios of the IgG–chelate conjugates using a colorimetric method with the Y(III)–Arsenazo III reagent. For DOTA-NHS and CHX-A"-DTPA, we used this colorimetric method to establish novel methods for the accurate measurement of the chelate/IgG molar ratios. Additionally, we succeeded in obtaining high reaction yields and high repeatability in these conjugation reactions by using stock solutions of the reactive chelating agents in dehydrated DMSO in combination with a dry-handling technique. Abbreviations DOTA 1,4,7,10-detraazacyclododecane-1,4,7,10-tetraacetic acid DTPA diethylenetriaminepentaacetic acid DOTA-NHS 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester NHS N-hydroxysuccinimide ester DTPA-di diethylenetriaminepentaacetic dianhydride p-SCN-Bn-CHX-A”-DTPA S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid CHX-A”-DTPA S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid, MALDI matrix-assisted laser desorption/ionization TOF time-of-flight D-PBS Dulbecco’s phosphate-buffered saline. r.t. room temperature A652 absorbance at 652 nm A280 absorbance at 280 nm Declarations Supplementary Information The online version contains supplementary information available at https:// . Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by Grant-in-Aid for Scientific Research (B) grant number 24K03286 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This study was also supported by a grant with grant number JP25yf0126003 from the Japan Agency for Medical Research and Development. Partial support for this study was forthcoming from the MSD Life Science Foundation, Public Interest Incorporated Foundation. Author contributions MY developed experimental plans for all the studies and carried out conjugation reactions and colorimetric assays of the obtained conjugates. MY was also a major contributor to the writing of the manuscript. KS contributed in syntheses and analyses of IgG–chelate conjugates. TK, YI, HO, and YM designed the IgG–chelate conjugates by paying special attention to the number of conjugated chelate molecules per IgG molecule. AT chose chelating reagents for the conjugation reactions and wrote about the number of conjugated chelate molecules per one IgG molecule. All authors read and approved the final manuscript. Acknowledgements For their valuable advice and technical assistance in mass spectroscopy measurements, we are indebted to Dr. Shushi Nagamori and Dr. Yumi Kanegae (the Advisory Board for Mass Spectrometry at Core Research Facilities in Center for Medical Science, the Jikei University School of Medicine), and to Ms. Erika Osada and Ms. Emi Tsuchitani (Core Research Facilities, the Jikei University School of Medicine) for their providing valuable advices and technical assistance in mass spectroscopy measurements. 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Inorg Chem. 1992;31:1095-1099. https://pubs.acs.org/doi/10.1021/ic00032a034 Winter G, Hamp-Goldstein C, Fischer G, Kletting P, Glatting G, Solbach C, Herrmann H, Sala E, Feuring M, Döhner H, Beer AJ, Bunjes D, Prasad V. Optimization of radiolabeling of a [90Y]Y-Anti-CD66-antibody for radioimmunotherapy before allogeneic hematopoietic cell transplantation. Cancers (Basel). 2023;15(14):3660. doi: 10.3390/cancers15143660. Tables Tables 1 to 6 are available in the Supplementary Files section. Supplementary Files Tablesummaryfinal2.pdf SupplementaryInformationfinal2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8683063","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581124296,"identity":"f0737901-f77c-479a-b8f0-b18b55b998dd","order_by":0,"name":"Masayuki Yokoyama","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-6053-1856","institution":"The Jikei University School of Medicine: Tokyo Jikeikai Ika Daigaku","correspondingAuthor":true,"prefix":"","firstName":"Masayuki","middleName":"","lastName":"Yokoyama","suffix":""},{"id":581124297,"identity":"7b3ea330-0849-4ba9-9870-eb27cd51a90c","order_by":1,"name":"Kouichi Shiraishi","email":"","orcid":"","institution":"Jikei University School of Medicine: Tokyo Jikeikai Ika Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Kouichi","middleName":"","lastName":"Shiraishi","suffix":""},{"id":581124298,"identity":"c2e6995e-d8ac-48e9-a829-931fdff56133","order_by":2,"name":"Teppei Komatsu","email":"","orcid":"","institution":"Jikei University School of Medicine: Tokyo Jikeikai Ika Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Teppei","middleName":"","lastName":"Komatsu","suffix":""},{"id":581124299,"identity":"1fd6fa68-1360-440f-903f-d9ace7045a5b","order_by":3,"name":"Yasuyuki Iguchi","email":"","orcid":"","institution":"Jikei University School of Medicine: Tokyo Jikeikai Ika Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Yasuyuki","middleName":"","lastName":"Iguchi","suffix":""},{"id":581124300,"identity":"eaf50d4d-50c2-4fe0-9c13-21dfc169e727","order_by":4,"name":"Hiroki Ohta","email":"","orcid":"","institution":"Jikei University School of Medicine: Tokyo Jikeikai Ika Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Ohta","suffix":""},{"id":581124301,"identity":"3662bc96-048b-4110-9374-d29a2f913be6","order_by":5,"name":"Yasuhiro Matsumura","email":"","orcid":"","institution":"Laboratory of Rin Institute Inc.","correspondingAuthor":false,"prefix":"","firstName":"Yasuhiro","middleName":"","lastName":"Matsumura","suffix":""},{"id":581124302,"identity":"574f23ce-451c-4f51-af7a-585992d93a63","order_by":6,"name":"Atsushi B Tsuji","email":"","orcid":"","institution":"National Institutes for Quantum Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"B","lastName":"Tsuji","suffix":""}],"badges":[],"createdAt":"2026-01-24 01:35:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8683063/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8683063/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101419219,"identity":"239c43a2-1716-47ea-a872-b3e9ae15394f","added_by":"auto","created_at":"2026-01-29 13:13:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":467618,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of temperature and time on the reactions involving the Y(III)–Arsenazo III solution and the DTPA and DOTA chelates. In Figs. 1a–1c, the black solid line represents the negative control at r.t., the red line represents 1.0 μM of DTPA, and the blue line represents 1.0 μM of DOTA. In Figs. 1b and 1c, the black dotted line represents the Y(III)–Arsenazo III solution in the absence of any chelate. \u003cstrong\u003e\u0026nbsp;(a) \u003c/strong\u003eReactions at r.t. for 10 min.\u003cstrong\u003e (b) \u003c/strong\u003eReactions at 80°C for 20 min. \u003cstrong\u003e(c) \u003c/strong\u003eReactions at 37°C for 3 days under room light. The red and blue lines completely overlap around the 652 nm peak.\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8683063/v1/b478c252b92349fab5304805.jpg"},{"id":101419285,"identity":"4e5570ab-857f-4baa-a2a5-462f0372a53a","added_by":"auto","created_at":"2026-01-29 13:13:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":491082,"visible":true,"origin":"","legend":"\u003cp\u003eColorimetric determination of [DOTA] with a Y(III)–Arsenazo III solution at 37°C for 3 days. \u003cstrong\u003e(a) \u003c/strong\u003eA standard curve of [DOTA] in the absence of IgG.\u003cstrong\u003e (b)\u003c/strong\u003e [DOTA]-dependent spectrum change in the presence of 0.12 μM of IgG. \u003cstrong\u003e(c) \u003c/strong\u003eA standard curve of [DOTA] in the presence of IgG, as derived from Fig. 2b.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8683063/v1/b347236f6e939e396d030af6.jpg"},{"id":101419263,"identity":"0f2bfc49-4df9-49eb-858d-4c3825ba6c1b","added_by":"auto","created_at":"2026-01-29 13:13:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":328881,"visible":true,"origin":"","legend":"\u003cp\u003eA standard curve of [DTPA] in the presence of 0.12 μM of IgG. \u0026nbsp;Y(III)–Arsenazo III solutions were reacted with various concentrations of DTPA in the presence of 0.12 μM of IgG at room temperature for 15 min.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8683063/v1/2afa093b2302547430b0571f.jpg"},{"id":101419287,"identity":"eac2f952-f918-4d26-958a-0114f0cf9df1","added_by":"auto","created_at":"2026-01-29 13:13:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":442765,"visible":true,"origin":"","legend":"\u003cp\u003eMass spectra of intact IgG and three IgG–DOTA conjugates. The black line: intact monoclonal IgG, the blue line: conjugate A1, the green line: conjugate A2, and the red line: conjugate A3.