Application of BODIPY-labeled erythromycin for macrolide detection by a fluorescence polarization immunoassay

Application of BODIPY-labeled erythromycin for macrolide detection by a fluorescence polarization immunoassay | 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 Application of BODIPY-labeled erythromycin for macrolide detection by a fluorescence polarization immunoassay L. I. Mukhametova, D. A. Arutyunyan, A. V. Shishkina, A. G. Tereshchenkov, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7808262/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Immune methods of analysis are widely used in laboratories in various fields of medicine, including enzyme immunoassay (ELISA), polarization fluorescence immunoassay (FPIA) and immunochromatographic analysis. The FPIA method allows for high specificity and sensitivity of low-molecular-weight analytes in a homogeneous medium without separation and has the ability to automate the analysis system, high accuracy, label stability, and speed and simplicity of analysis. In this work, a fluorescently labeled tracer for the determination of erythromycin with new BODIPY dye was obtained. A pair of immunoreagents (tracers and antibodies) for detecting these antibiotics were selected and characterized. The conditions for conducting FPIA were optimized, calibration curves were obtained, and the analytical characteristics of the FPIA were determined: detection limit, range of detectable concentrations, sensitivity and cross-reactivity. Using the developed FPIA methods for determining antibiotics most commonly used in veterinary medicine, water samples collected from reservoirs in Moscow and the Moscow region were tested. Thus, in this work, the FPIA method for determining erythromycin with a detection limit of 1.6 ng/ml was developed, the advantages of using the new fluorescent label BODIPY were shown, the accuracy of the method was verified by an introduced-found test, and real water samples were tested. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Macrolide antibiotics are a class of drugs used to treat bacterial infections [ 1 , 2 ]. In clinical practice, macrolides are among the four most frequently used antimicrobial drugs against gram-positive bacteria, some gram-negative bacteria and Mycoplasma and are also used as adjuvants for the prevention of a number of animal diseases. [ 3 , 4 ]. The chemical structure of macrolides consists of a lactone ring containing 14 to 16 carbon atoms with one or more sugar residues linked by a glycosidic bond (Fig. 1 ) [ 5 ]. The mechanism of their antimicrobial action is due to the disruption of protein synthesis on the ribosomes of the microbial cell and consists of inhibiting the synthesis of bacterial protein by binding to the 50S subunit of the ribosome, namely, by interacting with 23S rRNA [ 5 , 6 ]. Macrolides are classified by the World Health Organization (WHO) and the World Organization for Animal Health (OIE) as critically important for human and animal health. [ 7 , 8 ]. However, the use of macrolides is associated with certain side effects in humans, such as gastrointestinal disturbances, hypersensitivity reactions, rash and fever, transient hearing loss and possible gastrointestinal infections (ERYs), which have also been reported after the use of erythromycin. Some individuals may experience prolonged QT intervals, which can lead to cardiac arrhythmias [ 9 ]. Importantly, human health is directly related to the quality of food products, and veterinary drug residues constitute only one of many contamination factors. Although the use of antibiotics in livestock production is necessary to improve weight gain and feed efficiency, to prevent or treat diseases, their residues may be present in animal products [ 1 , 10 , 11 ]. Residue concentrations exceeding the permissible doses may be caused by failure to follow the technical instructions or dosage calculations provided by the manufacturer, failure to comply with the specified withdrawal period and the use of equipment contaminated with drugs. Antimicrobials are the main veterinary drugs that can potentially contaminate animal products, the most common of which is erythromycin. Erythromycin residues can lead to prolonged and undesirable effects when these substances are ingested in small quantities over a long period of time [ 1 , 10 ]. Therefore, antibiotic content is regulated and controlled on the basis of maximum residue levels (MRLs) or acceptable limits set in each country [ 12 ]. The maximum permissible concentration of ERY residues in milk is set at 40 µg/kg by EU countries and Customs Union countries [ 13 ]. WHO установил MRLs for erythromycin: fat, kidney, liver and muscle (chicken and turkey): 100 µg/kg; eggs (chicken): 50 µg/kg [ 14 ]. Therefore, food safety laboratories must test many samples because of the need to control the detection of antibiotics. Various instrumental methods have been developed for the determination of macrolide drugs and their metabolites in various food samples. [ 1 , 8 , 9 , 11 – 13 ]. However, these instrumental monitoring methods require highly qualified specialists and complex sample preprocessing, which complicates the analysis and increases its time. Thus, sensitive, specific, reliable and rapid analysis methods are essential for the effective monitoring of ERY in food products. Modern biosensor technologies for the determination of erythromycin have been developed [ 18 – 20 ]. However, immunoassay methods are increasingly used to detect toxic contaminants in food samples because of their sensitivity, selectivity and time savings, and they have become effective alternatives to instrumental methods. For example, enzyme-linked immunosorbent assay (ELISA) methods for determining erythromycin using rabbit polyclonal antiserum with a detection limit (LOD) of 0.04 ng/ml have been developed [ 21 , 22 ], as do monoclonal antibody (LOD 0.057 нг/мл) [ 23 ] and lateral flow immunoassays (LFAs) [ 24 , 25 ]. However, LFAs are semiquantitative methods for detecting toxicants; heterogeneous ELISAs require a long time and complex manipulations, whereas homogeneous immunoassays have shown great potential for simplifying routine tasks [ 26 ]. Polarization fluorescence immunoassay (FPIA) has several advantages, such as sensitivity, reliability, rapidity and the ability to analyze large numbers of samples. [ 26 , 27 ]. The principle of small molecule detection by FPIA is that a fluorescently labeled analyte (tracer) and the analyte to be determined compete for a limited number of binding sites on antibodies, which leads to a change in the fluorescence polarization (FP) value [ 26 , 27 ]. If there is no analyte in the test solution, the tracer binds to the specific antibody and forms a bulk antigen‒antibody complex with a slower mobility than the free tracer, resulting in a high FP signal. As the analyte concentration in the reaction mixture increases, the antibody will bind to the analyte; therefore, the concentration of free tracer will increase, and the FP signal will decrease. FPIA has attracted increasing attention and is often used to detect many small molecule compounds, such as veterinary drugs [ 28 , 29 ], pesticides [ 30 ] and toxins [ 31 , 32 ], in food and environmental samples. Currently, FPIA methods for determining tylosin are known [ 28 ] and ERY [ 29 ] in honey and milk, respectively. In these studies, fluorescein was used as a fluorescent label on the antigen, and the detection limits of the drugs were 34.7 ng/ml for tylosin and 14.08 ng/ml for erythromycin. In the literature, most FPIA methods use fluorescein as a fluorescent label, and this choice is justified by its unique properties: high quantum yield of 92%, fluorescence lifetime of 4.05 ns, chemical stability, and availability of reagents with various reactive groups (sulfhydryls, amino groups and carboxyl groups), which are relatively inexpensive and commercially available. In addition, most devices for measuring the FP signal are tuned to the radiation and emission wavelengths specifically for fluorescein. Currently, other dyes that are capable of absorbing and emitting in the same region as fluorescein are used. Currently, boron-based dyes are used, such as BODIPY. These dyes are advantageous in FP assays because they have longer excited-state lifetimes than fluorescein dyes do [ 33 – 35 ]. This extended lifetime allows FPs to be