Development and validation of a latex particle-enhanced turbidimetric immunoassay for fecal calprotectin Bioprocessing and Bioengineering

Development and validation of a latex particle-enhanced turbidimetric immunoassay for fecal calprotectin Bioprocessing and Bioengineering | 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 Development and validation of a latex particle-enhanced turbidimetric immunoassay for fecal calprotectin Bioprocessing and Bioengineering Zhe Zhou, Yu Li, Feihu Zhang, Shaodong Wang, Wenhua Huang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8411228/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2026 Read the published version in Biotechnology Letters → Version 1 posted 4 You are reading this latest preprint version Abstract Background Fecal calprotectin (FCP) is a crucial non-invasive biomarker for diagnosing and monitoring inflammatory bowel disease (IBD). However, the standard enzyme-linked immunosorbent assay (ELISA) is time-consuming and labor-intensive. This study aimed to develop and evaluate a rapid, automated latex particle-enhanced turbidimetric immunoassay (LTIA) for FCP quantification. Methods Recombinant human S100A8 and S100A9 proteins (the subunits of calprotectin) were expressed in E. coli and purified. Polyclonal antibodies were generated in rabbits by immunization with these proteins. The antibodies were then covalently coupled to carboxylated latex particles (188 nm) to create the LTIA reagent. The linear repeatability, within-lab precision and interference substances of the reagent were determined, The performance of this method was evaluated using clinical fecal samples (n = 104), including linearity and correlation with commercial ELISA Kit (B Ü Hlmann fCAl). Results The developed LTIA demonstrated excellent linearity within the range of 50–1500 µg/g (R² = 0.9910). A strong correlation was observed between the new LTIA and the reference ELISA method, with a correlation coefficient (r) of 0.9830. The regression equation was y = 1.0359x + 8.8159. Bland-Altman analysis confirmed good agreement between the two methods, with 95.19% of data points within the 95% limits of agreement. Conclusion The LTIA showed strong correlation with the established ELISA for FCP quantification, offering a rapid, automated alternative potentially suitable for high-throughput labs. This study, completing development from antibody to reagent, supports its translational potential. However, this study has several limitations. Limitations include a single-center sample set, limiting generalizability, the need for multicenter validation, and unreported key analytical parameters such as LOD, LOQ, and inter-assay variation. IBD Calprotectin Latex enhanced turbidimetric immunoassay Figures Figure 1 Figure 2 Figure 3 Introduction Inflammatory bowel disease (IBD) comprises a series of chronic systemic inflammatory disorders that impair the normal functions of the digestive system, and includes conditions such as Crohn’s disease and ulcerative colitis(Kaplan & Windsor, 2021 ). IBD has emerged as a serious global concern owing to its rising incidence across all continents, and has a significant impact on the healthcare system worldwide owing to the lack of effective therapeutic strategies(Bisgaard et al., 2022 ). Although endoscopy is the gold standard for diagnosing IBD, evaluating therapeutic efficacy, and detecting postoperative recurrence(Shen et al., 2025 ), its utility is limited by invasiveness, cost, and associated procedural risks(Liu et al., 2022 ). Fecal calprotectin is a well-established non-invasive biomarker for diagnosing IBD(Jukic et al., 2021 ). Furthermore, it plays a crucial role in monitoring disease activity, predicting future relapse, and guiding treatment decisions, thereby reducing the reliance on repeated endoscopy(Lamb et al., 2019 ). Calprotectin is a member of the S100 calcium-binding protein family and exists as a heterodimer composed of S100A8 and S100A9 subunits(Donato et al., 2013 ). Calprotectin is released by activated neutrophils, mediates direct antibacterial activity, and thereby plays a critical role in innate immunity(Edgeworth et al., 1991 ; Zygiel & Nolan, 2018 ). Calprotectin is widely distributed in various biological fluids, where its concentration is elevated in proportion to the severity of inflammation(Mellor et al., 2022 ). Under physiological conditions, its fecal concentration is approximately six-fold higher than that in plasma(Ayling & Kok, 2018 ). A fecal calprotectin (FCP) level below 50 µg/g is widely used as a diagnostic cut-off to distinguish irritable bowel syndrome (IBS) from inflammatory bowel disease (IBD)(Menees et al., 2015 ). Furthermore, the International Organisation for the Study of Inflammatory Bowel Disease (IOIBD) Selecting Therapeutic Targets in Inflammatory Bowel Disease (STAR) consensus recommends a treatment target of FCP < 150 µg/g(Turner et al., 2021 ). In China, fecal calprotectin (FCP) is commonly quantified using enzyme-linked immunosorbent assay (ELISA). However, ELISA is costly, time-consuming, and labor-intensive due to its multiple incubation and washing steps(Tomckowiack et al., 2023 ). To address these limitations, establishing a rapid, reliable, and automated alternative is imperative. Latex particle-enhanced turbidimetric immunoassay (LTIA) represents such a method(Deng et al., 2021 ). This technique employs antibody-coated latex particles that agglutinate in the presence of the specific analyte(Thakkar et al., 1991 ). The resultant aggregation increases turbidity in a concentration-dependent manner, which can be quantified by turbidimetry(Xia et al., 2017 ). Owing to its high efficiency and ease of automation, LTIA is well-suited for high-throughput clinical laboratories(Machida et al., 2015 ). In this study, we independently developed the assay from antibody production to final reagent formulation and optimized it for automated analyzers. This work provides a rapid and fully automated alternative method suitable for high-throughput clinical settings. Materials and Methods Construction of prokaryotic expression vectors of S100A8 and S100A9 The coding sequences of S100A8(NM_002964) and S100A9(NM_002965) were retrieved from the NCBI database( https://www.ncbi.nlm.nih.gov/gene ). Using the NCBI ORFfinder tool, the open reading frames (ORFs) were identified. Subsequently, the sequences were submitted to Nanjing GenScript Biotechnology Co., Ltd. for codon optimization and chemical synthesis. Following digestion with BamHI and XhoI, the synthesized gene was ligated into the linearized pET-28a vector, generating an expression construct designed to produce the recombinant protein with an N-terminal His-tag. The resulting recombinant plasmids, S100A8_pET-28a(+) and S100A9_pET-28a(+), were transformed into E. coli BL21(DE3) competent cells. Positive transformants were selected on LB agar plates supplemented with kanamycin (30 µg/mL). Single colonies were inoculated into LB liquid medium containing the same antibiotics (kanamycin 30 µg/mL) and cultured overnight at 37°C with shaking at 200 rpm. Recombinant plasmids were extracted using a commercial plasmid miniprep kit. The extracted plasmids were verified by DNA sequencing, which was performed by GENEWIZ (Suzhou) Biotechnology Co., Ltd., using the T7 terminator as the sequencing primer. Expression and purification of recombinant S100A8 and S100A9 proteins The transformed E. coli strains S100A8-BL21(DE3) and S100A9-BL21(DE3) were inoculated into 200 mL of LB liquid medium supplemented with 30 µg/mL kanamycin. Protein expression was induced with 0.05 mmol/L isopropyl-β-D-thiogalactoside (IPTG) when the cell density reached an OD600 = 0.5–0.6. Induction was carried out at 18°C for 20 hours. After induction, the cells were harvested by centrifugation at 2000 rpm for 15 min at 4°C. The pellet was resuspended in 5 mL of lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) supplemented with protease inhibitors, followed by thorough mixing via vortexing. Cell disruption was performed using an ultrasonic disruptor for 20 min, and the lysate was centrifuged at 4000 rpm for 15 min. The supernatant was collected and stored at − 20°C. The yield of recombinant protein was evaluated by 12% SDS-PAGE. For purification, the supernatant was filtered through a 0.22 µm membrane and loaded onto a Ni-NTA column. The column was washed with elution buffers containing imidazole at gradient concentrations (20, 50, 100, 150, 200, 250, 300, 500 mM, and 1 M) in a base buffer (50 mM NaH₂PO₄ and 300 mM NaCl (pH 8.0)). Protein elution was monitored using a UV monitor to detect the protein peak. Eluted protein fractions and the crude supernatant after cell lysis were analyzed by SDS-PAGE. Preparation and purification of polyclonal antibodies The purified recombinant S100A8 and S100A9 proteins were emulsified with Freund’s adjuvant and used to immunize New Zealand rabbits. The initial immunization (300 µg/rabbit) used complete adjuvant, followed by three booster injections (100 µg/rabbit) with incomplete adjuvant at 15-day