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8683063/v1/92a7d6f0bd779029503ccb3d.jpg"},{"id":103505112,"identity":"fcc71302-d303-4db5-b4b6-50eec0451be6","added_by":"auto","created_at":"2026-02-26 13:24:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2645590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8683063/v1/b9368973-1728-4f1e-8179-34aaa4bda8d3.pdf"},{"id":101419286,"identity":"667fcbfe-2a5c-4ba0-a22f-93d751dc080d","added_by":"auto","created_at":"2026-01-29 13:13:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":140478,"visible":true,"origin":"","legend":"","description":"","filename":"Tablesummaryfinal2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8683063/v1/022e29beab56d3bb1806eec6.pdf"},{"id":101419224,"identity":"38c13ea9-da28-4a55-8573-3e4053aa5e99","added_by":"auto","created_at":"2026-01-29 13:13:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6572547,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationfinal2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8683063/v1/89914e078e35d447298cef3c.docx"}],"financialInterests":"","formattedTitle":"Efficient Conjugation of Chelating Agents to IgG and Accurate Colorimetric Determination of the Chelate-to-IgG Molar Ratio.","fulltext":[{"header":"Background","content":"\u003cp\u003eChelate compounds are essential chemicals that serve importantly as a metal-ion coordinator of proteins for medical diagnoses and therapies. For example, the chelating agent DTPA (diethylenetriaminepentaacetic acid) and its derivatives have been widely used in both research and clinical applications. Likewise, the chelating agent DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and its derivatives have also been extensively used in these same applications owing to their higher coordination affinities than DTPA\u0026rsquo;s. This higher coordination affinity exhibited by DOTA is attributable to its cyclic chemical structure. Representative metal ions used for imaging diagnoses include \u003csup\u003e67\u003c/sup\u003eGa, \u003csup\u003e99m\u003c/sup\u003eTc, and \u003csup\u003e111\u003c/sup\u003e In, while \u003csup\u003e90\u003c/sup\u003eY, \u003csup\u003e177\u003c/sup\u003eLu, and \u003csup\u003e211\u003c/sup\u003eAt are used for radionuclide-based therapies. Additionally, Gd is clinically employed as a positive MRI contrast agent in coordinated structures within low-molecular-weight chelates.\u003c/p\u003e \u003cp\u003eTo conduct medical diagnoses and therapies involving metal ions, medical practitioners use conjugates consisting of proteins, chelating agents, and metal ions. The first step in preparing these conjugates is the conjugation of chelate compounds to the given protein. For avoidance of protein denaturation, this conjugation reaction must be conducted in either pure aqueous buffers or aqueous buffers containing small amounts of water-miscible organic solvents such as ethanol and DMSO. Consequently, the reaction yields for this type of conjugation are typically much lower than those obtained in the standard organic chemistry carried out in organic solvents. These lower reaction yields occur because active chemical species used for the conjugation\u0026mdash;such as N-hydroxysuccinimide esters, acid anhydrides, and phenyl isothiocyanates\u0026mdash;react not only with the primary amino groups of proteins but also with hydroxide ions (OH\u003csup\u003e\u0026minus;\u003c/sup\u003e) present in aqueous buffers. Moreover, these conjugation reactions are favored at higher pH because only the unprotonated form (\u0026ndash;NH2) of the primary amino group can react with these active chemical species. In contrast, the protonated form (\u0026ndash;NH3\u003csup\u003e+\u003c/sup\u003e) that is predominant at low pH cannot react with the active chemical species. This explains why reported reaction yields for chelate compounds were generally low, typically ranging from 2% to less than 15% (Abadi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alirezapour et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Arano et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Hoppman et al. 2011; Sudo et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Winter et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother potential problem in the conjugation of chelating agents to proteins is the poor reproducibility of the reaction yields. Most published papers report only a single value for the reaction yield, and to our knowledge, no paper has examined and reported the yield reproducibility.\u003c/p\u003e \u003cp\u003eIn previously published papers concerning the conjugation of chelating agents to proteins, an array of experimental procedures have been reported involving, for example, various kinds of buffers, pH levels, temperatures, reaction times, and concentrations of chelating agents and proteins. Therefore, many readers of these papers, including the authors of the present study, have had great difficulty ascertaining which reaction conditions are generally optimal. In the current paper, we aim to identify several experimental factors affecting the conjugation of chelating agents to proteins and we present a facile and reproducible procedure that achieves high reaction yields.\u003c/p\u003e \u003cp\u003eIt is well known that the coordination affinity for metal ions is higher in DOTA and its derivatives than in DTPA and its derivatives. This superior affinity constitutes an important advantage of DOTA-based chelates over the other types of chelates. However, the coordination reactions in which metal ions form complexes with DOTA and its derivatives proceed much more slowly than the corresponding coordination reactions involving other types of chelates, such as DTPA and its derivatives. The relative slowness of the kinetics for DOTA-based chelates not only hinders coordination reactions where metal ions serve as imaging markers and therapeutic agents, but also poses a technical problem for determining chelate concentrations via a Y\u0026sup3;⁺-based colorimetric agent, the Y(III)\u0026ndash;Arsenazo III complex reagent. We therefore examined both the reaction temperatures and reaction periods to optimize the colorimetric determination of the DOTA moieties conjugated to human IgG as a model protein.\u003c/p\u003e \u003cp\u003eWe encountered yet another technical problem when we measured the chelate-to-IgG (chelate/IgG) molar ratios for CHX-A\u0026rdquo;-DTPA-based IgG\u0026ndash;chelate conjugates. Present in the chemical structure of this chelating agent is one phenyl (benzene) ring, which\u0026mdash;because it can exhibit absorbance at 280 nm (A280)\u0026mdash;may significantly interfere with the A280 measurement of IgG concentrations, [IgG]. To address this problem, we performed three steps: (1) we synthesized a model compound that mimicked the CHX-A\u0026rdquo;-DTPA moiety conjugated to IgG, (2) we estimated how much the model compound contributed to absorbance at 280 nm, and (3) we obtained the true chelate/IgG molar ratio values of the IgG\u0026ndash;CHX-A\u0026rdquo;-DTPA conjugates.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eWe obtained a chelate compound, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS, Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) bearing an N-hydroxysuccinimide ester reactive group through an ordered synthesis made by Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). We purchased two commercially available chelate compounds; diethylenetriaminepentaacetic acid (DTPA, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and diethylenetriaminepentaacetic dianhydride (DTPA-di, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). We purchased a chelate compound, p-SCN-Bn-CHX-A\u0026rdquo;-DTPA (CHX-A\u0026rdquo;-DTPA, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) from Macrocyclics, Inc. (Plano, USA).\u003c/p\u003e \u003cp\u003eWe purchased the Arsenazo III (2,2'-[1,8-Dihydroxy-3,6-disulfo-2,7-naphthalene] bis(azo)dibenzenearsonic acid) reagent from FUJIFILM Wako Pure Chemical Corp. (Tokyo, Japan) and YCl3\u0026middot;6H2O from Sigma-Aldrich Japan G.K. (Tokyo, Japan). We prepared a colorimetric agent, the Y(III)\u0026ndash;Arsenazo III solution, according to a specific reference (Pippin et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). We created a 2-fold-concentrated Y(III)\u0026ndash;Arsenazo III stock solution containing 3.2 \u0026micro;M Y(III) and 10 \u0026micro;M Arsenazo III in 0.15 M sodium acetate buffer at pH 4.0.\u003c/p\u003e \u003cp\u003eWe also made the following two purchases: dehydrated DMSO from FUJIFILM Wako Pure Chemical Corp. (Tokyo, Japan); and human IgG (Product No. I4506) from Sigma-Aldrich Japan G.K. (Tokyo, Japan). Other reagents were of reagent grade. A monoclonal IgG1 was kindly provided by the Laboratory of RIN Institute Inc. (Tokyo, Japan).