sensitive to binding interactions across a broader range of molecular weights. The longer lifetime of BODIPY dyes means that they remain excited for a longer period. This allows for more rotational movement and depolarization before the fluorophore emits light. Consequently, BODIPY dyes can be used to detect binding events in molecules with a wider range of molecular weights than dyes with shorter lifetimes. In FP assays, if the fluorophore's rotational correlation time (which is related to molecular weight) is much shorter than its excited-state lifetime, the fluorescence polarization will not be significantly affected by binding. The longer lifetime of BODIPY allows it to be sensitive to smaller changes in rotational correlation time, making it useful for studying binding interactions with a wider range of molecular weights. FP analysis using antibiotic conjugates [ 36 ] or pesticides [ 37 ] with BODIPY is a convenient platform for studying the interactions of various drugs with ribosomes to determine the kinetic parameters of binding and modulation of their interactions. We hypothesized that the use of BODIPY dye as a fluorescent label on erythromycin would reduce the detection limit of the macrolide antibiotic group in food products by FPIA. The aim of this study was to develop a method for FPIA using a portable detector for the determination of erythromycin via a conjugate of the studied antibiotic with BODIPY and to test these methods for their determination in milk, water and honey. 2. Materials and methods The following reagents were used in this work: erythromycin (ERY, Fluka, Germany), clarithromycin (CLA), roxithromycin (ROX), azithromycin (AZI), dirithromycin (DIR), tylosin (TYL), BODIPY FL-C5 NHS Ester (succinimidyl ester) (Invitrogen, США), and the antiserum ACLA-PAb was received as described previously [21]. The FP signal was measured via a Sentry 200 portable fluorimeter (Ellie, USA) at an excitation wavelength of 495 nm, and emission was recorded at 530 nm. 2.1. The following buffer solutions were used : 10 mM phosphate buffer, pH 7.4, containing 0.15 M NaCl, 0.005% Tween 20 (PBS-T) and 25 mM borate buffer, pH 8.5 (BB). 2.2. Preparation of erythromycin conjugated with the fluorescent dye BODIPY (ERY-BDP). Synthesis was performed as described previously [36,38]. 9-(S)-Erythromycylamine A, obtained from erythromycin according to a previously described method [39], was dissolved in tetrahydrofuran and mixed with BODIPY FL-C5 succinimide ester under an argon atmosphere. After stirring at room temperature for 24 h, the solvent was removed in vacuo. The resulting residue was purified via HPLC. The concentration of the conjugate was determined via the molar absorption coefficient of the dye ε 504 in MeOH (BODIPY-FL-C5 succinimide ester) = 87000 M -1 cm -1 . 2.3. High-resolution liquid chromatography‒tandem mass spectrometry (LC‒MS/MS) analysis was conducted via a comprehensive UPLC‒MS/MS system comprising an Acquity UPLC chromatograph (Waters, USA) integrated with a Thermo Scientific TQD quadrupole mass spectrometer. The analysis was performed in positive ion mode employing electrospray ionization (ESI) methodology. Chromatographic separation was achieved via a BEH C18 column (Waters) with a particle size of 1.7 μm and dimensions of 50 mm × 2.1 mm. The mobile phase consisted of a linear gradient of 5–100% acetonitrile (CH3CN) in 20 mM formic acid (HCOOH), delivered at a flow rate of 0.5 mL/min at a column temperature of 35°C over a duration of 4 minutes. The photophysical properties of the analyte were characterized by its excitation (λex) and emission (λem) wavelengths in methanol, which were determined to be 504 nm and 511 nm, respectively. The retention time (tR) of the compound in the LC‒MS analysis was 1.50 minutes. The theoretical and experimental mass‒charge (m/z) ratios were calculated and measured as 1037.64 and 1037.20, respectively, for the protonated molecular ion [C53H87BF2N4O13 + H]+. 2.4. Determination of the working concentration of the ERY-BDP conjugate . A series of dilutions of the ERY-BDP conjugate were prepared in BB or PBS-T buffer solutions, and fluorescence polarization was measured via a Sentry-200 portable fluorimeter. The criterion for selecting the working concentration of the ERY-BDP conjugate was a stable FP value and an excess of the conjugate fluorescence intensity over the background signal value by 10 times. 2.5. Characterization of the interaction of the ERY-BDP conjugate with the aCLA-PAb antiserum. The working dilution of the aCLA-PAb antiserum was determined as follows. A series of antisera dilutions from 1:100 to 1:64000 in BB or PBS-T in a volume of 500 μl were prepared in borosilicate glass tubes. Five hundred microliters of the ERY-BDP conjugate solution at a concentration of 5 nM in the appropriate buffer was added, the mixture was incubated at room temperature for 5 min, and fluorescence polarization was measured via a Sentry-200 portable fluorimeter. The binding of ERY-BDP to nonimmune sera was studied similarly. All experiments were performed in duplicate, and three instrumental measurements were taken. The average values were calculated, and the FP dependences on the antiserum dilution were plotted. 2.6. Study of the kinetics of the interaction of ERY-BDP with the aCLA-PAb antiserum. Five hundred microliters of the antiserum at a 1:1000 dilution in BB was added to 500 μl of the ERY-BDP conjugate in the selected dilution, and the change in fluorescence polarization was measured for 25 minutes at 30-second intervals. All the experiments were performed in duplicate. The average values were calculated, and the FP dependences on time were plotted. 2.7. Conducting FPIA. Fifty microliters of standard erythromycin solutions in water (0, 0.1, 1, 3, 6, 10, 30, 100, 1000, or 10000 ng/ml) and 500 microliters of the ERY-BDP conjugate solution with a concentration of 5 nM in the test buffer were added to glass tubes. Then, 500 microliters of aCLA-PAb antiserum in the selected dilution was added, and after 10 min, the fluorescence polarization was recorded via a Sentry-200 portable detector. Each measurement was performed in triplicate, and three instrumental measurements were made. 2.8. Determination of the analytical characteristics of FPIA. The dependence of fluorescence polarization on the erythromycin concentration (calibration curve) was plotted on a semilogarithmic scale and approximated via a four-parameter sigmoid function: $$\:Y=B+\frac{A-B}{\left(1+{\left(\frac{x}{C}\right)}^{D}\right)}$$ 1 where x is the analyte concentration, y is the FP value, and A is the asymptotic maximum of the FP value, B is the asymptotic minimum (background value) of the FP value, and C is the inflection point of the curve in semilogarithmic coordinates (equal to 50% inhibition of the intensity), and D is the slope of the curve at the inflection point. The IC10, IC20, IC50, and IC80 values were calculated as concentrations that reduced the analytical signal by 10, 20, 50, and 80%, respectively. The IC10 value was estimated as the detection limit, and IC20–IC80 was the working range of the determined concentrations. Cross-reactivity values were calculated via the following formula: \(\:CR\left(\%\right)=\frac{{IC}_{50}\left(ERY\right)}{{IC}_{50}\left(CR\right)}*100\) (2). Preparation of honey, milk and water samples. Honey samples were prepared as described previously [ 28 ] with some modifications. One gram of honey was placed in 5 ml polypropylene tubes, and aliquots of 0 to 60 µl of the ERY solution at a concentration of 100 µg/ml were added. The samples were mixed in a vortex-1 vortex mixer (IKA, Germany) for 1 min. Then, 2250 µl of 5 M aqueous sodium acetate solution was added to each tube, the mixture was incubated for 30 min at 37°C, 2250 µl of acetonitrile was added, and the mixture was extracted and then centrifuged at 3500 × g for 10 min. 2000 µl of the upper organic layer was collected and evaporated until the solvent was removed, and the dry residue was redissolved in 500 µl of PBS and mixed. The resulting extract was analyzed. The milk samples were pretreated via simple dilution with PBS at a ratio of 1:9. Water samples from open water bodies were additionally filtered through 13 mm diameter 0.22 µm pore size syringe filters, and if necessary, the pH was adjusted to 7.4 by the addition of 1 M NaOH. Using a Sentry-200 portable detector, fluorescence polarization was recorded in triplicate for each sample, and erythromycin concentrations were determined on the basis of the FPIA calibration curve. The agreement between the injected and detected ERY concentrations was assessed as a percentage. 