intervals. Serum was collected 15 days after the final immunization. Polyclonal antibodies were purified from the serum using Protein A affinity chromatography. ELISA S100A8 and S100A9 proteins were diluted in a specific coating buffer to a concentration of 10 µg/mL. Each well of an ELISA plate was coated with 100 µL of the protein solution and incubated overnight at 4°C. The plate was then blocked with 1% bovine serum albumin (BSA) at 37°C for 2 hours to prevent non-specific binding. After blocking, 100 µL of rabbit serum at various dilution ratios (ranging from 1:100 to 1:1,280,000) was added to each well. Normal rabbit serum was used as a negative control. The plate was incubated at 37°C for 1 hour. Subsequently, horseradish peroxidase (HRP)-conjugated sheep anti-rabbit secondary antibody, diluted at 1:5,000, was added to each well, followed by another 1-hour incubation at 37°C. For detection, TMB substrate solution was added and the plate was incubated in the dark at 37°C for 30 minutes. The reaction was stopped by adding stop solution, and the optical density at 450 nm (OD450) was measured using an ELISA microplate reader. A preset cutoff value of 0.05 was used to define the negative control. The P/N ratio was calculated as the OD450 value of the sample divided by that of the negative control. A sample was considered positive when the P/N value was ≥ 2.1. Latex reagents The carboxyl series for covalent binding latex particle produced by JSR Corporation (Japan) with a diameter of 188 nm (IMMUTEX P0113, LOT. J5612K-01) was used. Antibody coupling is carried out according to the instructions of latex particles. A 250 µL aliquot of latex beads was diluted and mixed with 425 µL of HEPES buffer (50 mM, pH 7.2). Then, 125 µL each of S100A8 and S100A9 antibodies (10 mg/mL) was added, and the mixture was stirred at 25–37°C for 1 hour. Subsequently, 125 µL of EDC (10 mg/mL in HEPES buffer) was added, and the reaction was allowed to proceed for another hour at 25–37°C with continuous mixing. The mixture was centrifuged at 25,000 × g for 30 minutes, and the supernatant was discarded. The pellet was resuspended in 5 mL of a 5% BSA solution, thoroughly mixed, and incubated at 25–37°C for 1 hour to block non-specific sites. After a second centrifugation under the same conditions (25,000 × g, 30 minutes), the supernatant was removed. The resulting conjugate was resuspended in 5 mL of storage buffer (25 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween-20, pH 7.2) and stored at 2–8°C. Measurement procedure The quantification was performed using an LC-400 specific protein analyzer (Nanjing Laola Electronics Co., Ltd.). Samples and reagents were dispensed according to the manufacturer's instructions. A six-point calibration curve was generated using recombinant calprotectin at concentrations of 0, 50, 100, 250, 750, and 1500 µg/g. Fecal samples were diluted 1:500 in normal saline and centrifuged at 10,000 rpm for 5 minutes. The resulting supernatant was collected for analysis. Then, 200 µL of PB buffer (50 mM PB, 1% PVP, 0.05% Tween-20, pH 7.0) was dispensed into a tube, followed by the addition of 25 µL of latex reagent (0.2% latex in 50 mM PB, pH 7.0, containing 1% PVP and 0.05% Tween-20) to initiate the reaction. Subsequently, 4 µL of the experimental sample was added to the mixture and incubated at 37°C for 5 minutes. The absorbance at 750 nm was measured before and after the reaction, and the difference was calculated. Comparison with commercially available reagent kits The performance of the calprotectin immunolatex reagent was evaluated against a commercially available fCAL ELISA kit (BÜHLMANN Laboratories AG). The correlation between the two methods was assessed using linear regression analysis and visualized in a scatter plot. Clinical sample testing The stool sample came from Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School. The project will start and end from November 5, 2024 to March 30, 2025, and all subjects will sign written informed consent. The experimental protocols and procedures were approved by Nanjing Drum Tower Hospital (approval number: FM-M7-2023051901). According to the CLSI EP9-A2 standard formulated by the National Committee for clinical laboratory standardization of the United States, the correlation coefficient of the test results of the test method and the comparison method shall not be less than 0.95. It is estimated that the correlation coefficient of this test is more than 0.95, so the parameters are set as follows: a = 0.05, s = 1 (bilateral), β = 0.2, ρ 0 = 0.95, ρ 1 = 0.95. The sample size is calculated according to the sample size estimation formula of the correlation coefficient test (continuous variable). The sample size is 94 cases. Combined with regulatory requirements and statistical requirements, considering the 5% rejection rate, the final sample size of this clinical trial is determined to be no less than 100 cases. The detection result of calprotectin is not less than 30% of the total number of cases outside the reference range (abnormal value sample). Subgroup sample size requirements: 20 cases of Crohn's disease, 10 cases of ulcerative colitis. Statistical analyses A comprehensive statistical protocol was employed to establish the equivalence between the novel LTIA and the reference ELISA method. Correlation and Regression Analysis: Linear regression analysis (ordinary least squares) was performed on paired results from 104 clinical samples. The correlation coefficient (R), slope (b), and intercept (a) were calculated, with acceptance criteria set at R ≥ 0.95. The 95% confidence intervals (CI) for both the slope and intercept were also determined. Bland-Altman Analysis: Method agreement was assessed by plotting the differences between paired measurements against their means. The mean bias ( d ˉ) and standard deviation (SD) of the differences were computed. The 95% limits of agreement (LoA) were defined as d ˉ ± 1.96 SD. Satisfactory agreement was concluded if > 95% of data points resided within the LoA. Analysis at Medical Decision Levels: Clinical acceptability was evaluated at critical decision points (50 µg/g and 200 µg/g). The 95% CI of the expected bias at these concentrations was calculated and compared against pre-defined allowable error limits. The two methods were considered clinically equivalent at a given decision level if the entire CI of the expected bias fell within the allowable limit. Paired measurement data from both methods were assessed for outliers as specified in the EP9-A2 guideline. Any statistical outliers were removed, with the total proportion of data removal being limited to a maximum of 5%. The graphpad prism version 9.4.1 (graphpad, San Diego, California) was used for statistical analysis. p < 0.05, The difference was statistically significant. Results Preparation of S100A8 and S100A9 recombinant proteins and polyclonal antibodies SDS-PAGE analysis of the bacterial lysate after induction is shown in the figure below. A prominent protein band between 13 kDa and 20 kDa was observed in the induced group, indicating successful expression of the recombinant protein (Figure1.A). The target protein was predominantly present in the soluble fraction (supernatant) rather than in the pellet, suggesting that it was expressed in soluble form without forming inclusion bodies. To obtain highly purified target protein, the lysate was filtered through a 0.22 μm membrane. The His-tagged S100A8 and S100A9 proteins were then bound to a Ni-NTA affinity column. To determine the optimal imidazole concentration for elution, a gradient elution was performed using buffers containing 20, 50, 100, 150, 200, 250, 300, 500 mM, and 1 M imidazole (Figure1.B&C). Most contaminating proteins were removed with 100 mM imidazole, while the target protein was effectively eluted at 250 mM imidazole, which was identified as the optimal concentration. Under these conditions, a large amount of recombinant protein was successfully purified. The SDS-PAGE result of the purified protein is presented in the Figure1 D. The titer detection of indirect ELISA is shown in Figure.2 E. The titers of S100A8 and S100A9 antiserum after four immunizations are 1:320000 and 1:80000, indicating the success of rabbit immunization. Rabbit serum can be collected and purified. Calibration curve We used the calprotectin standard sample with the concentration of 50-1500 μ g/g to evaluate the calibration curve. As shown in Figure2, the standard curve is drawn with the expected value of calprotectin standard as the X axis and the measured od578 as the Y axis. For the latex reagent based on polyclonal antibody, the deviation from the theoretical value is less than 5% in the measurement range of 50-1500 μ g/g, indicating that there is no lack of parallelism and good linearity. The linear regression equation was y=0.0003670x-0.003354, R 2 =0.9901. Repeatability and within-lab precision A 20-day precision study was conducted under stable laboratory conditions. The experimental design included duplicate measurements of three distinct concentrations