\u003c/p\u003e \u003cp\u003eFor mass spectroscopy, we purchased sinapic acid (Product No. 198-13363) from FUJIFILM Wako Pure Chemical Corp. (Tokyo, Japan) and protein standard II (Part No. 8207234) from Bruker, Japan (Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe conjugation of chelating agents to immunoglobulin G (IgG): Preparation of IgG–chelate conjugates\u003c/h3\u003e\n\u003cp\u003eWe added 100 \u0026micro;L of human IgG solution (10.0 mg/mL in serine) to a 1.5 mL Eppendorf tube. Then, we added 100 \u0026micro;L of either 0.10 M borate-KCl buffer or 0.20 M HEPES buffer. Most commonly, we used the former buffer at pH 8.5. Next, we added a chelating-agent solution (5 mM) in dehydrated DMSO. For DOTA-NHS, we stirred this reaction mixture at 4\u0026deg;C for 2.0 h, followed by reaction at room temperature (r.t.) for 1.5 h. For DTPA-di and CHX-A\u0026rdquo;-DTPA, the reaction took place at 25\u0026deg;C for 2.0 h. After the reactions, we purified the reaction mixtures by means of gel-filtration chromatography using a PD MidiTrap\u0026trade; G-25 column (Cytiva, Tokyo, Japan). We collected a 0.5\u0026ndash;1.5 mL fraction eluted with Dulbecco\u0026rsquo;s phosphate-buffered saline (D-PBS) to remove unreacted low-molecular-weight chelating reagents.\u003c/p\u003e\n\u003ch3\u003eColorimetric determination of the chelate concentrations\u003c/h3\u003e\n\u003cp\u003eWe performed colorimetric assays of chelate concentrations using the Y(III)\u0026ndash;Arsenazo III reagent solution. To this end, we followed (with some modifications) the steps presented in the previously mentioned reference (Pippin et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). In the presence of chelate compounds, the Y\u003csup\u003e3+\u003c/sup\u003e ion transfers from the Arsenazo III moiety to the chelate, altering the visible (VIS) light spectrum; we used absorbance at 652 nm (A652) to determine the chelate concentration.\u003c/p\u003e \u003cp\u003eWe added 450 \u0026micro;L of the 2-fold concentrated Y(III)\u0026ndash;Arsenazo III solution to a 1.5 mL Eppendorf tube. To it, we then added (450 \u0026ndash; x) \u0026micro;L of 0.15 M sodium acetate buffer at pH 4.0, followed by x \u0026micro;L of the IgG\u0026ndash;chelate conjugate solution in D-PBS. Consequently, the total volume of the reaction mixture was 900 \u0026micro;L. The added volume (x \u0026micro;L, typically 20\u0026ndash;30 \u0026micro;L) of the IgG\u0026ndash;chelate solution was adjusted on the basis of absorbance at 280 nm (A280). Our reason for taking this step was to maintain, throughout one series of experiments, a fixed [IgG] ranging from 0.03 to 0.12 \u0026micro;M in the 900-\u0026micro;L reaction mixture.\u003c/p\u003e \u003cp\u003eIn parallel, we prepared a standard curve of A652 values plotted against chelate concentrations. For this standard curve, we added 450 \u0026micro;L of the 2-fold concentrated Y(III)\u0026ndash;Arsenazo III solution to a 1.5 mL Eppendorf tube. To it, we then added (450 \u0026ndash; x \u0026ndash; y) \u0026micro;L of 0.15 M sodium acetate buffer at pH 4.0, followed, first, by x \u0026micro;L of an intact IgG solution in saline at 2.7\u0026ndash;5.4 \u0026micro;M and, thereafter, by y \u0026micro;L of a chelate solution in 0.15 M sodium acetate buffer at pH 4.0 (Added in this order). The total volume of the reaction mixture was 900 \u0026micro;L. The x \u0026micro;L of intact IgG solution was set for the same IgG concentration as that of the IgG\u0026ndash;chelate conjugates, and the y \u0026micro;L of the chelate solution varied in a range extending from 0.2 to 1.2 \u0026micro;M in the 900 \u0026micro;L solution. Chelates used for this standard-curve preparation were DOTA for the IgG\u0026ndash;DOTA-NHS conjugate and DTPA for both the IgG\u0026ndash;DTPA-di and the IgG\u0026ndash;CHX-A\u0026rdquo;-DTPA conjugates.\u003c/p\u003e \u003cp\u003eThe reaction-duration conditions for IgG\u0026ndash;DTPA-di and IgG\u0026ndash;CHX-A\u0026rdquo;-DTPA were r.t. and 10 min. In these cases, the VIS spectra of IgG\u0026ndash;chelate conjugates were unchanged over the 10-min reaction period.\u003c/p\u003e \u003cp\u003eReaction conditions for IgG\u0026ndash;DOTA-NHS were 37\u0026deg;C and 3 days. These conditions were optimized after pre-examination at various temperatures and time periods. Details of this pre-examination will be described in the Results section.\u003c/p\u003e\n\u003ch3\u003eMeasurements\u003c/h3\u003e\n\u003cp\u003eWe recorded UV-VIS spectra using a V-750 UV-Visible Spectrophotometer (JASCO International Co. Ltd., Tokyo, Japan). To determine IgG concentration ([IgG]), we measured absorbance at 280 nm (A280). In the colorimetric assay with the Y(III)\u0026ndash;Arsenazo III solution, we measured absorbance at 652 nm (A652). In actual measurement procedures, we subtracted background absorbance at 600 nm for the IgG assay and at 800 nm for the colorimetric assay to compensate for minute changes in quartz-cell conditions in each measurement. Therefore, (A280 \u0026ndash; A600) values and (A652 \u0026ndash; A800) values were used in the IgG assays and the colorimetric chelate assays, respectively, although only A280 and A652 are described in the rest of this paper.\u003c/p\u003e \u003cp\u003eWe obtained mass spectra of intact IgG and IgG\u0026ndash;DOTA-NHS conjugates by means of MALDI-TOF mass spectroscopy using an Autoflex Speed (Bruker Daltonics GmbH, Germany). After preparing protein solutions in Milli Q water containing 0.10 vol.% TFA at approximately 1 mg protein/mL, we mixed the protein solution with a saturated sinapic acid solution in absolute ethanol containing 0.10 vol.% of TA30 (TA30: 30 vol.% acetonitrile and 0.07 vol.% TFA in MilliQ water). The mixing volume ratio was 1:1. We deposited 0.5 \u0026micro;L of the saturated sinapic acid solution onto the appropriate spot on a ground-steel MALDI target plate to dry. Then, we deposited 1.0 \u0026micro;L of a protein sample solution onto the same spot, which was covered with the dried sinapic acid layer. After the sample solution dried, we measured three MALDI-TOF mass spectra from one spot on the target plate: three spots were measured for each sample. Consequently, nine measurements were conducted for one sample. We obtained the average\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (S.D.) of the most intense molecular ion peak from a total of nine spectra.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOptimization of reaction conditions for the colorimetric assay of DOTA.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe first performed a colorimetric DOTA assay (the chemical structure of DOTA is shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) with the Y(III)\u0026ndash;Arsenazo III solution. In performing this assay, we carefully referred to the reaction conditions reported for a DTPA derivative in a previous study (Pippin et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). As shown in Fig.\u0026nbsp;1a, after a 10-min r.t. reaction, A652 was significantly lower for 1 \u0026micro;M DTPA (red line) than for the control (black line), which involved only the Y(III)\u0026ndash;Arsenazo III solution. By contrast, A652 for DOTA (blue line) was nearly identical to A652 for the control under these same reaction conditions. The rigid tetraazacyclododecane structure of DOTA prevented the Y\u003csup\u003e3+\u003c/sup\u003e ion coordination reaction from proceeding at r.t.. It has been well documented that coordination rates of Gd\u003csup\u003e3+\u003c/sup\u003e metal ions to DOTA are very low (Sherry et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Studies along these lines have also uncovered evidence of low coordination rates for 14 kinds of lanthanide metal ions, including Y\u003csup\u003e3+\u003c/sup\u003e (Kodama et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) and for 11 kinds of divalent metal ions, including Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, and Cu\u003csup\u003e2+\u003c/sup\u003e (Kasprzyk et al. 1982). As a result of these low coordination rates, reaction yields of radionucleotides conjugated to DOTA were not quantitative (100%) for \u003csup\u003e90\u003c/sup\u003eY\u003csup\u003e3+\u003c/sup\u003e (Li et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Sugyo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003csup\u003e111\u003c/sup\u003eIn\u003csup\u003e3+\u003c/sup\u003e (Suzuki et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and \u003csup\u003e90\u003c/sup\u003eY\u003csup\u003e3+\u003c/sup\u003e and \u003csup\u003e111\u003c/sup\u003eIn\u003csup\u003e3+\u003c/sup\u003e (Li et al. 2023) under mild conditions (r.t. to 40\u0026deg;C), whereas a quantitative reaction yield is commonly obtained with DTPA under the mild conditions. Taking these results and reported facts into consideration, we examined the reaction of DOTA at 80\u0026deg;C for 20 min: as shown in Fig.