3. Results and Discussion 3.1. Preparation of immunoreagents and optimization of the FPIA method. Macrolide antibiotics are used in agriculture for the treatment and prevention of diseases in animals and are found in food products. Simple and fast methods are needed for their determination. FPIA does not require lengthy manipulations, takes only a few minutes and can be carried out outside the laboratory. Since various macrolide antibiotics are used in veterinary medicine, such as erythromycin, roxithromycin, clarithromycin, and tylosin, we selected polyclonal antibodies with broad specificity (aCLA-PAb) to develop a method for determining macrolides in food products. This antiserum has broad cross-reactivity and allows the determination of several macrolides [ 21 ]. For the test system under development, a conjugate of erythromycin with BODIPY dye was obtained (Fig. 2 a) [ 36 ]. To introduce a fluorescent label into the erythromycin molecule, the keto group at position C9 (Fig. 1 ) was chosen and stereospecifically converted into a 9-(S)-amino group. This allowed the resulting 9-(S)-erythromycinamine to be acylated under mild conditions via BODIPY FL-C5 succinimide ester, and the resulting conjugate was obtained in good yield without prior protection of the functional groups of the parent antibiotic. The optimal concentration of the conjugate was determined by examining the FP values and fluorescence intensity at different concentrations of ERY-BDP (Fig. 2 b). Then, the binding of the antiserum was studied, and the time to equilibrium was investigated by studying the binding of aCLA-PAb to ERY-BDP (final concentration of 2.5 nM) for 20 min. The binding reached equilibrium in approximately 2–3 min (data not shown). During this time period, the total fluorescence intensity did not change, indicating that the formation of the ERY-BDP complex with antibodies did not cause significant quenching of the fluorescence intensity. Initially, we tested the interaction of the ERY-BDP conjugate with the rabbit immune antiserum aCLA-PAb and nonimmune antisera obtained against nicotinic acid (aNIC-PAb) and tylosin (aTYL-PAb). As shown in Fig. 3 (a), at pH 8.5, binding of the ERY-BDP conjugate to aCLA-PAb is observed: the FP signal increases, and equilibrium in the reaction system occurs in just 2–3 min. As the dilution of the antiserum increases, the FP signal decreases. When nonimmune sera aNIC-PAb and aTYL-PAb were tested, an increase in the FP signal was also observed, but it was lower than that for the immune antiserum. Thus, ERY-BDP at pH 8.5 nonspecifically binds to serum proteins. With dilution of the antiserum, a decrease in the FP signal was observed. However, when the pH was reduced to 7.4, the FP signal for the ERY-BDP*aCLA-PAb complex increased, whereas for the ERY-BDP pairs with aNIC-PAb and aTYL-PAb, it decreased (Fig. 3 b). The change in the FP signal between the immune and nonimmune sera at a dilution of 200 mP was approximately 50 mP (Fig. 3 b). The dependence of the FP signal change at constant concentrations of the tracer and antiserum on the erythromycin concentration was determined for the determination of erythromycin by the FPIA method at pH 8.5 and 7.4 (Fig. 4 ). The detection limit at two pH values was 1.6 ng/ml, but the detection range at pH 7.4 was wider, from 5 to 300 ng/ml, whereas that at pH 8.5 ranged from 5 to 130 ng/ml. Thus, a decrease in the pH of the buffer solution made it possible to expand the range of drug determination. 3.2. Specificity of the FPIA To evaluate the specificity of the constructed FPIA (expressed by its CR), macrolides (DIR, ROX, CLA, TYL and AZI) were tested. For the assessment of cross-reactivity, the binding capacity of ERY was taken as a reference (100%). The cross-reactivity dependencies presented for the studied macrolides demonstrate the differences during the assay for the mutual determination of macrolides and the variability of the values (Table 2 ). As shown in Fig. 5 and Table 1 , the FPIA method showed an obvious CR with macrolide antibiotics with 14-member lactone rings, i.e., ERY (100%), ROX (100.9%), CLA (146%), and DIR (6.7%), and with the 15-membered ketolide AZI (206%). Another macrolide antibiotic, TYL, containing 16-membered lactone rings, was not recognized by the antibody (CR < 0.1%). Electrical characteristics and molecular shape are the main determinants of antibody–antigen recognition [ 40 ]. The quantity of lactone ring atoms and substituent groups in this study unavoidably altered the macrolide antibiotic's structure and electron distribution, which means that they have a significant impact on antibody recognition. High antibody affinities can result from the macrolide's 14-membered lactone ring. Analysis of the obtained data indicates that this antibody is specific to the common carbohydrate components of these macrolides. However, some structural features of the drugs also influence their cross-reactivity. Although dirithromycin is also a 14-membered macrolide obtained by the condensation of erythromycylamine and methoxyacetaldehyde and is a C9-oxazine derivative of erythromycylamine, similar in structure to erythromycin, DIR is poorly recognized by the antibody. The lower IC50 values for CLA and ROX are because their structures are closer to the immunizing hapten than to ERY [ 21 ]. However, the structural differences between the AZI macrocycle and the 14-membered lactone ring demonstrated an IC50 two times lower than those of ERY, CLA and ROX. Table 1 Cross-reactivity, IC50 values, and limits of detection for macrolides. Macrolide IC50, ng/mL CR% LOD, ng/mL Erythromycin 51 100 1.6 Azithromycin 25 206 2.5 Roxithromycin 51 100 3.2 Clarithromycin 59 146 5.0 Dirithromycin 766 6.7 383 Tylosin >>100000 <<0.1 - 3.3. Recovery tests in milk and water samples The FPIA method for determining ERY developed by us has a fairly low detection limit of 1.6 ng/mL, which is sufficient for analyzing ERY in food products, such as honey and milk. We have demonstrated the possibility of determining tylosin in honey by the FPIA method [ 28 ]. However, in honey samples contaminated with erythromycin, the degree of ERY recovery did not exceed 59%, which may indicate that ERY is not stable in acidic environments and is destroyed by rearrangement of the lactone ring, which leads to the formation of inactive degradation products (anhydro-ERY, pseudo-ERY and enol ether of ERY) [ 41 ], which cannot be recognized by the obtained antibodies. Thus, the developed FPIA is capable of detecting only the native form of ERY and cannot be used to detect erythromycin in honey because of its rapid destruction. A simple, inexpensive, safe and effective method of sample pretreatment to reduce the influence of its components (matrix effect) is sample dilution via an analytical buffer. Homogeneous FPIA allows the determination of toxicants in milk after simple dilution of samples with a research buffer. It has been shown that by diluting a milk sample 10 times, the influence of milk components on the analysis is completely eliminated [ 42 ]. The MPL of ERY residues in milk is set at 40 µg/kg [ 13 ]. Thus, the FPIA we developed is capable of determining this concentration in milk. To test the developed method, three milk samples from different manufacturers were purchased from supermarkets, to which known concentrations of ERY were added, and after dilution of the milk samples, they were tested via the FPIA method. The results are presented in Table 2 . Table 2 FPIA recovery test of erythromycin in milk and water. Sample Added, ng/ml Found, ng/ml Recovery, % Milk sample 1 40 42 ± 2 105 60 64 ± 4 106 100 95 ± 5 95 Milk sample 2 40 38 ± 1 95 60 62 ± 3 103 100 102 ± 7 102 Milk sample 3 40 36 ± 3 90 60 58 ± 2 96 100 110 ± 6 110 Distilled water 5 6 ± 1 120 10 11 ± 2 110 30 28 ± 1 93 River water 5 4 ± 1 80 10 9 ± 2 90 30 32 ± 3 106 In addition, the FPIA method was tested on samples of distilled water and water taken from an open reservoir. The absence of an admixture of macrolide antibiotics was preliminarily checked. Sample preparation for water is quite simple, and only filtration through 0.22 µm filters is needed. As seen from the presented results, the method determines erythromycin in milk samples with an accuracy of 90–110% in water from 80–120%. 