across two analytical runs per day. This setup allowed for the calculation of within-run and within-laboratory coefficients of variation (CV), with the acceptance criteria for both set at ≤10%, based on regulatory guidelines and the performance of analogous products. Statistical analysis of the 80 results was performed in accordance with the Grubbs' method for outlier detection, which identified no outliers. A subsequent variance components analysis (decomposing SS, DF, and MS) was conducted to quantify repeatability and within-laboratory precision. The results yielded a repeatability CV of 4.9% and a within-laboratory CV of 4.7%, both satisfying the acceptance criteria of ≤10%. (Table 1) Interference substances The interference samples with different concentrations were detected. When ferrous sulfate (<0.11mg/50mg fecal), 5-aminosalicylic acid (<5.21mg/50mg fecal), prednisone acetate tablets (<0.31mg/50mg fecal), and hemoglobin (<1.25mg/50mg fecal), the result deviation was less than 10%. (Table 2) Correlation coefficient The Fcal ELISA Kit (B Ü Hlmann laboratories Ag) was used to test 104 clinical samples, and the latex reagent was used as the control.From the linear regression chart (Figure3.A), R 2 =0.9664, r=0.9830>0.95. The slope (b)=1.036, intercept (a)=8.836, the 95% confidence interval of R is 0.9751-0.9885, and the 95% confidence interval of slope is 0.9980-1.0738; The 95% confidence interval of intercept was -9.3509-26.9898 Bland-Altman analysis for absolute difference yielded a mean bias of 19.10 with 95% limits of agreement (LoA) ranging from -128.82 to 167.03. Among the 104 paired measurements, 5 points (4.81%) fell outside these limits, while 95.19% were contained within, meeting the predefined acceptability criterion (>95%). Analysis of the ratio of the means indicated a relative bias of 1.975, with 95% LoA from -8.723 to 12.673. Here, 98.08% (102/104) of data points resided within the limits. Both analyses confirm good agreement between the LTIA and ELISA methods. (Figure 3. B&C). At the medical decision level of 50 µg/g, the expected bias of the LTIA method was 10.615, which fell well within the pre-defined clinically acceptable range of -40 to 30. Similarly, at 200 µg/g, the expected bias was 16, also residing within the acceptable range of -80 to 50. These results confirm that the bias of the new method is clinically acceptable at both critical decision points (Table 3). Outlier analysis was performed in accordance with the EP9-A2 guideline. The analysis yielded a mean absolute difference ∣Y−X∣ of 46.08 and a mean relative difference ∣Y−X∣/X of 1.175. No outliers were identified from the 106 sample comparisons, resulting in an outlier proportion of 0% (0/106), which satisfies the criterion of being less than 5%. Discussion IBD represents a significant global public health challenge(Borowitz, 2022 ). Early screening and accurate monitoring of therapeutic efficacy are crucial for improving patient outcomes(Zabotti et al., 2024 ). Although endoscopy remains the gold standard for diagnosing IBD, its invasive nature precludes its use as a routine screening tool(Shen et al., 2025 ). Fecal calprotectin has emerged as a well-established biomarker, not only for distinguishing IBD from IBS but also for exhibiting a positive correlation with IBD severity(Liu et al., 2022 ). However, the current standard detection method, ELISA, is costly and not readily amenable to automation(Xia et al., 2022 ). Therefore, developing a novel, sensitive, quantitative immunoassay for fecal calprotectin is of considerable clinical value. In this study, we developed a robust latex-enhanced immunoturbidimetric assay (LTIA) for FCP. Rabbit polyclonal antibodies were raised against the S100A8 and S100A9 subunits of calprotectin and covalently conjugated to carboxylated latex particles (188 nm diameter). The assay is designed for compatibility with common automated biochemical analyzers, enabling rapid, accurate, and high-throughput testing. Performance validation demonstrated excellent analytical characteristics: a wide linear range (0–1500 µg/g) with strong correlation (R² = 0.9910), and impressive precision (within-run CV: 4.9%; within-laboratory CV: 4.7%). The method showed excellent agreement with a commercial ELISA kit and exhibited strong resistance to interference from common substances relevant to IBD patients, including ferrous sulfate, 5-aminosalicylic acid, prednisone, and hemoglobin. A key advancement of this study lies in its integrated approach.(Liu et al., 2025 ) We not only independently developed the assay from antibody production to final kit formulation but also validated its performance against the reference method according to the stringent CLSI EP9-A2 standard. This combination of complete process control and standardized clinical validation directly facilitates the translation of the technology into a commercially viable product. This study has several limitations that should be acknowledged. First, key methodological validation parameters, including the limit of detection (LOD), lower limit of quantification (LOQ), dilution linearity, and reagent stability, remain to be established in future work. Second, the clinical samples used were derived from a single center, which may limit the generalizability of the findings. Future work is planned to advance this research. First, we will initiate a multi-center validation study to externally verify the assay's performance across different settings. Second, we will systematically investigate long-term reagent stability and establish the remaining analytical performance characteristics to ensure the assay's readiness for routine clinical use. In conclusion, we have successfully developed and validated a LTIA-based kit for FCP quantification that is fully operable on automated biochemical analyzers. This novel assay demonstrates performance parity with the conventional ELISA method while offering significant advantages in speed, simplicity, and cost-effectiveness—key drivers for clinical adoption. Its superior analytical performance, combined with operational efficiency and robust anti-interference capability, positions this kit as a highly promising in vitro diagnostic (IVD) product with clear commercial potential for widespread application in clinical laboratories. Declarations Ethics approval and consent to participate All the procedures and studies involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The experimental protocols and procedures were approved by Nanjing Drum Tower Hospital (approval number: FM-M7-2023051901). The stool sample was collected from Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School. The project was conducted from November 5, 2024 to March 30, 2025, with written informed consent obtained from all participants in accordance with ethical guidelines. Consent for publication Written informed consent for publication was obtained from all participants. Competing interests The authors declare that they have no competing interests. Consent to participate Informed consent was obtained from all individual participants included in the study. Consent to publish The authors affirm that human research participants provided informed consent for publication. Funding The study was financially supported by the National Key R&D Program of China (grant number: 2022YFB4600600) and the Guangdong Basic and Applied Basic Research Foundation (grant number: 2020B1515120001). Author Contribution Z.Z.: data acquisition, analysis, interpretation, and manuscript preparation; Y.L. and F.Z.: data acquisition and analysis; S.W. and W.H.: conceptualization, study design, supervision, data interpretation, and funding. All the authors participated in preparing the manuscript and approved its final version before submission. Acknowledgements Not applicable. Data Availability The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. References Ayling, R. M., & Kok, K. (2018). Fecal Calprotectin. Adv Clin Chem , 87 , 161–190. https://doi.org/10.1016/bs.acc.2018.07.005 Bisgaard, T. H., Allin, K. H., Keefer, L., Ananthakrishnan, A. N., & Jess, T. (2022). Depression and anxiety in inflammatory bowel disease: epidemiology, mechanisms and treatment. Nat Rev Gastroenterol Hepatol , 19 (11), 717–726. https://doi.org/10.1038/s41575-022-00634-6 Borowitz, S. M. (2022). The epidemiology of inflammatory bowel disease: Clues to pathogenesis? Front Pediatr , 10 , 1103713. https://doi.org/10.3389/fped.2022.1103713 Deng, X., Zeng, M., Wang, X., Liu, J., Ma, Y., Wang, X., & Xu, L. (2021). Preparation and characterization of cyclic citrullinated peptide-immobilized latex beads for measurement of anti-citrillinated protein antibody through latex particle-enhanced turbidimetric immunoassay. J Chromatogr A , 1642 , 462000. https://doi.org/10.1016/j.chroma.2021.462000 Donato, R., Cannon, B. R., Sorci, G., Riuzzi, F., Hsu, K., Weber, D. J., & Geczy, C. L. (2013). Functions of S100 proteins. Curr Mol Med , 13 (1), 24–57. Edgeworth, J., Gorman, M., Bennett, R., Freemont, P., & Hogg, N. (1991). Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells. J Biol Chem , 266 (12), 7706–7713. Jukic, A., Bakiri, L., Wagner, E. F., Tilg, H., & Adolph, T. E. (2021). Calprotectin: from biomarker to biological function. Gut , 70 (10), 1978–1988. https://doi.org/10.1136/gutjnl-2021-324855 Kaplan, G. G., & Windsor, J. W. (2021). The four epidemiological stages in the global evolution of inflammatory bowel disease. Nat Rev Gastroenterol Hepatol , 18 (1), 56–66. https://doi.org/10.1038/s41575-020-00360-x Lamb, C. A., Kennedy, N. A., Raine, T., Hendy, P. A., Smith, P. J., Limdi, J. K., Hayee, B., Lomer, M. C. E., Parkes, G. C., Selinger, C., Barrett, K. J., Davies, R. J., Bennett, C., Gittens, S., Dunlop, M. G., Faiz, O., Fraser, A., Garrick, V., Johnston, P. D.,… Hawthorne, A. B. (2019). British Society of Gastroenterology consensus guidelines on the management of inflammatory bowel disease in adults. Gut , 68 (Suppl 3), s1–s106. https://doi.org/10.1136/gutjnl-2019-318484 Liu, D., Saikam, V., Skrada, K. A., Merlin, D., & Iyer, S. S. (2022). Inflammatory bowel disease biomarkers. Med Res Rev , 42 (5), 1856–1887. https://doi.org/10.1002/med.21893 Liu, Y., Li, M., Zhang, H., Gao, L., Liu, J., Hou, Y., & Xu, J. (2025). Development of a fully automated latex-enhanced immunoturbidimetric method for quantitative serum Lp(a) measurement. Biotechnol Lett , 47 (2), 31. https://doi.org/10.1007/s10529-025-03564-w Machida, T., Miyashita, K., Sone, T., Tanaka, S., Nakajima, K., Saito, M., Stanhope, K., Havel, P., Sumino, H., & Murakami, M. (2015). Determination of serum lipoprotein lipase using a latex particle-enhanced turbidimetric immunoassay with an automated analyzer. Clin Chim Acta , 442 , 130–135. https://doi.org/10.1016/j.cca.2015.01.016 Mellor, L. F., Gago-Lopez, N., Bakiri, L., Schmidt, F. N., Busse, B., Rauber, S., Jimenez, M., Megías, D., Oterino-Sogo, S., Sanchez-Prieto, R., Grivennikov, S., Pu, X., Oxford, J., Ramming, A., Schett, G., & Wagner, E. F. (2022). Keratinocyte-derived S100A9 modulates neutrophil infiltration and affects psoriasis-like skin and joint disease. Ann Rheum Dis , 81 (10), 1400–1408. https://doi.org/10.1136/annrheumdis-2022-222229 Menees, S. B., Powell, C., Kurlander, J., Goel, A., & Chey, W. D. (2015). A meta-analysis of the utility of C-reactive protein, erythrocyte sedimentation rate, fecal calprotectin, and fecal lactoferrin to exclude inflammatory bowel disease in adults with IBS. Am J Gastroenterol , 110 (3), 444–454. https://doi.org/10.1038/ajg.2015.6 Shen, B., Abreu, M. T., Cohen, E. R., Farraye, F. A., Fischer, M., Feuerstadt, P., Kapur, S., Ko, H. M., Kochhar, G. S., Liu, X., Mahadevan, U., McBride, D. L., Navaneethan, U., Regueiro, M., Ritter, T., Sharma, P., & Lichtenstein, G. R. (2025). Endoscopic diagnosis and management of adult inflammatory bowel disease: a consensus document from the American Society for Gastrointestinal Endoscopy IBD Endoscopy Consensus Panel. Gastrointest Endosc , 101 (2), 295–314. https://doi.org/10.1016/j.gie.2024.08.034 Thakkar, H., Davey, C. L., Medcalf, E. A., Skingle, L., Craig, A. R., Newman, D. J., & Price, C. P. (1991). Stabilization of turbidimetric immunoassay by covalent coupling of antibody to latex particles. Clin Chem , 37 (7), 1248–1251. Tomckowiack, C., Ramirez-Reveco, A., Henríquez, C., & Salgado, M. (2023). Development and evaluation of polyclonal antibody-based antigen detection ELISA and dot blot assays as a less costly diagnostic alternative for pathogenic Leptospira infection. Acta Trop , 238 , 106782. https://doi.org/10.1016/j.actatropica.2022.106782 Turner, D., Ricciuto, A., Lewis, A., D'Amico, F., Dhaliwal, J., Griffiths, A. M., Bettenworth, D., Sandborn, W. J., Sands, B. E., Reinisch, W., Schölmerich, J., Bemelman, W., Danese, S., Mary, J. Y., Rubin, D., Colombel, J. F., Peyrin-Biroulet, L., Dotan, I., Abreu, M. T., & Dignass, A. (2021). STRIDE-II: An Update on the Selecting Therapeutic Targets in Inflammatory Bowel Disease (STRIDE) Initiative of the International Organization for the Study of IBD (IOIBD): Determining Therapeutic Goals for Treat-to-Target strategies in IBD. Gastroenterology , 160 (5), 1570–1583. https://doi.org/10.1053/j.gastro.2020.12.031 Xia, L. Y., Tang, Y. N., Zhang, J., Dong, T. Y., & Zhou, R. X. (2022). Advances in the DNA Nanotechnology for the Cancer Biomarkers Analysis: Attributes and Applications. Semin Cancer Biol , 86 (Pt 2), 1105–1119. https://doi.org/10.1016/j.semcancer.2021.12.012 Xia, Y., Shen, H., Zhu, Y., Xu, H., Li, Z., & Si, J. (2017). A sensitive three monoclonal antibodies based automatic latex particle-enhanced turbidimetric immunoassay for Golgi protein 73 detection. Sci Rep , 7 , 40090. https://doi.org/10.1038/srep40090 Zabotti, A., Cabas, N., Cacioppo, S., Zoratti, C., Giovannini, I., Berretti, D., Luchetti, M. M., De Vita, S., Quartuccio, L., Terrosu, G., & Marino, M. (2024). The Challenge of IBD-Related Arthritis Screening Questionnaires in Early and Predominantly Entheseal Phenotypes. Rheumatol Ther , 11 (5), 1321–1331. https://doi.org/10.1007/s40744-024-00709-7 Zygiel, E. M., & Nolan, E. M. (2018). Transition Metal Sequestration by the Host-Defense Protein Calprotectin. Annu Rev Biochem , 87 , 621–643. https://doi.org/10.1146/annurev-biochem-062917-012312 Tables Table 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table13.docx Cite Share Download PDF Status: Published Journal Publication published 24 Apr, 2026 Read the published version in Biotechnology Letters → Version 1 posted Editorial decision: Revision requested 04 Jan, 2026 Editor assigned by journal 22 Dec, 2025 Submission checks completed at journal 22 Dec, 2025 First submitted to journal 20 Dec, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8411228","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":569132112,"identity":"1024bac1-a371-435a-b2fe-7fab51b3b900","order_by":0,"name":"Zhe Zhou","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Zhou","suffix":""},{"id":569132120,"identity":"630926af-8e86-4aed-918e-abd8a53b9ea3","order_by":1,"name":"Yu Li","email":"","orcid":"","institution":"Northwestern Polytechnical University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Li","suffix":""},{"id":569132125,"identity":"c05c8877-ad85-4755-9958-5fe1a7e64181","order_by":2,"name":"Feihu Zhang","email":"","orcid":"","institution":"Guangdong Medical College","correspondingAuthor":false,"prefix":"","firstName":"Feihu","middleName":"","lastName":"Zhang","suffix":""},{"id":569132128,"identity":"202d393c-1042-4c72-a62c-409a8a2e6dcc","order_by":3,"name":"Shaodong Wang","email":"","orcid":"","institution":"Nanjing General Hospital of Nanjing Military Command","correspondingAuthor":false,"prefix":"","firstName":"Shaodong","middleName":"","lastName":"Wang","suffix":""},{"id":569132130,"identity":"094edf39-333b-41c4-a4b4-3f29fa7b9f74","order_by":4,"name":"Wenhua Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYBACAyQG44MEAxs5Nv7mA0RrYTb4UJFmzCdxLIFoLWySM84cTpzHkGOATweDuUTys4df22zygIwH0rxth9PbGM4YMPyo2IZTi+WMNHNj2ba0YiDDwJi3LT23jbmtgLHnzG3cDruRYCYtue1w4oYbCQbJvG3WuW0MhzcwM7bh05L+DajlP1BL+ofDvG3M6WwMCQYEtOSYSX7cdgCoJcewccYZ5wQ2hhQCWs68KZNm/JecuOHMm2IGYCAbtgED+SBevxxP3yb544xd4obj6dt/AKNSXr6/+eCDHxW4tYAAMw+6yAG86oGA8QchFaNgFIyCUTCyAQDytGAhym742wAAAABJRU5ErkJggg==","orcid":"","institution":"Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Wenhua","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-12-20 09:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8411228/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8411228/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10529-026-03733-5","type":"published","date":"2026-04-24T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101904276,"identity":"8ca8eb2a-394a-4bc9-a21c-ca7d8a8c30d3","added_by":"auto","created_at":"2026-02-04 20:13:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2935600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation of S100A8 and S100A9 recombinant proteins and polyclonal antibodies.\u003c/strong\u003e(A) SDS-PAGE identification of recombinant proteins. (B) Purification of S100A8 by SDS-PAGE. (C) Purification of S100A8 by SDS-PAGE. (D) SDS-PAGE analysis of bulk protein purification. (E) Determination of antiserum titer by indirect ELISA.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8411228/v1/68194ea42a03636971bf2f8e.png"},{"id":101904274,"identity":"4af8a3fa-590b-4808-abd2-82a017fffee6","added_by":"auto","created_at":"2026-02-04 20:13:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStandard curve for latex reagent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe absorbance represents the average value of 5 calibration samples± 2 SD, plotted against the concentration of the calibration samples(n=3).