\u0026nbsp;1b, the degree of reduction in A652 was almost the same for 1 \u0026micro;M of DOTA (blue line) as for 1 \u0026micro;M of DTPA (red line). Additionally, as shown in Fig. S2, DTPA and DOTA provided the same degree of reductions after a 20-min reaction at 80\u0026deg;C when the concentrations of these two chelates were identical at 0.5, 1.0, and 2.0 \u0026micro;M. We thus concluded that the reduction in A652 resulted from the elimination of the Y\u003csup\u003e3+\u003c/sup\u003e ion from the Arsenazo III moiety. We further concluded that chelate-coordinated Y\u003csup\u003e3+\u003c/sup\u003e ions exhibited no absorbance at 652 nm irrespective of the differences between the chemical structure of DTPA and that of DOTA.\u003c/p\u003e \u003cp\u003eThe high temperature (80\u0026deg;C) examined in the above-mentioned results is suitable for DOTA determination in the absence of proteins; however, it is not applicable to protein-chelate conjugates because proteins are easily denatured at such a high temperature. Therefore, we examined reactions at 37\u0026deg;C for 3 days. Figure\u0026nbsp;1c shows the spectrum changes with 1.0 \u0026micro;M of DTPA (red line) and 1.0 \u0026micro;M of DOTA (blue line). Both lines completely overlap at about 652 nm. This fact indicates that these reaction conditions (37\u0026deg;C, 3 days) seem appropriate for the DOTA systems. However, absorbance at 652 nm (A652) of a control (37\u0026deg;C, 3 days, black dotted line) was lower than A652 of the negative control (r.t., 10 min, black solid line). This comparison indicates that the reduction in A652 occurring over 3 days of incubation at 37\u0026deg;C was due possibly to the effects of ambient room light on the Y(III)\u0026ndash;Arsenazo III reagent over this lengthy period of time. To test this possibility, we compared the behavior of the Y(III)\u0026ndash;Arsenazo III solution in the dark and under room light for 3 days at 37\u0026deg;C. Fig. S3a shows that A652 for the lighted sample at 37\u0026deg;C for 3 days (blue line) was markedly lower than A652 for the control at r.t. for 10 min (black line), while A652 for the darkened sample at 37\u0026deg;C for 3 days (red line) was essentially identical to A652 for the previously mentioned negative control (indeed, the two lines almost completely overlapped). For the conditions involving a temperature of 37\u0026deg;C and a duration of 3 days, the reaction had to proceed in the dark as our aim was to avoid A652 values lower than the negative control. Thanks to this basic finding, we made sure that all the results presented in Fig.\u0026nbsp;2 and Fig.\u0026nbsp;3 and in Fig. S3b through Fig. S5 were obtained in the dark. Under these dark conditions, we successfully confirmed a well-correlated linear relationship between [DOTA] and A652 (see Fig.\u0026nbsp;2a).\u003c/p\u003e \u003cp\u003eIn the next step, we examined reaction periods over time (the period extended from 10 min to 7 days). For 1.0 \u0026micro;M of DOTA, absorbance at about 550 nm progressively increased and absorbance at 652 nm progressively decreased (Fig. S3b) during the period beginning at the 10-min marker, passing through the 1-day marker, and ending at the 3-day marker (with the isosbestic point observed at ~\u0026thinsp;580 nm); however, during the period beginning at the 3-day marker, passing through the 5-day marker, and ending at the 7-day marker, absorbance progressively decreased at about 550 nm and at 652 nm (with no isosbestic point observed). We surmised that the substantial degradation of the Y(III)\u0026ndash;Arsenazo III reagent was due to the lengthy incubation periods (i.e., periods over 3 days in length) at 37\u0026deg;C. After performing the above experiments, we concluded that the optimal conditions for [DOTA] determination were a 3-day duration, a 37\u0026deg;C temperature, and a darkened setting. Under these conditions, we successfully obtained a standard curve of A652 values plotted against [DOTA] with a high R\u003csup\u003e2\u003c/sup\u003e value (0.9788), as shown in Fig.\u0026nbsp;2a.\u003c/p\u003e \u003cp\u003eNext, we set out to determine the optimal reaction conditions for IgG\u0026ndash;DOTA-NHS conjugates. In the reaction conditions involving a temperature of 80\u0026deg;C and a duration of 10 min, we observed that peaks at 652 nm would markedly shift to longer wavelengths (red shift) in the presence of IgG. Fig. S4a presents this shift: the red solid line shows 0.5 \u0026micro;M [DOTA] in the presence of 0.20 \u0026micro;M IgG and the blue solid line shows 1.0 \u0026micro;M [DOTA] in the presence of 0.20 \u0026micro;M IgG. This peak red shift is an easily identifiable sign of IgG denaturation. As shown in Fig. S4b, the peak shift was also clearly observed at 56\u0026deg;C, which is a temperature commonly used for complement inactivation of serum. In contrast, the reaction at 37\u0026deg;C did not exhibit the peak shift, as demonstrated by the curve of the blue solid line in Fig. S4b. When the reaction was performed at 37\u0026deg;C for 3 days, the peak shift was not observed, as shown in Fig.\u0026nbsp;2b. Therefore, we have concluded that 37\u0026deg;C is an appropriate choice for the IgG\u0026ndash;DOTA-NHS assays.\u003c/p\u003e \u003cp\u003eWe then attempted to establish a standard curve for [DOTA] in the presence of IgG. As shown in Fig. S5, we found that the presence of IgG was associated with relatively low A652 values even for a short period of incubation in the Y(III)\u0026ndash;Arsenazo III solution. The sky-blue line (DOTA\u0026thinsp;+\u0026thinsp;IgG, 37\u0026deg;C, 1h) illustrates this finding. Absorbance around 550 nm was lower for the sky-blue line than for the negative control (black solid line). This finding indicates that the spectrum change was closely linked not to a transfer of Y(III) ions from the Arsenazo III moiety to the chelating agent, but to the Y(III)\u0026ndash;Arsenazo III molecules\u0026rsquo; adsorption to and interaction with the IgG molecules. The same pattern and degree of spectrum change were observed in an experiment we conducted with +\u0026thinsp;IgG at r.t. for 10 min (see the green line in Fig. S5). This finding indicates that IgG\u0026rsquo;s adsorption to and interaction with IgG occurred much faster than the Y(III)\u0026rsquo;s transfer from the Arsenazo III moiety to DOTA. A652 for the sky-blue line (+\u0026thinsp;IgG, 37\u0026deg;C, 1 h) was lower than A652 for the green line (+\u0026thinsp;IgG, r.t., 10 min). However, A652 for the positive control (80\u0026deg;C, 10 min) was even lower than A652 for the 1-hour reaction at 37\u0026deg;C (sky-blue line).\u003c/p\u003e \u003cp\u003eBased on all the above-mentioned results, we set the standard reaction conditions for [DOTA] determination in the presence of IgG at 37\u0026deg;C for 3 days in the dark. As shown in Fig.\u0026nbsp;2b, we observed that absorbances at 652 nm and absorbances at about 550 nm were markedly lower for all the colored lines ([DOTA]\u0026thinsp;=\u0026thinsp;0.2, 0.4, 0.6, 0.8, 1.0 \u0026micro;M) than for the negative control (black solid line). More specifically, these differences in absorbance occurred because the Y(III)\u0026ndash;Arsenazo III reagent interacted with and adsorbed to the IgG. We observed [DOTA]-dependent spectrum changes in A652 and in absorbance at about 550 nm for the five colored lines. A652 was lower and absorbance at about 550 nm was higher for the colored lines than those of the black dotted line ([DOTA]\u0026thinsp;=\u0026thinsp;0 \u0026micro;M). From the A652 values presented in Fig.\u0026nbsp;2b, we successfully obtained a standard, strongly linear curve depicting the relationship between A652 and [DOTA]. (see Fig.\u0026nbsp;2c). The high R\u003csup\u003e2\u003c/sup\u003e value (0.984) of the standard curve proved the strong linearity. When we measured concentrations of the DOTA moiety conjugated to IgG, we set [IgG] at a fixed value both for the standard curve and for the IgG\u0026ndash;DOTA-NHS conjugate samples. This fixed [IgG] value ranged from 0.06 \u0026micro;M to 0.12 \u0026micro;M. From this range, we applied a particular value to each experiment in consideration of the DOTA-NHS/IgG molar ratio estimated for each experiment: the smaller the estimated DOTA-NHS/IgG molar ratio was, the larger the [IgG] was.\u003c/p\u003e \u003cp\u003eAs a consequence of these experiments and measurements, we successfully obtained the DOTA-NHS/IgG molar ratios of the IgG\u0026ndash;DOTA-NHS conjugates from the [DOTA-NHS] that we had obtained in the colorimetric assay and the [IgG] that we had fixed in all the measurements of the standard curve and the conjugates.