4. Conclusion Thus, a highly sensitive and rapid FPIA was developed for the detection of the macrolide antibiotic erythromycin. FPIA requires highly specific antibodies and a fluorescently labeled analyte. In this work, the boron-based fluorescent label BODIPY was used to obtain fluorescently labeled erythromycin. The FPIA technique was optimized: the pH of the reaction buffer and the concentrations of the immunoreagents were selected. A competitive FPIA format was used to determine ERY, and the analysis time was only 5 min. The achieved ERY detection limit of 1.6 ng/mL allows for analysis of food products and water bodies. The developed FPIA was successfully used to determine ERY in water and milk, with a recovery of 80–120%. The use of a portable analyzer allows for out-of-laboratory monitoring of the ERY content and environmental monitoring outside the laboratory. Declarations Authors’ contributions L.M.: Writing original draft, Validation, Methodology, Data curation, Visualization, Funding acquisition. D.A.: Writing original draft, Methodology, Software, Software, Supervision, Resources. A.S.: Investigation, Data curation, Visualization. I.G.: Investigation, Validation, Data curation. M.B.: Investigation, Validation, Data curation. A.T.: Investigation, Validation, Data curation. N.S.: Investigation, Validation, Data curation. S.A.: Writing, Review and Editing, Methodology, Investigation, Supervision, Funding acquisition. All authors read and approved the final manuscript. Acknowledgment We would like to thank V. N. Tashlitsky for conducting the LC‒MS analysis. CONFLICT OF INTEREST The authors of this work declare that they have no conflicts of interest. Funding This work was carried out with the financial support of M.V. Lomonosov Moscow State University (Project number 1210415000398 “Molecular Design, Structural and Functional Analysis and Regulation of Enzyme Systems, Cell Structures and Bionanomaterials: Fundamentals and Applications in Technology, Medicine, and Environmental Protection”). References Tong, Y.; Guo, J.; Li, F.; Lai, K.P.; Mo, J. Antibiotic Erythromycin in Fish: Pharmacokinetics, Effects, and Health Risks. Environ. Pollut. 2025 , 373 , 126203, doi:10.1016/j.envpol.2025.126203. Macrolide Antibiotics. In Comprehensive Medicinal Chemistry II ; Elsevier, 2007; pp. 519–566 ISBN 978-0-08-045044-5. Gajic, I.; Tomic, N.; Lukovic, B.; Jovicevic, M.; Kekic, D.; Petrovic, M.; Jankovic, M.; Trudic, A.; Mitic Culafic, D.; Milenkovic, M.; et al. A Comprehensive Overview of Antibacterial Agents for Combating Multidrug-Resistant Bacteria: The Current Landscape, Development, Future Opportunities, and Challenges. 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Arutyunyan, D.A.; Mukhametova, L.I.; Shanin, I.A.; Kondakov, S.E.; Eremin, S.A. Using Fluorescence Polarization Immunoassay for the Determination of Flunixin in Milk. J. Anal. Chem. 2025 , 80 , 1030–1039, doi:10.1134/S1061934825700376. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Dec, 2025 Reviews received at journal 28 Nov, 2025 Reviews received at journal 17 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers invited by journal 09 Nov, 2025 Editor assigned by journal 11 Oct, 2025 Submission checks completed at journal 08 Oct, 2025 First submitted to journal 08 Oct, 2025 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. 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1","display":"","copyAsset":false,"role":"figure","size":285732,"visible":true,"origin":"","legend":"\u003cp\u003eStructures of macrolide antibiotics: azithromycin (a), erythromycin, clarithromycin, roxithromycin and dirithromycin (b) and tylosin (c).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7808262/v1/d7e29bc54d4fbf40e09f33e0.png"},{"id":96203277,"identity":"6e0ad56d-ce0b-4896-9621-68d8207edf64","added_by":"auto","created_at":"2025-11-18 16:52:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":257157,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Conjugate ERY-BDP\u003cstrong\u003e \u003c/strong\u003eand (b) changes in fluorescence polarization at pH 7.4 (1) and pH 8.5 (2) and fluorescence intensity atpH 7.4 (3) and pH 8.5 (4) of the tracers at different concentrations (25°C).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7808262/v1/36453d20108035f3757eba53.png"},{"id":96203280,"identity":"7c2fd4d3-0bac-4587-b3ec-160182b6ed65","added_by":"auto","created_at":"2025-11-18 16:52:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":128956,"visible":true,"origin":"","legend":"\u003cp\u003eTesting of the immune antiserum aCLA-PAb (1) and antisera obtained against other preparations aNIC-PAb (2) and aTYL-PAb (3) with the ERY-BDP conjugate (final concentration 2.5 nM) and at various dilutions at pH 8.5 (a) and pH 7.4 (b), 25°C. All the experiments were repeated three times.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7808262/v1/d9f16ca0e16c0fcc0adaa361.png"},{"id":96203285,"identity":"d6122600-9d3b-42b8-a573-176b0f604ec0","added_by":"auto","created_at":"2025-11-18 16:52:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132635,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curves for erythromycin determination:aCLA-PAb (finish dilution 400 times), ERY-BDP\u003cstrong\u003e \u003c/strong\u003e(конечная концентрация 2.5 нМ) при рН 8.5 (1) и рН 7.4 (2), 25°C. All experiments were performed in duplicate.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7808262/v1/d1a1e0ecce6bb377d2266564.png"},{"id":96250433,"identity":"0bf8079e-3bfb-4980-95ae-59b951c500ab","added_by":"auto","created_at":"2025-11-19 07:38:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":188829,"visible":true,"origin":"","legend":"\u003cp\u003eGroup-specific FPIA standard curves for the determination of 14-membered macrolide antibiotics, TYL and AZI in PBS. Each point of the curves represents the mean value from four experiments.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7808262/v1/be76c85c389476fffed13cc0.png"},{"id":96257042,"identity":"4c9c1e16-1629-4786-bb5e-85f012c1c49d","added_by":"auto","created_at":"2025-11-19 07:51:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1763381,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7808262/v1/befcec91-dc1e-4e76-9c83-a6719c2ae72a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Application of BODIPY-labeled erythromycin for macrolide detection by a fluorescence polarization immunoassay","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMacrolide antibiotics are a class of drugs used to treat bacterial infections [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In clinical practice, macrolides are among the four most frequently used antimicrobial drugs against gram-positive bacteria, some gram-negative bacteria and Mycoplasma and are also used as adjuvants for the prevention of a number of animal diseases. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The chemical structure of macrolides consists of a lactone ring containing 14 to 16 carbon atoms with one or more sugar residues linked by a glycosidic bond (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The mechanism of their antimicrobial action is due to the disruption of protein synthesis on the ribosomes of the microbial cell and consists of inhibiting the synthesis of bacterial protein by binding to the 50S subunit of the ribosome, namely, by interacting with 23S rRNA [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Macrolides are classified by the World Health Organization (WHO) and the World Organization for Animal Health (OIE) as critically important for human and animal health. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the use of macrolides is associated with certain side effects in humans, such as gastrointestinal disturbances, hypersensitivity reactions, rash and fever, transient hearing loss and possible gastrointestinal infections (ERYs), which have also been reported after the use of erythromycin. Some individuals may experience prolonged QT intervals, which can lead to cardiac arrhythmias [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eImportantly, human health is directly related to the quality of food products, and veterinary drug residues constitute only one of many contamination factors. Although the use of antibiotics in livestock production is necessary to improve weight gain and feed efficiency, to prevent or treat diseases, their residues may be present in animal products [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Residue concentrations exceeding the permissible doses may be caused by failure to follow the technical instructions or dosage calculations provided by the manufacturer, failure to comply with the specified withdrawal period and the use of equipment contaminated with drugs. Antimicrobials are the main veterinary drugs that can potentially contaminate animal products, the most common of which is erythromycin. Erythromycin residues can lead to prolonged and undesirable effects when these substances are ingested in small quantities over a long period of time [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, antibiotic content is regulated and controlled on the basis of maximum residue levels (MRLs) or acceptable limits set in each country [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe maximum permissible concentration of ERY residues in milk is set at 40 \u0026micro;g/kg by EU countries and Customs Union countries [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. WHO установил MRLs for erythromycin: fat, kidney, liver and muscle (chicken and turkey): 100 \u0026micro;g/kg; eggs (chicken): 50 \u0026micro;g/kg [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, food safety laboratories must test many samples because of the need to control the detection of antibiotics. Various instrumental methods have been developed for the determination of macrolide drugs and their metabolites in various food samples. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, these instrumental monitoring methods require highly qualified specialists and complex sample preprocessing, which complicates the analysis and increases its time. Thus, sensitive, specific, reliable and rapid analysis methods are essential for the effective monitoring of ERY in food products. Modern biosensor technologies for the determination of erythromycin have been developed [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, immunoassay methods are increasingly used to detect toxic contaminants in food samples because of their sensitivity, selectivity and time savings, and they have become effective alternatives to instrumental methods. For example, enzyme-linked immunosorbent assay (ELISA) methods for determining erythromycin using rabbit polyclonal antiserum with a detection limit (LOD) of 0.04 ng/ml have been developed [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], as do monoclonal antibody (LOD 0.057 нг/мл) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and lateral flow immunoassays (LFAs) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, LFAs are semiquantitative methods for detecting toxicants; heterogeneous ELISAs require a long time and complex manipulations, whereas homogeneous immunoassays have shown great potential for simplifying routine tasks [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePolarization fluorescence immunoassay (FPIA) has several advantages, such as sensitivity, reliability, rapidity and the ability to analyze large numbers of samples. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The principle of small molecule detection by FPIA is that a fluorescently labeled analyte (tracer) and the analyte to be determined compete for a limited number of binding sites on antibodies, which leads to a change in the fluorescence polarization (FP) value [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. If there is no analyte in the test solution, the tracer binds to the specific antibody and forms a bulk antigen‒antibody complex with a slower mobility than the free tracer, resulting in a high FP signal. As the analyte concentration in the reaction mixture increases, the antibody will bind to the analyte; therefore, the concentration of free tracer will increase, and the FP signal will decrease. FPIA has attracted increasing attention and is often used to detect many small molecule compounds, such as veterinary drugs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], pesticides [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and toxins [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], in food and environmental samples. Currently, FPIA methods for determining tylosin are known [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and ERY [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] in honey and milk, respectively. In these studies, fluorescein was used as a fluorescent label on the antigen, and the detection limits of the drugs were 34.7 ng/ml for tylosin and 14.08 ng/ml for erythromycin. In the literature, most FPIA methods use fluorescein as a fluorescent label, and this choice is justified by its unique properties: high quantum yield of 92%, fluorescence lifetime of 4.05 ns, chemical stability, and availability of reagents with various reactive groups (sulfhydryls, amino groups and carboxyl groups), which are relatively inexpensive and commercially available. In addition, most devices for measuring the FP signal are tuned to the radiation and emission wavelengths specifically for fluorescein. Currently, other dyes that are capable of absorbing and emitting in the same region as fluorescein are used. Currently, boron-based dyes are used, such as BODIPY. These dyes are advantageous in FP assays because they have longer excited-state lifetimes than fluorescein dyes do [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This extended lifetime allows FPs to be sensitive to binding interactions across a broader range of molecular weights. The longer lifetime of BODIPY dyes means that they remain excited for a longer period. This allows for more rotational movement and depolarization before the fluorophore emits light. Consequently, BODIPY dyes can be used to detect binding events in molecules with a wider range of molecular weights than dyes with shorter lifetimes. In FP assays, if the fluorophore's rotational correlation time (which is related to molecular weight) is much shorter than its excited-state lifetime, the fluorescence polarization will not be significantly affected by binding. The longer lifetime of BODIPY allows it to be sensitive to smaller changes in rotational correlation time, making it useful for studying binding interactions with a wider range of molecular weights. FP analysis using antibiotic conjugates [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] or pesticides [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] with BODIPY is a convenient platform for studying the interactions of various drugs with ribosomes to determine the kinetic parameters of binding and modulation of their interactions. We hypothesized that the use of BODIPY dye as a fluorescent label on erythromycin would reduce the detection limit of the macrolide antibiotic group in food products by FPIA. The aim of this study was to develop a method for FPIA using a portable detector for the determination of erythromycin via a conjugate of the studied antibiotic with BODIPY and to test these methods for their determination in milk, water and honey.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eThe following reagents were used in this work: erythromycin (ERY, Fluka, Germany), clarithromycin (CLA), roxithromycin (ROX), azithromycin (AZI), dirithromycin (DIR), tylosin (TYL), BODIPY FL-C5 NHS Ester (succinimidyl ester) (Invitrogen, США), and the antiserum ACLA-PAb was received as described previously [21].\u003c/p\u003e\n\u003cp\u003eThe FP signal was measured via a Sentry 200 portable fluorimeter (Ellie, USA) at an excitation wavelength of 495 nm, and emission was recorded at 530 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1. The following buffer solutions were used\u003c/strong\u003e: 10 mM phosphate buffer, pH 7.4, containing 0.15 M NaCl, 0.005% Tween 20 (PBS-T) and 25 mM borate buffer, pH 8.5 (BB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. \u003c/strong\u003e\u003cstrong\u003ePreparation of erythromycin \u003c/strong\u003e\u003cstrong\u003econjugated\u003c/strong\u003e\u003cstrong\u003e with \u003c/strong\u003e\u003cstrong\u003ethe \u003c/strong\u003e\u003cstrong\u003efluorescent dye BODIPY (ERY-BDP).\u003c/strong\u003e Synthesis was performed as described previously [36,38]. 