\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8411228/v1/2f60a00d8fe6cad22a241cbd.png"},{"id":101943189,"identity":"dbf698a5-2873-45ac-a675-a7a7a94ffd07","added_by":"auto","created_at":"2026-02-05 09:41:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":124222,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStandard curve for latex reagent \u003c/strong\u003e(A) Correlation analysis of the results obtained by LTIA and ELISA(n=104). (B) Bland-Altman plot (absolute difference) mean bias=19.10 ,95% limits of agreement [-128.82,167.03] (n=104). (C) Bland-Altman plot (ratio) mean bias=2.082 ,95% limits of agreement [-10.36,14.52] (n=104)\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-8411228/v1/e6d33e55477796cc4f870622.png"},{"id":107927741,"identity":"ad9650d3-0842-4759-bc13-74415f7dc9ed","added_by":"auto","created_at":"2026-04-27 16:02:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3166816,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8411228/v1/1d4d71a7-c48e-4cd4-8db0-6c1c198f6039.pdf"},{"id":101904273,"identity":"e6747953-d213-4063-92d5-de504e81d70b","added_by":"auto","created_at":"2026-02-04 20:13:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28344,"visible":true,"origin":"","legend":"","description":"","filename":"Table13.docx","url":"https://assets-eu.researchsquare.com/files/rs-8411228/v1/d3db43248e8ba6ee635dee6f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and validation of a latex particle-enhanced turbidimetric immunoassay for fecal calprotectin Bioprocessing and Bioengineering","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInflammatory bowel disease (IBD) comprises a series of chronic systemic inflammatory disorders that impair the normal functions of the digestive system, and includes conditions such as Crohn\u0026rsquo;s disease and ulcerative colitis(Kaplan \u0026amp; Windsor, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). IBD has emerged as a serious global concern owing to its rising incidence across all continents, and has a significant impact on the healthcare system worldwide owing to the lack of effective therapeutic strategies(Bisgaard et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although endoscopy is the gold standard for diagnosing IBD, evaluating therapeutic efficacy, and detecting postoperative recurrence(Shen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), its utility is limited by invasiveness, cost, and associated procedural risks(Liu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Fecal calprotectin is a well-established non-invasive biomarker for diagnosing IBD(Jukic et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, it plays a crucial role in monitoring disease activity, predicting future relapse, and guiding treatment decisions, thereby reducing the reliance on repeated endoscopy(Lamb et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Calprotectin is a member of the S100 calcium-binding protein family and exists as a heterodimer composed of S100A8 and S100A9 subunits(Donato et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Calprotectin is released by activated neutrophils, mediates direct antibacterial activity, and thereby plays a critical role in innate immunity(Edgeworth et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Zygiel \u0026amp; Nolan, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Calprotectin is widely distributed in various biological fluids, where its concentration is elevated in proportion to the severity of inflammation(Mellor et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Under physiological conditions, its fecal concentration is approximately six-fold higher than that in plasma(Ayling \u0026amp; Kok, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A fecal calprotectin (FCP) level below 50 \u0026micro;g/g is widely used as a diagnostic cut-off to distinguish irritable bowel syndrome (IBS) from inflammatory bowel disease (IBD)(Menees et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, the International Organisation for the Study of Inflammatory Bowel Disease (IOIBD) Selecting Therapeutic Targets in Inflammatory Bowel Disease (STAR) consensus recommends a treatment target of FCP\u0026thinsp;\u0026lt;\u0026thinsp;150 \u0026micro;g/g(Turner et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn China, fecal calprotectin (FCP) is commonly quantified using enzyme-linked immunosorbent assay (ELISA). However, ELISA is costly, time-consuming, and labor-intensive due to its multiple incubation and washing steps(Tomckowiack et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To address these limitations, establishing a rapid, reliable, and automated alternative is imperative. Latex particle-enhanced turbidimetric immunoassay (LTIA) represents such a method(Deng et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This technique employs antibody-coated latex particles that agglutinate in the presence of the specific analyte(Thakkar et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). The resultant aggregation increases turbidity in a concentration-dependent manner, which can be quantified by turbidimetry(Xia et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Owing to its high efficiency and ease of automation, LTIA is well-suited for high-throughput clinical laboratories(Machida et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we independently developed the assay from antibody production to final reagent formulation and optimized it for automated analyzers. This work provides a rapid and fully automated alternative method suitable for high-throughput clinical settings.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of prokaryotic expression vectors of S100A8 and S100A9\u003c/h2\u003e \u003cp\u003eThe coding sequences of S100A8(NM_002964) and S100A9(NM_002965) were retrieved from the NCBI database(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/gene\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/gene\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Using the NCBI ORFfinder tool, the open reading frames (ORFs) were identified. Subsequently, the sequences were submitted to Nanjing GenScript Biotechnology Co., Ltd. for codon optimization and chemical synthesis. Following digestion with BamHI and XhoI, the synthesized gene was ligated into the linearized pET-28a vector, generating an expression construct designed to produce the recombinant protein with an N-terminal His-tag.\u003c/p\u003e \u003cp\u003eThe resulting recombinant plasmids, S100A8_pET-28a(+) and S100A9_pET-28a(+), were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) competent cells. Positive transformants were selected on LB agar plates supplemented with kanamycin (30 \u0026micro;g/mL). Single colonies were inoculated into LB liquid medium containing the same antibiotics (kanamycin 30 \u0026micro;g/mL) and cultured overnight at 37\u0026deg;C with shaking at 200 rpm.\u003c/p\u003e \u003cp\u003eRecombinant plasmids were extracted using a commercial plasmid miniprep kit. The extracted plasmids were verified by DNA sequencing, which was performed by GENEWIZ (Suzhou) Biotechnology Co., Ltd., using the T7 terminator as the sequencing primer.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExpression and purification of recombinant S100A8 and S100A9 proteins\u003c/h3\u003e\n\u003cp\u003eThe transformed E. coli strains S100A8-BL21(DE3) and S100A9-BL21(DE3) were inoculated into 200 mL of LB liquid medium supplemented with 30 \u0026micro;g/mL kanamycin. Protein expression was induced with 0.05 mmol/L isopropyl-β-D-thiogalactoside (IPTG) when the cell density reached an OD600\u0026thinsp;=\u0026thinsp;0.5\u0026ndash;0.6. Induction was carried out at 18\u0026deg;C for 20 hours.\u003c/p\u003e \u003cp\u003eAfter induction, the cells were harvested by centrifugation at 2000 rpm for 15 min at 4\u0026deg;C. The pellet was resuspended in 5 mL of lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) supplemented with protease inhibitors, followed by thorough mixing via vortexing. Cell disruption was performed using an ultrasonic disruptor for 20 min, and the lysate was centrifuged at 4000 rpm for 15 min. The supernatant was collected and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. The yield of recombinant protein was evaluated by 12% SDS-PAGE.\u003c/p\u003e \u003cp\u003eFor purification, the supernatant was filtered through a 0.22 \u0026micro;m membrane and loaded onto a Ni-NTA column. The column was washed with elution buffers containing imidazole at gradient concentrations (20, 50, 100, 150, 200, 250, 300, 500 mM, and 1 M) in a base buffer (50 mM NaH₂PO₄ and 300 mM NaCl (pH 8.0)). Protein elution was monitored using a UV monitor to detect the protein peak. Eluted protein fractions and the crude supernatant after cell lysis were analyzed by SDS-PAGE.\u003c/p\u003e\n\u003ch3\u003ePreparation and purification of polyclonal antibodies\u003c/h3\u003e\n\u003cp\u003eThe purified recombinant S100A8 and S100A9 proteins were emulsified with Freund\u0026rsquo;s adjuvant and used to immunize New Zealand rabbits. The initial immunization (300 \u0026micro;g/rabbit) used complete adjuvant, followed by three booster injections (100 \u0026micro;g/rabbit) with incomplete adjuvant at 15-day intervals. Serum was collected 15 days after the final immunization. Polyclonal antibodies were purified from the serum using Protein A affinity chromatography.