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDOTA-NHS conjugation to IgG\u003c/h2\u003e \u003cp\u003eIn the current study, we prepared a stock solution of DOTA-NHS in dehydrated DMSO and allowed the DOTA-NHS to react with the primary amino group of the IgG molecule by using the \u0026ldquo;dry-handling technique\u0026rdquo; described in detail in the Supplementary Information. Table\u0026nbsp;1 summarizes the results of the DOTA conjugation reaction to IgG. Across all tested pH values and all DOTA-NHS/IgG feed molar ratios, we obtained reaction yields of 43% or more for the DOTA-NHS. These values for the DOTA-NHS conjugation to proteins are greater than previously reported values (Abadi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alirezapour et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; D'Huyvetter et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hoppmann et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rossin et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sudo et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) for the DOTA-NHS conjugation reaction to proteins (Table S2 summarizes the previously reported DOTA-NHS reaction yields.).\u003c/p\u003e \u003cp\u003eComparisons of the reaction pH levels reveal that reaction yields did not change markedly for the pH range of 8.3\u0026ndash;9.2 in a 0.05 M borate-KCl buffer, as shown in Runs 1\u0026ndash;5 of Table\u0026nbsp;1. By fixing the reaction pH at 8.5, we examined conjugation reactions three times at a DOTA-NHS/IgG feed molar ratio of 4.0 and 12.0, as summarized in Runs 6\u0026ndash;11. We obtained high reaction yields with small standard deviations. The conjugation procedure proved to have good reproducibility in terms of the DOTA-NHS reaction yields.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eComparing two methods for DOTA-NHS/IgG molar-ratio determinations\u003c/h2\u003e \u003cp\u003eIn addition to the colorimetric method, we determined DOTA-NHS/IgG molar ratios of IgG\u0026ndash;DOTA-NHS conjugates by means of mass spectroscopy (Cassells et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rossin et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For this study, we used a monoclonal IgG that was kindly supplied by the Laboratory of RIN Institute Inc. (Tokyo, Japan). For this monoclonal IgG, we used three DOTA-NHS/IgG feed ratios (4.0, 10.0, and 20.0) and obtained three IgG\u0026ndash;DOTA-NHS conjugates, which we coded as A1, A2, and A3 (for a summary, see Table\u0026nbsp;2). Figure\u0026nbsp;4 presents mass spectra of intact IgG and three IgG\u0026ndash;DOTA conjugates; A1, A2 and A3. By subtracting the mass-to-charge ratio of the intact IgG from the mass-to-charge ratios of the conjugates, we obtained the products\u0026rsquo; DOTA-NHS/IgG molar ratios using a molecular weight (386.4) of one DOTA moiety conjugated to IgG. Table\u0026nbsp;3 compares these \u0026ldquo;mass spectroscopy\u0026rdquo; DOTA-NHS/IgG molar-ratio values with the \u0026ldquo;colorimetric assay\u0026rdquo; DOTA-NHS/IgG molar-ratio values. The two sets of ratio values are in good agreement with each other.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConjugation of DTPA-di to IgG and measurements of the DTPA-di/IgG molar ratios of the IgG\u0026ndash;DTPA-di conjugates\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor conjugates obtained with the DTPA-di reagent, we prepared a standard curve of [DTPA]. In the DTPA experiment, the reaction readily yielded a reduction in A652 in the Y(III)\u0026ndash;Arsenazo III solution at r.t. for a very short period, such as 15 min. As shown in Fig.\u0026nbsp;3, we obtained a standard, strongly linear curve depicting the relationship between A652 and [DTPA] in the presence of IgG. The large R\u003csup\u003e2\u003c/sup\u003e value (0.9996) proved the strong linearity. When we measured the chelate-to-IgG (chelate/IgG) molar ratios, we set [IgG] at a fixed value for both the standard curve of DTPA and the IgG\u0026ndash;DTPA-di conjugate samples. These fixed [IgG] values were in a range of 0.06\u0026ndash;0.12 \u0026micro;M. On the standard curve depicting the DTPA in the presence of a fixed concentration of IgG, the reduction in A652, which was due to the Y(III)\u0026ndash;Arsenazo III reagent\u0026rsquo;s adsorption to and interaction with IgG, was much smaller than the corresponding reduction that is observable on the DOTA\u0026rsquo;s standard curve. We can observe this relative smallness by comparing the x-intercept presented in Fig.\u0026nbsp;2c (0.0605 for the DOTA standard curve) and the corresponding x-intercept presented in Fig.\u0026nbsp;3 (0.0986 for the DTPA standard curve). This smallness stems from the fact that the DTPA experiments were conducted at a lower reaction temperature, and for a much shorter reaction period than was the case for the DOTA experiments.\u003c/p\u003e \u003cp\u003eWe conjugated the DTPA moiety to IgG using the DTPA-di reagent at various pH levels in two buffer systems. Results are summarized in Runs 1\u0026ndash;11 of Table\u0026nbsp;4. By using the [DTPA] standard curve, we measured the DTPA-di/IgG molar ratios of the IgG\u0026ndash;DTPA-di conjugates. We prepared Runs 1\u0026ndash;11 with a DTPA-di/IgG feed molar ratio of 10.0 and obtained large DTPA-di/IgG product molar ratios of 7.0 or more. When the DTPA-di/IgG feed molar ratio was reduced from 10, considerably high reaction yields were also obtained, as shown in Runs 12 and 13 at pH 8.5. These reaction yield values are much greater than previously reported values for the conjugation of DTPA-di to proteins (Arano et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; McLarty et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Scollard et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) (see Table S2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConjugation of CHX-A\u0026rdquo;-DTPA to IgG and measurements of the CHX-A\u0026rdquo;-DTPA /IgG molar ratios of the IgG\u0026ndash;CHX-A\u0026rdquo;-DTPA conjugate\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor conjugates obtained with the CHX-A\u0026rdquo;-DTPA chelating reagent, we used a standard curve of [DTPA]. When we determined chelate/IgG molar ratios, we set two fixed values: a fixed value for [IgG] for the standard DTPA curve, and a fixed value for the volume of CHX-A\"-DTPA conjugate samples. Below, we explain our reason for fixing the latter value.\u003c/p\u003e \u003cp\u003eAfter conjugation of the CHX-A\u0026rdquo;-DTPA reagent to IgG, one phenyl ring is present for each CHX-A\u0026rdquo;-DTPA moiety conjugated to IgG. This phenyl ring may affect A280-based [IgG] measurement results by biasing them upward, leading to underestimation of the chelate/IgG ratio, if there is no correction of the estimated [IgG]. To correct for this potential bias with respect to the CHX-A\"-DTPA/IgG molar ratios, we relied on a model compound that simulated the CHX-A\"-DTPA moiety conjugated to IgG. A chemical structure of this model compound is shown in scheme (1) of Fig. S6. Details of this correction process are provided in the Supplementary Information (the text and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;5 summarizes the post-correction CHX-A\"-DTPA/IgG molar ratios obtained at various pH values. Reaction yields of the CHX-A\u0026rdquo;-DTPA reagent ranged from 32% to 45%. These values, despite being smaller than those of the DOTA-NHS and DTPA-di reagents, are still reasonably high for reaction yields of chelating agents working in aqueous buffers. The reaction yields that we obtained for the CHX-A\"-DTPA reagent were either much greater than previously reported values (Price et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Winter et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) or approximately equal to them (Cassells et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Strand et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eThe “dry-handling technique” for chelating-agent solutions and its effect on reaction yields\u003c/h3\u003e\n\u003cp\u003eIn following the handling procedures for chelating-agent solutions, we strictly maintained dry (dehydrated) and inert environments (see the Supplementary Information for details). Because we were unsure whether or not this dry-handling technique would sufficiently raise the reaction yields of the chelating agents, we carried out a set of experiments comparing the dry-handling technique with the normal-handling technique. In the normal-handling technique, we prepared 5-mM chelating-agent solutions by diluting the 50-mM chelating-agent solutions in dehydrated DMSO with reagent-grade (not dehydrated) DMSO. Therefore, the 5-mM chelate solutions prepared according to the normal-handling technique contained 90% reagent-grade DMSO. Additionally, before using them, we incubated (at r.t. for 1 day) three kinds of 5-mM chelate stock solutions containing 90% reagent-grade DMSO. If water contaminated in the reagent-grade DMSO reacts significantly with the chelating agents during this 1-day incubation, the reaction yields of these chelating agents should be lower than the reaction yields obtained in the dry-handling technique. Results are summarized in Table\u0026nbsp;6. For DOTA-NHS, the dehydrated environment that we created for the dry-handling technique provided markedly higher reaction yields than did the normal environment, even though the difference did not reach statistical significance (p\u0026thinsp;=\u0026thinsp;0.09, n\u0026thinsp;=\u0026thinsp;3) in an unpaired Student\u0026rsquo;s t-test. No marked differences between the reaction yields for the dehydrated environment and those for the normal environment were observed for the other reagents, DTPA-di and CHX-A\"-DTPA.