9-(S)-Erythromycylamine A, obtained from erythromycin according to a previously described method [39], was dissolved in tetrahydrofuran and mixed with BODIPY FL-C5 succinimide ester under an argon atmosphere. After stirring at room temperature for 24 h, the solvent was removed in vacuo. The resulting residue was purified via HPLC. The concentration of the conjugate was determined via the molar absorption coefficient of the dye \u0026epsilon;\u003csub\u003e504\u003c/sub\u003e in MeOH (BODIPY-FL-C5 succinimide ester) = 87000 M\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. \u003c/strong\u003e \u003cstrong\u003eHigh-resolution liquid chromatography‒tandem mass spectrometry\u003c/strong\u003e (LC‒MS/MS) analysis was conducted via a comprehensive UPLC‒MS/MS system comprising an Acquity UPLC chromatograph (Waters, USA) integrated with a Thermo Scientific TQD quadrupole mass spectrometer. The analysis was performed in positive ion mode employing electrospray ionization (ESI) methodology. Chromatographic separation was achieved via a BEH C18 column (Waters) with a particle size of 1.7 \u0026mu;m and dimensions of 50 mm \u0026times; 2.1 mm. The mobile phase consisted of a linear gradient of 5\u0026ndash;100% acetonitrile (CH3CN) in 20 mM formic acid (HCOOH), delivered at a flow rate of 0.5 mL/min at a column temperature of 35\u0026deg;C over a duration of 4 minutes. The photophysical properties of the analyte were characterized by its excitation (\u0026lambda;ex) and emission (\u0026lambda;em) wavelengths in methanol, which were determined to be 504 nm and 511 nm, respectively. The retention time (tR) of the compound in the LC‒MS analysis was 1.50 minutes. The theoretical and experimental mass‒charge (m/z) ratios were calculated and measured as 1037.64 and 1037.20, respectively, for the protonated molecular ion [C53H87BF2N4O13 + H]+.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Determination of the working concentration of the ERY-BDP conjugate\u003c/strong\u003e. A series of dilutions of the ERY-BDP conjugate were prepared in BB or PBS-T buffer solutions, and fluorescence polarization was measured via a Sentry-200 portable fluorimeter. The criterion for selecting the working concentration of the ERY-BDP conjugate was a stable FP value and an excess of the conjugate fluorescence intensity over the background signal value by 10 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Characterization of the interaction of the ERY-BDP conjugate with the aCLA-PAb antiserum. \u003c/strong\u003eThe working dilution of the aCLA-PAb antiserum was determined as follows. A series of antisera dilutions from 1:100 to 1:64000 in BB or PBS-T in a volume of 500 \u0026mu;l were prepared in borosilicate glass tubes. Five hundred microliters of the ERY-BDP conjugate solution at a concentration of 5 nM in the appropriate buffer was added, the mixture was incubated at room temperature for 5 min, and fluorescence polarization was measured via a Sentry-200 portable fluorimeter. The binding of ERY-BDP to nonimmune sera was studied similarly. All experiments were performed in duplicate, and three instrumental measurements were taken. The average values were calculated, and the FP dependences on the antiserum dilution were plotted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Study of the kinetics of the interaction of ERY-BDP with the aCLA-PAb antiserum. \u003c/strong\u003eFive hundred microliters of the antiserum at a 1:1000 dilution in BB was added to 500 \u0026mu;l of the ERY-BDP conjugate in the selected dilution, and the change in fluorescence polarization was measured for 25 minutes at 30-second intervals. All the experiments were performed in duplicate. The average values were calculated, and the FP dependences on time were plotted.\u003c/p\u003e\n\u003cp id=\"_Toc231338810\"\u003e\u003cstrong\u003e2.7. Conducting FPIA. \u003c/strong\u003eFifty microliters of standard erythromycin solutions in water (0, 0.1, 1, 3, 6, 10, 30, 100, 1000, or 10000 ng/ml) and 500 microliters of the ERY-BDP conjugate solution with a concentration of 5 nM in the test buffer were added to glass tubes. Then, 500 microliters of aCLA-PAb antiserum in the selected dilution was added, and after 10 min, the fluorescence polarization was recorded via a Sentry-200 portable detector. Each measurement was performed in triplicate, and three instrumental measurements were made.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. Determination of \u003c/strong\u003e\u003cstrong\u003ethe \u003c/strong\u003e\u003cstrong\u003eanalytical characteristics of FPIA. \u003c/strong\u003eThe dependence of fluorescence polarization on the erythromycin concentration (calibration curve) was plotted on a semilogarithmic scale and approximated via a four-parameter sigmoid function:\u003c/p\u003e\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:Y=B+\\frac{A-B}{\\left(1+{\\left(\\frac{x}{C}\\right)}^{D}\\right)}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003ex\u003c/em\u003e is the analyte concentration, \u003cem\u003ey\u003c/em\u003e is the FP value, and \u003cem\u003eA\u003c/em\u003e is the asymptotic maximum of the FP value, \u003cem\u003eB\u003c/em\u003e is the asymptotic minimum (background value) of the FP value, and \u003cem\u003eC\u003c/em\u003e is the inflection point of the curve in semilogarithmic coordinates (equal to 50% inhibition of the intensity), and \u003cem\u003eD\u003c/em\u003e is the slope of the curve at the inflection point.\u003c/p\u003e\u003cp\u003eThe IC10, IC20, IC50, and IC80 values were calculated as concentrations that reduced the analytical signal by 10, 20, 50, and 80%, respectively. The IC10 value was estimated as the detection limit, and IC20\u0026ndash;IC80 was the working range of the determined concentrations. Cross-reactivity values were calculated via the following formula:\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:CR\\left(\\%\\right)=\\frac{{IC}_{50}\\left(ERY\\right)}{{IC}_{50}\\left(CR\\right)}*100\\)\u003c/span\u003e\u003c/span\u003e (2).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of honey, milk and water samples.\u003c/b\u003e Honey samples were prepared as described previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] with some modifications. One gram of honey was placed in 5 ml polypropylene tubes, and aliquots of 0 to 60 \u0026micro;l of the ERY solution at a concentration of 100 \u0026micro;g/ml were added. The samples were mixed in a vortex-1 vortex mixer (IKA, Germany) for 1 min. Then, 2250 \u0026micro;l of 5 M aqueous sodium acetate solution was added to each tube, the mixture was incubated for 30 min at 37\u0026deg;C, 2250 \u0026micro;l of acetonitrile was added, and the mixture was extracted and then centrifuged at 3500 \u0026times; g for 10 min. 2000 \u0026micro;l of the upper organic layer was collected and evaporated until the solvent was removed, and the dry residue was redissolved in 500 \u0026micro;l of PBS and mixed. The resulting extract was analyzed.\u003c/p\u003e\u003cp\u003eThe milk samples were pretreated via simple dilution with PBS at a ratio of 1:9. Water samples from open water bodies were additionally filtered through 13 mm diameter 0.22 \u0026micro;m pore size syringe filters, and if necessary, the pH was adjusted to 7.4 by the addition of 1 M NaOH.\u003c/p\u003e\u003cp\u003eUsing a Sentry-200 portable detector, fluorescence polarization was recorded in triplicate for each sample, and erythromycin concentrations were determined on the basis of the FPIA calibration curve. The agreement between the injected and detected ERY concentrations was assessed as a percentage.\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Preparation of immunoreagents and optimization of the FPIA method.