\u003c/p\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eS100A8 and S100A9 proteins were diluted in a specific coating buffer to a concentration of 10 \u0026micro;g/mL. Each well of an ELISA plate was coated with 100 \u0026micro;L of the protein solution and incubated overnight at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe plate was then blocked with 1% bovine serum albumin (BSA) at 37\u0026deg;C for 2 hours to prevent non-specific binding. After blocking, 100 \u0026micro;L of rabbit serum at various dilution ratios (ranging from 1:100 to 1:1,280,000) was added to each well. Normal rabbit serum was used as a negative control. The plate was incubated at 37\u0026deg;C for 1 hour.\u003c/p\u003e \u003cp\u003eSubsequently, horseradish peroxidase (HRP)-conjugated sheep anti-rabbit secondary antibody, diluted at 1:5,000, was added to each well, followed by another 1-hour incubation at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eFor detection, TMB substrate solution was added and the plate was incubated in the dark at 37\u0026deg;C for 30 minutes. The reaction was stopped by adding stop solution, and the optical density at 450 nm (OD450) was measured using an ELISA microplate reader.\u003c/p\u003e \u003cp\u003eA preset cutoff value of 0.05 was used to define the negative control. The P/N ratio was calculated as the OD450 value of the sample divided by that of the negative control. A sample was considered positive when the P/N value was \u0026ge;\u0026thinsp;2.1.\u003c/p\u003e\n\u003ch3\u003eLatex reagents\u003c/h3\u003e\n\u003cp\u003eThe carboxyl series for covalent binding latex particle produced by JSR Corporation (Japan) with a diameter of 188 nm (IMMUTEX P0113, LOT. J5612K-01) was used. Antibody coupling is carried out according to the instructions of latex particles. A 250 \u0026micro;L aliquot of latex beads was diluted and mixed with 425 \u0026micro;L of HEPES buffer (50 mM, pH 7.2). Then, 125 \u0026micro;L each of S100A8 and S100A9 antibodies (10 mg/mL) was added, and the mixture was stirred at 25\u0026ndash;37\u0026deg;C for 1 hour. Subsequently, 125 \u0026micro;L of EDC (10 mg/mL in HEPES buffer) was added, and the reaction was allowed to proceed for another hour at 25\u0026ndash;37\u0026deg;C with continuous mixing. The mixture was centrifuged at 25,000 \u0026times; g for 30 minutes, and the supernatant was discarded. The pellet was resuspended in 5 mL of a 5% BSA solution, thoroughly mixed, and incubated at 25\u0026ndash;37\u0026deg;C for 1 hour to block non-specific sites. After a second centrifugation under the same conditions (25,000 \u0026times; g, 30 minutes), the supernatant was removed. The resulting conjugate was resuspended in 5 mL of storage buffer (25 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween-20, pH 7.2) and stored at 2\u0026ndash;8\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement procedure\u003c/h2\u003e \u003cp\u003eThe quantification was performed using an LC-400 specific protein analyzer (Nanjing Laola Electronics Co., Ltd.). Samples and reagents were dispensed according to the manufacturer's instructions. A six-point calibration curve was generated using recombinant calprotectin at concentrations of 0, 50, 100, 250, 750, and 1500 \u0026micro;g/g.\u003c/p\u003e \u003cp\u003eFecal samples were diluted 1:500 in normal saline and centrifuged at 10,000 rpm for 5 minutes. The resulting supernatant was collected for analysis. Then, 200 \u0026micro;L of PB buffer (50 mM PB, 1% PVP, 0.05% Tween-20, pH 7.0) was dispensed into a tube, followed by the addition of 25 \u0026micro;L of latex reagent (0.2% latex in 50 mM PB, pH 7.0, containing 1% PVP and 0.05% Tween-20) to initiate the reaction. Subsequently, 4 \u0026micro;L of the experimental sample was added to the mixture and incubated at 37\u0026deg;C for 5 minutes. The absorbance at 750 nm was measured before and after the reaction, and the difference was calculated.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparison with commercially available reagent kits\u003c/h3\u003e\n\u003cp\u003eThe performance of the calprotectin immunolatex reagent was evaluated against a commercially available fCAL ELISA kit (B\u0026Uuml;HLMANN Laboratories AG). The correlation between the two methods was assessed using linear regression analysis and visualized in a scatter plot.\u003c/p\u003e\n\u003ch3\u003eClinical sample testing\u003c/h3\u003e\n\u003cp\u003eThe stool sample came from Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School. The project will start and end from November 5, 2024 to March 30, 2025, and all subjects will sign written informed consent. The experimental protocols and procedures were approved by Nanjing Drum Tower Hospital (approval number: FM-M7-2023051901).\u003c/p\u003e \u003cp\u003e According to the CLSI EP9-A2 standard formulated by the National Committee for clinical laboratory standardization of the United States, the correlation coefficient of the test results of the test method and the comparison method shall not be less than 0.95. It is estimated that the correlation coefficient of this test is more than 0.95, so the parameters are set as follows: a\u0026thinsp;=\u0026thinsp;0.05, s\u0026thinsp;=\u0026thinsp;1 (bilateral), β\u0026thinsp;=\u0026thinsp;0.2, ρ 0\u0026thinsp;=\u0026thinsp;0.95, ρ 1\u0026thinsp;=\u0026thinsp;0.95. The sample size is calculated according to the sample size estimation formula of the correlation coefficient test (continuous variable). The sample size is 94 cases. Combined with regulatory requirements and statistical requirements, considering the 5% rejection rate, the final sample size of this clinical trial is determined to be no less than 100 cases.\u003c/p\u003e \u003cp\u003eThe detection result of calprotectin is not less than 30% of the total number of cases outside the reference range (abnormal value sample). Subgroup sample size requirements: 20 cases of Crohn's disease, 10 cases of ulcerative colitis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eA comprehensive statistical protocol was employed to establish the equivalence between the novel LTIA and the reference ELISA method. Correlation and Regression Analysis: Linear regression analysis (ordinary least squares) was performed on paired results from 104 clinical samples. The correlation coefficient (R), slope (b), and intercept (a) were calculated, with acceptance criteria set at R\u0026thinsp;\u0026ge;\u0026thinsp;0.95. The 95% confidence intervals (CI) for both the slope and intercept were also determined. Bland-Altman Analysis: Method agreement was assessed by plotting the differences between paired measurements against their means. The mean bias (\u003cem\u003ed\u003c/em\u003eˉ) and standard deviation (SD) of the differences were computed. The 95% limits of agreement (LoA) were defined as \u003cem\u003ed\u003c/em\u003eˉ \u0026plusmn; 1.96 SD. Satisfactory agreement was concluded if\u0026thinsp;\u0026gt;\u0026thinsp;95% of data points resided within the LoA. Analysis at Medical Decision Levels: Clinical acceptability was evaluated at critical decision points (50 \u0026micro;g/g and 200 \u0026micro;g/g). The 95% CI of the expected bias at these concentrations was calculated and compared against pre-defined allowable error limits. The two methods were considered clinically equivalent at a given decision level if the entire CI of the expected bias fell within the allowable limit. Paired measurement data from both methods were assessed for outliers as specified in the EP9-A2 guideline. Any statistical outliers were removed, with the total proportion of data removal being limited to a maximum of 5%.\u003c/p\u003e \u003cp\u003eThe graphpad prism version 9.4.1 (graphpad, San Diego, California) was used for statistical analysis. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, The difference was statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003ch3\u003e\u003cstrong\u003e\u003cem\u003ePreparation of S100A8 and S100A9 recombinant proteins and polyclonal antibodies\u003c/em\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eSDS-PAGE analysis of the bacterial lysate after induction is shown in the figure below. A prominent protein band between 13 kDa and 20 kDa was observed in the induced group, indicating successful expression of the recombinant protein (Figure1.A). The target protein was predominantly present in the soluble fraction (supernatant) rather than in the pellet, suggesting that it was expressed in soluble form without forming inclusion bodies.\u003c/p\u003e\n\u003cp\u003eTo obtain highly purified target protein, the lysate was filtered through a 0.22 \u0026mu;m membrane. The His-tagged S100A8 and S100A9 proteins were then bound to a Ni-NTA affinity column. To determine the optimal imidazole concentration for elution, a gradient elution was performed using buffers containing 20, 50, 100, 150, 200, 250, 300, 500 mM, and 1 M imidazole (Figure1.B\u0026amp;C). Most contaminating proteins were removed with 100 mM imidazole, while the target protein was effectively eluted at 250 mM imidazole, which was identified as the optimal concentration.