\u003c/p\u003e \u003cp\u003e1) Reactions were conducted at a chelating agent/IgG molar ratio of 10.0 in a 50 mM pH 8.5 borate-KCl buffer. The reaction conditions were: 0\u0026deg;C for 2.0 h and r.t. for 1.5 h for DOTA-NHS, and 25\u0026deg;C for 2.0 h for DTPA-di and CHX-A\u0026rdquo;-DTPA. For each reagent, we carried out three runs, and from them, we calculated the averages and standard deviations.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eConjugation reactions involving chelating agents are an essential process for the metal-ion labeling of proteins. This labelling allows for protein-biodistribution analyses and pharmacokinetic analyses in animal experiments and human clinical practices. Additionally, metal-ion labeling can endow proteins with critical cancer-fighting cytotoxic functions. In these experimental and clinical applications, chemical conjugation reactions that involve facile procedures, high yields, and high repeatability are desirable. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAn assay method for determining the chelate-to-protein (chelate/protein) molar ratio is a critical factor in these conjugation reactions, as this ratio governs the capacity of the chelating agents to label metal ions and to preserve the proteins\u0026rsquo; original biological functions. If the ratios are too small, the diagnostic and therapeutic functions of the metal ions will be deficient. If the ratios are too large, the desired biological functions of the proteins may be compromised or even entirely extinguished owing to excessive protein modification by the chelating moieties. In general, three assay methods are available for measurements of the chelate/protein molar ratios: \u003cstrong\u003e(1) the radioisotope method, (2) the mass spectroscopic method,\u003c/strong\u003e and \u003cstrong\u003e(3) the colorimetric method\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(1) The radioisotope method:\u0026nbsp;\u003c/strong\u003eKnown amounts of a mixture of a nonradioactive (\u0026ldquo;cold\u0026rdquo;) metal ion and a radioactive metal ion of the same element are coordinated into a protein-chelate conjugate. In this feed of both the metal ions, the total ion mole number must be greater than the expected chelate\u0026rsquo;s mole number. After the coordination reaction of these ions into the conjugate is complete, the chelate/protein molar ratio is calculated from the molar ratio of the coordinated radioactive metal ion to the uncoordinated radioactive metal ion, as measured by means of thin-layer chromatography\u0026nbsp;(Arano et al. 1996; McLarty et al. 2009; Meares et al. 1984; Oskar et al.2021; Scollard et al. 2011; Strand et al. 2021; Sudo et al. 2023; Tang et al. 2005; Timmermand et al. 2021), high-performance liquid chromatography, or other methods. The radioisotope method requires a facility where radioactive materials can be handled.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(2) The mass spectroscopic method\u003c/strong\u003e (Cassells et al. 2021; D\u0026apos;Huyvetter et al. 2012; Hoppmann et al. 2011; Price et al. 2016; Rossin et al.\u0026nbsp;2011;Sugyo et al. 2015;): \u0026nbsp;Molecular weights of an intact protein and a protein\u0026ndash;chelate conjugate are measured typically by means of MALDI-TOF mass spectroscopy. The chelate/protein molar ratio is calculated from the difference between the two molecular weights. The mass spectroscopic method requires an expensive analytical instrument: a mass spectrometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(3) The colorimetric method\u003c/strong\u003e (Abadi et al. 2021; Alirezapour et al. 2013; Pippin et al. 1992; Winter et al. 2023): The amount of chelate is measured by the use of a chemical colorimetric reagent. One well-known example of a colorimetric reagent is the reagent that we used in the current study: \u0026nbsp;the Y(III)\u0026ndash;Arsenazo III reagent. Y\u003csup\u003e3+\u003c/sup\u003e ions are coordinated within the Arsenazo III moieties. The coordination affinity of Y\u003csup\u003e3+\u003c/sup\u003e ions in the Arsenazo III moiety is much lower than the affinities in such representative chelate compounds as DOTA and DTPA. Therefore, in the presence of these chelates, the Y\u003csup\u003e3+\u003c/sup\u003e ion transfers from the Arsenazo III moiety to the chelate moieties. This transfer causes a change in the VIS light spectrum, and this change at 652 nm can be measured with a spectrometer. Consequently, one can calculate the amount of the chelate by calculating the precise reduction in absorbance at 652 nm. \u0026nbsp;(A652). The colorimetric method can be performed if a relatively inexpensive instrument, a UV-VIS spectrometer, is available. However, reaction conditions for the transfer reaction must be optimized for specific chelate compounds and specific proteins.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the present study, we examined the colorimetric method in conjunction with the Y(III)\u0026ndash;Arsenazo III reagent and then compared some of these results with our mass-spectroscopy results. For DTPA-di-derived IgG\u0026ndash;DTPA conjugates, we easily performed the quantitative colorimetric analysis of the DTPA-moiety/IgG molar ratio by following the reaction conditions (r.t., 15 min.) described in a previously published paper (Pippin et al. 1992).\u003c/p\u003e\n\u003cp\u003eIn conducting the colorimetric assay for IgG\u0026ndash;DOTA-NHS conjugates derived from DOTA-NHS, we had to identify and resolve several technical problems. Initially, we planned to conduct the assay on the basis of two papers published by the same research group (Abadi et al. 2021 and Alirezapour et al. 2013). However, neither of the papers provided any information whatsoever regarding the two most critical reaction conditions: temperature and duration. Therefore, to obtain a standard curve for [DOTA] determination, we conducted our first attempted assay at r.t. for 15 min. However, as shown in Fig. 1a, no change in the VIS light spectrum was observed under these reaction conditions for DOTA in the Y(III)\u0026ndash;Arsenazo III solution. \u0026nbsp;A well-known disadvantage of DOTA and its derivatives is the slowness with which they react to metal ions (Kasprzyk\u0026nbsp;et al. 1982; Kodama et al. 1991; Li et al. 2023; Sherry et al. 1989; Sugyo et al. 2015; Suzuki et al. 2024; Wang et al. 1992). Therefore, for the reaction between DOTA and the Y(III)\u0026ndash;Arsenazo III reagent, we tried high temperatures at 80\u0026deg;C and 56\u0026deg;C, and we found a [DOTA]-dependent colorimetric change of the Y(III)\u0026ndash;Arsenazo III reagent at these high temperatures. (see Fig. 1b, Figs. S4a, and S4b) However, at these high temperatures, we observed an unfavorable 652 nm peak shift toward longer wavelengths (red shift). We speculated that the red shift was probably due to protein denaturing at these high temperatures. Consequently, we again sought to optimize the reaction conditions for the [DOTA] determination, this time by fixing the temperature at 37\u0026deg;C and the duration at 3 days. From this experiment, we successfully obtained the same degree of reduction in A652\u0026nbsp;values as we had obtained for the [DTPA] determination, but we avoided any red shift at 652 nm. This last experiment strongly indicates that the reduction in A652\u0026nbsp;was due to a transfer of Y\u003csup\u003e3+\u003c/sup\u003e ions from Arsenazo III moieties to any chelate possessing a Y\u003csup\u003e3+\u003c/sup\u003e-ion coordination affinity higher than the one associated with Arsenazo III moieties. Furthermore, this fact indicates that the standard curve obtained with a parent chelate compound (e.g., DTPA and DOTA) can be used to determine the chelate derivatives that are found in IgG\u0026ndash;chelate conjugates. For example, an intact DOTA molecule possesses 8 functional groups for coordination, while the DOTA derivative moiety that was conjugated to IgG possesses only 7 functional groups for coordination, as one functional group is lost in the conjugation of the DOTA-NHS to IgG.