\u003c/h2\u003e\u003cp\u003eMacrolide antibiotics are used in agriculture for the treatment and prevention of diseases in animals and are found in food products. Simple and fast methods are needed for their determination. FPIA does not require lengthy manipulations, takes only a few minutes and can be carried out outside the laboratory. Since various macrolide antibiotics are used in veterinary medicine, such as erythromycin, roxithromycin, clarithromycin, and tylosin, we selected polyclonal antibodies with broad specificity (aCLA-PAb) to develop a method for determining macrolides in food products. This antiserum has broad cross-reactivity and allows the determination of several macrolides [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For the test system under development, a conjugate of erythromycin with BODIPY dye was obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To introduce a fluorescent label into the erythromycin molecule, the keto group at position C9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was chosen and stereospecifically converted into a 9-(S)-amino group. This allowed the resulting 9-(S)-erythromycinamine to be acylated under mild conditions via BODIPY FL-C5 succinimide ester, and the resulting conjugate was obtained in good yield without prior protection of the functional groups of the parent antibiotic. The optimal concentration of the conjugate was determined by examining the FP values and fluorescence intensity at different concentrations of ERY-BDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Then, the binding of the antiserum was studied, and the time to equilibrium was investigated by studying the binding of aCLA-PAb to ERY-BDP (final concentration of 2.5 nM) for 20 min. The binding reached equilibrium in approximately 2\u0026ndash;3 min (data not shown). During this time period, the total fluorescence intensity did not change, indicating that the formation of the ERY-BDP complex with antibodies did not cause significant quenching of the fluorescence intensity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInitially, we tested the interaction of the ERY-BDP conjugate with the rabbit immune antiserum aCLA-PAb and nonimmune antisera obtained against nicotinic acid (aNIC-PAb) and tylosin (aTYL-PAb). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a), at pH 8.5, binding of the ERY-BDP conjugate to aCLA-PAb is observed: the FP signal increases, and equilibrium in the reaction system occurs in just 2\u0026ndash;3 min. As the dilution of the antiserum increases, the FP signal decreases. When nonimmune sera aNIC-PAb and aTYL-PAb were tested, an increase in the FP signal was also observed, but it was lower than that for the immune antiserum. Thus, ERY-BDP at pH 8.5 nonspecifically binds to serum proteins. With dilution of the antiserum, a decrease in the FP signal was observed. However, when the pH was reduced to 7.4, the FP signal for the ERY-BDP*aCLA-PAb complex increased, whereas for the ERY-BDP pairs with aNIC-PAb and aTYL-PAb, it decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The change in the FP signal between the immune and nonimmune sera at a dilution of 200 mP was approximately 50 mP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe dependence of the FP signal change at constant concentrations of the tracer and antiserum on the erythromycin concentration was determined for the determination of erythromycin by the FPIA method at pH 8.5 and 7.4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The detection limit at two pH values was 1.6 ng/ml, but the detection range at pH 7.4 was wider, from 5 to 300 ng/ml, whereas that at pH 8.5 ranged from 5 to 130 ng/ml. Thus, a decrease in the pH of the buffer solution made it possible to expand the range of drug determination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Specificity of the FPIA\u003c/h2\u003e\u003cp\u003eTo evaluate the specificity of the constructed FPIA (expressed by its CR), macrolides (DIR, ROX, CLA, TYL and AZI) were tested. For the assessment of cross-reactivity, the binding capacity of ERY was taken as a reference (100%). The cross-reactivity dependencies presented for the studied macrolides demonstrate the differences during the assay for the mutual determination of macrolides and the variability of the values (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the FPIA method showed an obvious CR with macrolide antibiotics with 14-member lactone rings, i.e., ERY (100%), ROX (100.9%), CLA (146%), and DIR (6.7%), and with the 15-membered ketolide AZI (206%). Another macrolide antibiotic, TYL, containing 16-membered lactone rings, was not recognized by the antibody (CR\u0026thinsp;\u0026lt;\u0026thinsp;0.1%). Electrical characteristics and molecular shape are the main determinants of antibody\u0026ndash;antigen recognition [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The quantity of lactone ring atoms and substituent groups in this study unavoidably altered the macrolide antibiotic's structure and electron distribution, which means that they have a significant impact on antibody recognition. High antibody affinities can result from the macrolide's 14-membered lactone ring. Analysis of the obtained data indicates that this antibody is specific to the common carbohydrate components of these macrolides. However, some structural features of the drugs also influence their cross-reactivity. Although dirithromycin is also a 14-membered macrolide obtained by the condensation of erythromycylamine and methoxyacetaldehyde and is a C9-oxazine derivative of erythromycylamine, similar in structure to erythromycin, DIR is poorly recognized by the antibody. The lower IC50 values for CLA and ROX are because their structures are closer to the immunizing hapten than to ERY [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the structural differences between the AZI macrocycle and the 14-membered lactone ring demonstrated an IC50 two times lower than those of ERY, CLA and ROX.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCross-reactivity, IC50 values, and limits of detection for macrolides.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMacrolide\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC50, ng/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCR%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLOD, ng/mL\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eErythromycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAzithromycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e206\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRoxithromycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClarithromycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e146\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDirithromycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e766\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e383\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTylosin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026gt;100000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026lt;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Recovery tests in milk and water samples\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe FPIA method for determining ERY developed by us has a fairly low detection limit of 1.6 ng/mL, which is sufficient for analyzing ERY in food products, such as honey and milk. We have demonstrated the possibility of determining tylosin in honey by the FPIA method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, in honey samples contaminated with erythromycin, the degree of ERY recovery did not exceed 59%, which may indicate that ERY is not stable in acidic environments and is destroyed by rearrangement of the lactone ring, which leads to the formation of inactive degradation products (anhydro-ERY, pseudo-ERY and enol ether of ERY) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], which cannot be recognized by the obtained antibodies. Thus, the developed FPIA is capable of detecting only the native form of ERY and cannot be used to detect erythromycin in honey because of its rapid destruction.\u003c/p\u003e\u003cp\u003eA simple, inexpensive, safe and effective method of sample pretreatment to reduce the influence of its components (matrix effect) is sample dilution via an analytical buffer. Homogeneous FPIA allows the determination of toxicants in milk after simple dilution of samples with a research buffer. It has been shown that by diluting a milk sample 10 times, the influence of milk components on the analysis is completely eliminated [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The MPL of ERY residues in milk is set at 40 \u0026micro;g/kg [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, the FPIA we developed is capable of determining this concentration in milk. To test the developed method, three milk samples from different manufacturers were purchased from supermarkets, to which known concentrations of ERY were added, and after dilution of the milk samples, they were tested via the FPIA method. The results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFPIA recovery test of erythromycin in milk and water.