\u003c/p\u003e\n\u003cp\u003eUnder these conditions, a large amount of recombinant protein was successfully purified. The SDS-PAGE result of the purified protein is presented in the Figure1 D.\u003c/p\u003e\n\u003cp\u003eThe titer detection of indirect ELISA is shown in Figure.2 E. The titers of S100A8 and S100A9 antiserum after four immunizations are 1:320000 and 1:80000, indicating the success of rabbit immunization. Rabbit serum can be collected and purified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCalibration curve\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used the calprotectin standard sample with the concentration of 50-1500 \u0026mu; g/g to evaluate the calibration curve. As shown in Figure2, the standard curve is drawn with the expected value of calprotectin standard as the X axis and the measured od578 as the Y axis. For the latex reagent based on polyclonal antibody, the deviation from the theoretical value is less than 5% in the measurement range of 50-1500 \u0026mu; g/g, indicating that there is no lack of parallelism and good linearity. The linear regression equation was y=0.0003670x-0.003354, R\u003csup\u003e2\u003c/sup\u003e=0.9901.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRepeatability and within-lab precision\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 20-day precision study was conducted under stable laboratory conditions. The experimental design included duplicate measurements of three distinct concentrations across two analytical runs per day. This setup allowed for the calculation of within-run and within-laboratory coefficients of variation (CV), with the acceptance criteria for both set at \u0026le;10%, based on regulatory guidelines and the performance of analogous products.\u0026nbsp;Statistical analysis of the 80 results was performed in accordance with the Grubbs\u0026apos; method for outlier detection, which identified no outliers. A subsequent variance components analysis (decomposing SS, DF, and MS) was conducted to quantify repeatability and within-laboratory precision. The results yielded a repeatability CV of 4.9% and a within-laboratory CV of 4.7%, both satisfying the acceptance criteria of \u0026le;10%. (Table 1)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInterference substances\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interference samples with different concentrations were detected. When ferrous sulfate (\u0026lt;0.11mg/50mg fecal), 5-aminosalicylic acid (\u0026lt;5.21mg/50mg fecal), prednisone acetate tablets (\u0026lt;0.31mg/50mg fecal), and hemoglobin (\u0026lt;1.25mg/50mg fecal), the result deviation was less than 10%. (Table 2)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCorrelation coefficient\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Fcal ELISA Kit (B \u0026Uuml; Hlmann laboratories Ag) was used to test 104 clinical samples, and the latex reagent was used as the control.From the linear regression chart (Figure3.A), R\u003csup\u003e2\u003c/sup\u003e=0.9664, r=0.9830\u0026gt;0.95. The slope (b)=1.036, intercept (a)=8.836, the 95% confidence interval of R is 0.9751-0.9885, and the 95% confidence interval of slope is 0.9980-1.0738; The 95% confidence interval of intercept was -9.3509-26.9898 Bland-Altman analysis for absolute difference yielded a mean bias of 19.10 with 95% limits of agreement (LoA) ranging from -128.82 to 167.03. Among the 104 paired measurements, 5 points (4.81%) fell outside these limits, while 95.19% were contained within, meeting the predefined acceptability criterion (\u0026gt;95%). Analysis of the ratio of the means indicated a relative bias of 1.975, with 95% LoA from -8.723 to 12.673. Here, 98.08% (102/104) of data points resided within the limits. Both analyses confirm good agreement between the LTIA and ELISA methods. (Figure\u003cins cite=\"mailto:Lin-PC\" datetime=\"2025-10-24T10:17\"\u003e\u0026nbsp;\u003c/ins\u003e3. B\u0026amp;C). At the medical decision level of 50 \u0026micro;g/g, the expected bias of the LTIA method was 10.615, which fell well within the pre-defined clinically acceptable range of -40 to 30. Similarly, at 200 \u0026micro;g/g, the expected bias was 16, also residing within the acceptable range of -80 to 50. These results confirm that the bias of the new method is clinically acceptable at both critical decision points (Table 3). Outlier analysis was performed in accordance with the EP9-A2 guideline. The analysis yielded a mean absolute difference ∣Y\u0026minus;X∣ of 46.08 and a mean relative difference ∣Y\u0026minus;X∣/X of 1.175. No outliers were identified from the 106 sample comparisons, resulting in an outlier proportion of 0% (0/106), which satisfies the criterion of being less than 5%.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIBD represents a significant global public health challenge(Borowitz, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Early screening and accurate monitoring of therapeutic efficacy are crucial for improving patient outcomes(Zabotti et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although endoscopy remains the gold standard for diagnosing IBD, its invasive nature precludes its use as a routine screening tool(Shen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Fecal calprotectin has emerged as a well-established biomarker, not only for distinguishing IBD from IBS but also for exhibiting a positive correlation with IBD severity(Liu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the current standard detection method, ELISA, is costly and not readily amenable to automation(Xia et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, developing a novel, sensitive, quantitative immunoassay for fecal calprotectin is of considerable clinical value.\u003c/p\u003e \u003cp\u003eIn this study, we developed a robust latex-enhanced immunoturbidimetric assay (LTIA) for FCP. Rabbit polyclonal antibodies were raised against the S100A8 and S100A9 subunits of calprotectin and covalently conjugated to carboxylated latex particles (188 nm diameter). The assay is designed for compatibility with common automated biochemical analyzers, enabling rapid, accurate, and high-throughput testing. Performance validation demonstrated excellent analytical characteristics: a wide linear range (0\u0026ndash;1500 \u0026micro;g/g) with strong correlation (R\u0026sup2; = 0.9910), and impressive precision (within-run CV: 4.9%; within-laboratory CV: 4.7%). The method showed excellent agreement with a commercial ELISA kit and exhibited strong resistance to interference from common substances relevant to IBD patients, including ferrous sulfate, 5-aminosalicylic acid, prednisone, and hemoglobin.\u003c/p\u003e \u003cp\u003eA key advancement of this study lies in its integrated approach.(Liu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) We not only independently developed the assay from antibody production to final kit formulation but also validated its performance against the reference method according to the stringent CLSI EP9-A2 standard. This combination of complete process control and standardized clinical validation directly facilitates the translation of the technology into a commercially viable product.\u003c/p\u003e \u003cp\u003eThis study has several limitations that should be acknowledged. First, key methodological validation parameters, including the limit of detection (LOD), lower limit of quantification (LOQ), dilution linearity, and reagent stability, remain to be established in future work. Second, the clinical samples used were derived from a single center, which may limit the generalizability of the findings. Future work is planned to advance this research. First, we will initiate a multi-center validation study to externally verify the assay's performance across different settings. Second, we will systematically investigate long-term reagent stability and establish the remaining analytical performance characteristics to ensure the assay's readiness for routine clinical use.\u003c/p\u003e \u003cp\u003eIn conclusion, we have successfully developed and validated a LTIA-based kit for FCP quantification that is fully operable on automated biochemical analyzers. This novel assay demonstrates performance parity with the conventional ELISA method while offering significant advantages in speed, simplicity, and cost-effectiveness\u0026mdash;key drivers for clinical adoption. Its superior analytical performance, combined with operational efficiency and robust anti-interference capability, positions this kit as a highly promising \u003cem\u003ein vitro\u003c/em\u003e diagnostic (IVD) product with clear commercial potential for widespread application in clinical laboratories.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All the procedures and studies involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The experimental protocols and procedures were approved by Nanjing Drum Tower Hospital (approval number: FM-M7-2023051901). The stool sample was collected from Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School. The project was conducted from November 5, 2024 to March 30, 2025, with written informed consent obtained from all participants in accordance with ethical guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003e Written informed consent for publication was obtained from all participants.