\u003c/p\u003e\n\u003cp\u003eWe found that, when present, IgG reduced the levels of A652\u0026nbsp;in the Y(III)\u0026ndash;Arsenazo III solution, as shown in Fig. 2bs and Fig. S5. This reduction in A652\u0026nbsp;occurred even in the DTPA-based assay systems involving relatively short reaction periods, such as 15 min. In order to deal with this reduction in A652, we\u0026nbsp;maintained a fixed concentration of IgG ([IgG]) throughout all the colorimetric measurements for both the standard-curve samples and the conjugate samples. Concentrations of the chelate moieties, [chelate moiety], in the IgG\u0026ndash;chelate conjugates were obtained in colorimetric measurements, while the concentration of IgG, [IgG], in these same conjugates was obtained from absorbance measurements at 280 nm. Accordingly, the chelate/IgG molar ratio can be obtained from these [chelate moiety] and [IgG] values.\u003c/p\u003e\n\u003cp\u003eWe encountered another technical problem in the colorimetric determination of the chelate/IgG molar ratios for IgG\u0026ndash;CHX-A\u0026quot;-DTPA conjugates derived from CHX-A\u0026quot;-DTPA. Because the CHX-A\u0026quot;-DTPA reagent contains one phenyl (benzene) ring structure, and because this phenyl ring remains in the CHX-A\u0026quot;-DTPA derivative of the IgG\u0026ndash;CHX-A\u0026quot;-DTPA conjugate, the UV-absorption property of the phenyl ring may interfere with A280\u0026nbsp;determination of [IgG] values. In order to solve this problem, we synthesized a model compound that, derived from the CHX-A\u0026quot;-DTPA reagent and ethylenediamine, could simulate the chemical structure of the CHX-A\u0026quot;-DTPA moiety conjugated to IgG. Then, we obtained a standard curve of the model compound\u0026rsquo;s A280\u0026nbsp;plotted against the [model compound], as shown in Figure S7b. From the reduction in A652, we obtained the [CHX-A\u0026quot;-DTPA moiety] of the IgG\u0026ndash;CHX-A\u0026quot;-DTPA conjugates. We then calculated the contribution of the CHX-A\u0026quot;-DTPA moiety to the A280\u0026nbsp;values of the conjugates by using the standard curve of the model compound. Accordingly, by subtracting the calculated A280\u0026nbsp;value of the CHX-A\u0026quot;-DTPA moiety from the initially measured A280\u0026nbsp;value that contained both absorbance of IgG and absorbance of the CHX-A\u0026quot;-DTPA moiety, we could compensate for the true A280\u0026nbsp;value representing only [IgG].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, we examined possible effects of impurities contaminating in DMSO and ethylenediamine reagents on the A280\u0026nbsp;measurements. As described closely in Supplemental Information and Fig. S8, these effects were turned out to be negligible.\u003c/p\u003e\n\u003cp\u003eYet another problem presented itself, this time regarding the IgG\u0026ndash;CHX-A\u0026quot;-DTPA conjugate. The true [IgG] in the IgG\u0026ndash;CHX-A\u0026quot;-DTPA conjugate solutions was unknown prior to the colorimetric assay owing to the presence of one phenyl ring from the conjugated CHX-A\u0026quot;-DTPA moiety, as described above. Therefore, we conducted the colorimetric assay\u0026nbsp;by adding a fixed volume (24 mL) of the conjugate sample solutions into the Y(III)\u0026ndash;Arsenazo III solution, irrespective of the samples\u0026rsquo; A280\u0026nbsp;values. Because of this fixed volume,\u0026nbsp;a potential problem arose: a difference between the [IgG] set for the standard curve and the true [IgG] of the conjugate in the colorimetric assay might cause an error in the estimation of the chelate/IgG molar ratios. We examined this potential problem (as described in the text of Supplemental Information and Figure S9) and determined that any errors stemming from this problem would be negligible.\u003c/p\u003e\n\u003cp\u003eThanks to the many carefully executed steps described above, we successfully established the colorimetric assay methods for three kinds of chelate-IgG conjugates with the Y(III)\u0026ndash;Arsenazo III reagent.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We compared the chelate/IgG molar ratios obtained from the colorimetric method with the chelate/IgG molar ratios obtained from the mass spectroscopic method. As summarized in Table 3, these molar-ratio values were in good agreement with each other. Therefore, researchers can safely choose either of these two methods, with the right choice dependent on, for example, the availability of instruments in a lab and the lab\u0026rsquo;s relative familiarity with the methods and their specific techniques.\u003c/p\u003e\n\u003cp\u003eUsing the colorimetric assays with the Y(III)\u0026ndash;Arsenazo III reagent, we analyzed the chelate/IgG molar ratios for three kinds of reactive chelating agents: DOTA-NHS, DTPA-di, and CHX-A\u0026quot;-DTPA. We conducted a comparison between our study\u0026rsquo;s reaction yields for these three kinds of chelating agents (see Table 6) and previous studies\u0026rsquo; reported reaction yields (see Table S2). The values of our study\u0026rsquo;s yields are much higher than the values reported in the previous studies, with only two exceptions (both of which were for CHX-A\u0026quot;-DTPA:\u0026nbsp;Cassells et al. (2021)\u0026nbsp;and\u0026nbsp;Strand et al. (2021). These comparisons demonstrate that we have successfully established conjugation procedures achieving very high reaction yields for the three kinds of chelating agents studied herein. We believe the central reason for these high reaction yields was our use of chelate stock solutions in dehydrated DMSO. Each of the three chelating agents involved a unique reactive group: DOTA-NHS used N-hydroxysuccinimide ester, DTPA-di used acid anhydride, and CHX-A\u0026quot;-DTPA used phenyl thioisocyanate. Regarding conjugation to proteins in aqueous buffers, all these reactive groups favor high pH levels because only proteins\u0026rsquo; free amino groups (\u0026ndash;NH2), which are the favorable amino-group form at high pH levels, can react with these reactive groups, and because the protonated form of the\u0026nbsp;amino group (\u0026ndash;NH3\u003csup\u003e+\u003c/sup\u003e), which is the favorable form at low pH levels, cannot react with the reactive groups of the chelating agents. That is why we frequently carried out the conjugation reactions of the chelating agents at high pH levels, such as 8.5 and 9.2.\u003c/p\u003e\n\u003cp\u003eWhen the conjugation reactions of these chelating agents are conducted in pure aqueous buffers that contain no DMSO, the first step in the multi-step synthesizing procedures must be to dissolve the chelating agents in the buffers. During this dissolution step, considerable amounts of the chelating agents may react with hydroxide ions, resulting in a substantial loss of these reagents before they react with the primary amino groups of the proteins. In particular, this side reaction occurs aggressively at high pH levels, where high concentrations of hydroxide ions exist. Furthermore, the time period required for complete dissolution can vary substantially depending on an experimenter\u0026rsquo;s technical skill. In sum, the longer the period, the larger the reagent loss. Therefore, significant variation in the dissolution period can lead to significant variation in the reaction yields of the chelating agents. We used a synthesizing procedure in which we added the chelating-agent solutions (in dehydrated DMSO) to the IgG solutions in aqueous buffers. In this procedure, the chelating agents started their reactions as soon as the addition of the chelating-agent solutions to the IgG solutions was complete. The rapid onset of the reactions enabled us to avoid any loss of the chelating agents that would have arisen from their side reactions with hydroxide ions during the dissolution step. The result was a series of high reaction yields. Additionally, we believe that, because we can evade technical differences among experimenters in our synthetic procedure, we obtained a narrow band of inconsistent variation\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ein the reaction yields of the chelating agents. Therefore, in our conjugation of the three chelating agents to IgG, we achieved both high reaction yields and high reaction-yield repeatability.\u003c/p\u003e\n\u003cp\u003eIn some previously published papers (Arano et al. 1996; Cassells et al. 2021; Sugyo et al. 2015), chelating-agent solutions were used in the conjugation of either DMSO or dehydrated DMSO to proteins. However, these papers omit important details about the reaction procedures or fail to examine the reaction yields of the chelating agents relative to the optimum pH levels of the reaction buffers. Additionally, we elucidate the effects that dehydrated solvents and dry-handling techniques can have on the reaction yields. Therefore, we believe the results of the current paper constitute very valuable technical information for researchers conducting research on the conjugation of chelating agents to proteins, particularly in the field of nuclear medicine.