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdded, ng/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFound, ng/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRecovery, %\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eMilk sample 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e105\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e64\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e106\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e95\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eMilk sample 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e38\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e62\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e103\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e102\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e102\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eMilk sample 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e36\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e58\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e110\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e110\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDistilled water\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e110\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eRiver water\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e32\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e106\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn addition, the FPIA method was tested on samples of distilled water and water taken from an open reservoir. The absence of an admixture of macrolide antibiotics was preliminarily checked. Sample preparation for water is quite simple, and only filtration through 0.22 \u0026micro;m filters is needed. As seen from the presented results, the method determines erythromycin in milk samples with an accuracy of 90\u0026ndash;110% in water from 80\u0026ndash;120%.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThus, a highly sensitive and rapid FPIA was developed for the detection of the macrolide antibiotic erythromycin. FPIA requires highly specific antibodies and a fluorescently labeled analyte. In this work, the boron-based fluorescent label BODIPY was used to obtain fluorescently labeled erythromycin. The FPIA technique was optimized: the pH of the reaction buffer and the concentrations of the immunoreagents were selected. A competitive FPIA format was used to determine ERY, and the analysis time was only 5 min. The achieved ERY detection limit of 1.6 ng/mL allows for analysis of food products and water bodies. The developed FPIA was successfully used to determine ERY in water and milk, with a recovery of 80\u0026ndash;120%. The use of a portable analyzer allows for out-of-laboratory monitoring of the ERY content and environmental monitoring outside the laboratory.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.M.: Writing original draft, Validation, Methodology, Data curation, Visualization, Funding acquisition. D.A.: Writing original draft, Methodology, Software, Software, Supervision, Resources. A.S.: Investigation, Data curation, Visualization. I.G.: Investigation, Validation, Data curation. M.B.: Investigation, Validation, Data curation. A.T.: Investigation, Validation, Data curation. N.S.: Investigation, Validation, Data curation. S.A.: Writing, Review and Editing, Methodology, Investigation, Supervision, Funding acquisition. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank V. N. Tashlitsky for conducting the LC‒MS analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCONFLICT OF INTEREST\u003c/p\u003e\n\u003cp\u003eThe authors of this work declare that they have no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was carried out with the financial support of M.V. Lomonosov Moscow State University (Project number 1210415000398 \u0026ldquo;Molecular Design, Structural and Functional Analysis and Regulation of Enzyme Systems, Cell Structures and Bionanomaterials: Fundamentals and Applications in Technology, Medicine, and Environmental Protection\u0026rdquo;).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTong, Y.; Guo, J.; Li, F.; Lai, K.P.; Mo, J. Antibiotic Erythromycin in Fish: Pharmacokinetics, Effects, and Health Risks. \u003cem\u003eEnviron. Pollut.\u003c/em\u003e \u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e373\u003c/em\u003e, 126203, doi:10.1016/j.envpol.2025.126203.\u003c/li\u003e\n\u003cli\u003eMacrolide Antibiotics. 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Chem.\u003c/em\u003e \u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e80\u003c/em\u003e, 1030\u0026ndash;1039, doi:10.1134/S1061934825700376.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"one-health-advances","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [One Health Advances](https://onehealthadv.biomedcentral.com/)","snPcode":"44280","submissionUrl":"https://submission.springernature.com/new-submission/44280/3","title":"One Health Advances","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7808262/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7808262/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImmune methods of analysis are widely used in laboratories in various fields of medicine, including enzyme immunoassay (ELISA), polarization fluorescence immunoassay (FPIA) and immunochromatographic analysis. The FPIA method allows for high specificity and sensitivity of low-molecular-weight analytes in a homogeneous medium without separation and has the ability to automate the analysis system, high accuracy, label stability, and speed and simplicity of analysis. In this work, a fluorescently labeled tracer for the determination of erythromycin with new BODIPY dye was obtained. A pair of immunoreagents (tracers and antibodies) for detecting these antibiotics were selected and characterized. The conditions for conducting FPIA were optimized, calibration curves were obtained, and the analytical characteristics of the FPIA were determined: detection limit, range of detectable concentrations, sensitivity and cross-reactivity. Using the developed FPIA methods for determining antibiotics most commonly used in veterinary medicine, water samples collected from reservoirs in Moscow and the Moscow region were tested. Thus, in this work, the FPIA method for determining erythromycin with a detection limit of 1.6 ng/ml was developed, the advantages of using the new fluorescent label BODIPY were shown, the accuracy of the method was verified by an introduced-found test, and real water samples were tested.\u003c/p\u003e","manuscriptTitle":"Application of BODIPY-labeled erythromycin for macrolide detection by a fluorescence polarization immunoassay","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 16:51:58","doi":"10.21203/rs.3.rs-7808262/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-12T00:23:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-28T10:27:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-17T15:30:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232582279904218366825629006104353011561","date":"2025-11-11T15:42:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94616279716629627458655046851806174905","date":"2025-11-10T08:56:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-09T11:00:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-11T05:22:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-09T02:44:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"One Health Advances","date":"2025-10-08T13:05:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"one-health-advances","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [One Health Advances](https://onehealthadv.biomedcentral.com/)","snPcode":"44280","submissionUrl":"https://submission.springernature.com/new-submission/44280/3","title":"One Health Advances","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f763ec7-c120-47a2-8182-30f5ca2d6da4","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-27T07:09:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 16:51:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7808262","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7808262","identity":"rs-7808262","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}