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, we examined the conjugation of three chelating agents (DOTA-NHS, DTPA-di, and CHX-A\"-DTPA) to human IgG and measured the chelate/IgG molar ratios of the IgG\u0026ndash;chelate conjugates using a colorimetric method with the Y(III)\u0026ndash;Arsenazo III reagent. For DOTA-NHS and CHX-A\"-DTPA, we used this colorimetric method to establish novel methods for the accurate measurement of the chelate/IgG molar ratios. Additionally, we succeeded in obtaining high reaction yields and high repeatability in these conjugation reactions by using stock solutions of the reactive chelating agents in dehydrated DMSO in combination with a dry-handling technique.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDOTA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1,4,7,10-detraazacyclododecane-1,4,7,10-tetraacetic acid\u003c/p\u003e\n\u003cp\u003eDTPA diethylenetriaminepentaacetic acid\u003c/p\u003e\n\u003cp\u003eDOTA-NHS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester\u003c/p\u003e\n\u003cp\u003eNHS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;N-hydroxysuccinimide ester\u003c/p\u003e\n\u003cp\u003eDTPA-di\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;diethylenetriaminepentaacetic dianhydride\u003c/p\u003e\n\u003cp\u003ep-SCN-Bn-CHX-A\u0026rdquo;-DTPA\u0026nbsp; S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid\u003c/p\u003e\n\u003cp\u003eCHX-A\u0026rdquo;-DTPA S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMALDI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;matrix-assisted laser desorption/ionization\u003c/p\u003e\n\u003cp\u003eTOF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;time-of-flight\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD-PBS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Dulbecco\u0026rsquo;s phosphate-buffered saline.\u003c/p\u003e\n\u003cp\u003er.t. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;room temperature\u003c/p\u003e\n\u003cp\u003eA652 absorbance at 652 nm\u003c/p\u003e\n\u003cp\u003eA280 absorbance at 280 nm\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary information available at https:// .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Grant-in-Aid for Scientific Research (B) grant number 24K03286 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This study was also supported by a grant with grant number JP25yf0126003 from the Japan Agency for Medical Research and Development. Partial support for this study was forthcoming from the MSD Life Science Foundation, Public Interest Incorporated Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMY developed experimental plans for all the studies and carried out conjugation reactions and colorimetric assays of the obtained conjugates. MY was also a major contributor to the writing of the manuscript. KS contributed in syntheses and analyses of IgG\u0026ndash;chelate conjugates. TK, YI, HO, and YM designed the IgG\u0026ndash;chelate conjugates by paying special attention to the number of conjugated chelate molecules per IgG molecule. AT chose chelating reagents for the conjugation reactions and wrote about the number of conjugated chelate molecules per one IgG molecule. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor their valuable advice and technical assistance in mass spectroscopy measurements, we are indebted to Dr. Shushi Nagamori and Dr. Yumi Kanegae (the Advisory Board for Mass Spectrometry at Core Research Facilities in Center for Medical Science, the Jikei University School of Medicine), and to Ms. Erika Osada and Ms. Emi Tsuchitani (Core Research Facilities, the Jikei University School of Medicine) for their providing valuable advices and technical assistance in mass spectroscopy measurements.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAbadi SA, Alirezapour B, Kertész I, Rasaee MJ, Mohammadnejad J, Paknejad M, Yousefnia H, Zolghadri S. Preparation, quality control, and biodistribution assessment of [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors. 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Evaluation of efficacy of radioimmunotherapy with 90Y-labeled fully human anti-transferrin receptor monoclonal antibody in pancreatic cancer mouse models. PLoS One. 2015;10:e0123761. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123761\u003c/p\u003e\n\u003cp\u003eSuzuki H, Matsukawa M, Madokoro R, Terasaka Y, Kannaka K, Uehara T. Reduction of the hepatic radioactivity levels of [111In]In-DOTA-labeled antibodies via cleavage of a linkage metabolized in lysosomes. Nucl Med Biol. 2024;132-133:108910. doi: 10.1016/j.nucmedbio.2024.108910. \u003c/p\u003e\n\u003cp\u003eTang Y, Wang J, Scollard DA, Mondal H, Holloway C, Kahn HJ, Reilly RM. Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using (111)In-trastuzumab (Herceptin) Fab fragments. Nucl Med Biol. 2005;32(1):51-8.doi: 10.1016/j.nucmedbio.2004.08.003.\u003c/p\u003e\n\u003cp\u003eTimmermand OV, Örbom A, Altai M, Zedan W, Holmqvist B, Safi M, Tran TA, Strand S-E, and Strand J. A Conjugation strategy to modulate antigen binding and FcRn interaction leads to improved tumor targeting and radioimmunotherapy efficacy with an antibody targeting prostate-specific antigen. Cancers. 2021;13:3469. https://doi.org/10.3390/cancers13143469.\u003c/p\u003e\n\u003cp\u003eWang X, Jin T, Comblin V, Lopez-Mut A, Merciny E, and Desreux J F. A Kinetic Investigation of the lanthanide DOTA chelates. stability and rates of formation and of dissociation of a macrocyclic Gadolinium(III) polyaza polycarboxylic MRI contrast agent. Inorg Chem. 1992;31:1095-1099. https://pubs.acs.org/doi/10.1021/ic00032a034\u003c/p\u003e\n\u003cp\u003eWinter G, Hamp-Goldstein C, Fischer G, Kletting P, Glatting G, Solbach C, Herrmann H, Sala E, Feuring M, Döhner H, Beer AJ, Bunjes D, Prasad V. Optimization of radiolabeling of a [90Y]Y-Anti-CD66-antibody for radioimmunotherapy before allogeneic hematopoietic cell transplantation. Cancers (Basel). 2023;15(14):3660. doi: 10.3390/cancers15143660.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"chelates, chelating agents, conjugates, metal ions, DOTA-NHS, DTPA-di, CHX-A”-DTPA, Arsenazo III","lastPublishedDoi":"10.21203/rs.3.rs-8683063/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8683063/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChelate conjugation is an essential step for metal-ion labelling of proteins, and the chelate-to-protein (chelate/protein) molar ratio of the resulting protein-chelate conjugate critically influences the conjugate\u0026rsquo;s functions. Therefore, both controlling and measuring the chelate/protein molar ratio are important technical considerations. Among available methods for chelate/protein determination, a colorimetric method using the Y(III)\u0026ndash;Arsenazo III reagent is convenient because it requires neither large-scale facilities nor expensive instruments. We examined this assay method for three representative chelating agents (DOTA-NHS, DTPA-di, and CHX-A\u0026rdquo;-DTPA). Concerning the conjugation reactions of chelating agents, high and reproducible reaction yields are desired. However, the reported yields have varied widely, and no standardized, high-yield, and reproducible procedure has been established.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe established new measurement methods to determine the chelate-to-protein molar ratio for DOTA-NHS and CHX-A\u0026rdquo;-DTPA using human IgG as the target protein for conjugation. We also established a conjugation procedure that affords high conjugation-reaction yields for these chelating agents. We achieved these outcomes both by preparing stock solutions of the chelating agents in dehydrated DMSO and by employing the \u0026ldquo;dry-handling technique\u0026rdquo; throughout the conjugation procedure. Through the combination of these two practices, we successfully obtained high reproducibility and high yields in the reactions.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe establishment of the two measurement methods for the chelate-to-protein molar ratio and the conjugation procedure delivering the above yields may substantially contribute to research and clinical applications of metal ion-labeled proteins in many fields of science and medicine, particularly in the field of nuclear medicine.\u003c/p\u003e","manuscriptTitle":"Efficient Conjugation of Chelating Agents to IgG and Accurate Colorimetric Determination of the Chelate-to-IgG Molar Ratio.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 13:10:54","doi":"10.21203/rs.3.rs-8683063/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"76b8a7e6-3457-4f8e-8d74-e78fa415b03e","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T07:36:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 13:10:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8683063","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8683063","identity":"rs-8683063","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}