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent to participate\u003c/h2\u003e \u003cp\u003e Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to publish\u003c/strong\u003e \u003cp\u003eThe authors affirm that human research participants provided informed consent for publication.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was financially supported by the National Key R\u0026amp;D Program of China (grant number: 2022YFB4600600) and the Guangdong Basic and Applied Basic Research Foundation (grant number: 2020B1515120001).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZ.Z.: data acquisition, analysis, interpretation, and manuscript preparation; Y.L. and F.Z.: data acquisition and analysis; S.W. and W.H.: conceptualization, study design, supervision, data interpretation, and funding. All the authors participated in preparing the manuscript and approved its final version before submission.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAyling, R. M., \u0026amp; Kok, K. (2018). Fecal Calprotectin. \u003cem\u003eAdv Clin Chem\u003c/em\u003e, \u003cem\u003e87\u003c/em\u003e, 161\u0026ndash;190. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/bs.acc.2018.07.005\u003c/span\u003e\u003cspan address=\"10.1016/bs.acc.2018.07.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBisgaard, T. H., Allin, K. H., Keefer, L., Ananthakrishnan, A. N., \u0026amp; Jess, T. (2022). Depression and anxiety in inflammatory bowel disease: epidemiology, mechanisms and treatment. \u003cem\u003eNat Rev Gastroenterol Hepatol\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(11), 717\u0026ndash;726. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41575-022-00634-6\u003c/span\u003e\u003cspan address=\"10.1038/s41575-022-00634-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorowitz, S. M. (2022). The epidemiology of inflammatory bowel disease: Clues to pathogenesis? \u003cem\u003eFront Pediatr\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e, 1103713. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fped.2022.1103713\u003c/span\u003e\u003cspan address=\"10.3389/fped.2022.1103713\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng, X., Zeng, M., Wang, X., Liu, J., Ma, Y., Wang, X., \u0026amp; Xu, L. (2021). Preparation and characterization of cyclic citrullinated peptide-immobilized latex beads for measurement of anti-citrillinated protein antibody through latex particle-enhanced turbidimetric immunoassay. \u003cem\u003eJ Chromatogr A\u003c/em\u003e, \u003cem\u003e1642\u003c/em\u003e, 462000. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chroma.2021.462000\u003c/span\u003e\u003cspan address=\"10.1016/j.chroma.2021.462000\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonato, R., Cannon, B. R., Sorci, G., Riuzzi, F., Hsu, K., Weber, D. J., \u0026amp; Geczy, C. L. (2013). Functions of S100 proteins. \u003cem\u003eCurr Mol Med\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(1), 24\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdgeworth, J., Gorman, M., Bennett, R., Freemont, P., \u0026amp; Hogg, N. (1991). Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells. \u003cem\u003eJ Biol Chem\u003c/em\u003e, \u003cem\u003e266\u003c/em\u003e(12), 7706\u0026ndash;7713.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJukic, A., Bakiri, L., Wagner, E. F., Tilg, H., \u0026amp; Adolph, T. E. (2021). Calprotectin: from biomarker to biological function. \u003cem\u003eGut\u003c/em\u003e, \u003cem\u003e70\u003c/em\u003e(10), 1978\u0026ndash;1988. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/gutjnl-2021-324855\u003c/span\u003e\u003cspan address=\"10.1136/gutjnl-2021-324855\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaplan, G. G., \u0026amp; Windsor, J. W. (2021). The four epidemiological stages in the global evolution of inflammatory bowel disease. \u003cem\u003eNat Rev Gastroenterol Hepatol\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(1), 56\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41575-020-00360-x\u003c/span\u003e\u003cspan address=\"10.1038/s41575-020-00360-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamb, C. A., Kennedy, N. A., Raine, T., Hendy, P. A., Smith, P. J., Limdi, J. K., Hayee, B., Lomer, M. C. E., Parkes, G. C., Selinger, C., Barrett, K. J., Davies, R. J., Bennett, C., Gittens, S., Dunlop, M. G., Faiz, O., Fraser, A., Garrick, V., Johnston, P. D.,\u0026hellip; Hawthorne, A. B. (2019). British Society of Gastroenterology consensus guidelines on the management of inflammatory bowel disease in adults. \u003cem\u003eGut\u003c/em\u003e, \u003cem\u003e68\u003c/em\u003e(Suppl 3), s1\u0026ndash;s106. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/gutjnl-2019-318484\u003c/span\u003e\u003cspan address=\"10.1136/gutjnl-2019-318484\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, D., Saikam, V., Skrada, K. A., Merlin, D., \u0026amp; Iyer, S. S. (2022). Inflammatory bowel disease biomarkers. \u003cem\u003eMed Res Rev\u003c/em\u003e, \u003cem\u003e42\u003c/em\u003e(5), 1856\u0026ndash;1887. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/med.21893\u003c/span\u003e\u003cspan address=\"10.1002/med.21893\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y., Li, M., Zhang, H., Gao, L., Liu, J., Hou, Y., \u0026amp; Xu, J. (2025). Development of a fully automated latex-enhanced immunoturbidimetric method for quantitative serum Lp(a) measurement. \u003cem\u003eBiotechnol Lett\u003c/em\u003e, \u003cem\u003e47\u003c/em\u003e(2), 31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10529-025-03564-w\u003c/span\u003e\u003cspan address=\"10.1007/s10529-025-03564-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachida, T., Miyashita, K., Sone, T., Tanaka, S., Nakajima, K., Saito, M., Stanhope, K., Havel, P., Sumino, H., \u0026amp; Murakami, M. (2015). Determination of serum lipoprotein lipase using a latex particle-enhanced turbidimetric immunoassay with an automated analyzer. \u003cem\u003eClin Chim Acta\u003c/em\u003e, \u003cem\u003e442\u003c/em\u003e, 130\u0026ndash;135. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cca.2015.01.016\u003c/span\u003e\u003cspan address=\"10.1016/j.cca.2015.01.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMellor, L. F., Gago-Lopez, N., Bakiri, L., Schmidt, F. N., Busse, B., Rauber, S., Jimenez, M., Meg\u0026iacute;as, D., Oterino-Sogo, S., Sanchez-Prieto, R., Grivennikov, S., Pu, X., Oxford, J., Ramming, A., Schett, G., \u0026amp; Wagner, E. F. (2022). Keratinocyte-derived S100A9 modulates neutrophil infiltration and affects psoriasis-like skin and joint disease. \u003cem\u003eAnn Rheum Dis\u003c/em\u003e, \u003cem\u003e81\u003c/em\u003e(10), 1400\u0026ndash;1408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/annrheumdis-2022-222229\u003c/span\u003e\u003cspan address=\"10.1136/annrheumdis-2022-222229\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMenees, S. B., Powell, C., Kurlander, J., Goel, A., \u0026amp; Chey, W. D. (2015). A meta-analysis of the utility of C-reactive protein, erythrocyte sedimentation rate, fecal calprotectin, and fecal lactoferrin to exclude inflammatory bowel disease in adults with IBS. \u003cem\u003eAm J Gastroenterol\u003c/em\u003e, \u003cem\u003e110\u003c/em\u003e(3), 444\u0026ndash;454. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ajg.2015.6\u003c/span\u003e\u003cspan address=\"10.1038/ajg.2015.6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen, B., Abreu, M. T., Cohen, E. R., Farraye, F. A., Fischer, M., Feuerstadt, P., Kapur, S., Ko, H. M., Kochhar, G. S., Liu, X., Mahadevan, U., McBride, D. L., Navaneethan, U., Regueiro, M., Ritter, T., Sharma, P., \u0026amp; Lichtenstein, G. R. (2025). Endoscopic diagnosis and management of adult inflammatory bowel disease: a consensus document from the American Society for Gastrointestinal Endoscopy IBD Endoscopy Consensus Panel. \u003cem\u003eGastrointest Endosc\u003c/em\u003e, \u003cem\u003e101\u003c/em\u003e(2), 295\u0026ndash;314. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.gie.2024.08.034\u003c/span\u003e\u003cspan address=\"10.1016/j.gie.2024.08.034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThakkar, H., Davey, C. L., Medcalf, E. A., Skingle, L., Craig, A. R., Newman, D. J., \u0026amp; Price, C. P. (1991). Stabilization of turbidimetric immunoassay by covalent coupling of antibody to latex particles. \u003cem\u003eClin Chem\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e(7), 1248\u0026ndash;1251.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomckowiack, C., Ramirez-Reveco, A., Henr\u0026iacute;quez, C., \u0026amp; Salgado, M. (2023). Development and evaluation of polyclonal antibody-based antigen detection ELISA and dot blot assays as a less costly diagnostic alternative for pathogenic Leptospira infection. \u003cem\u003eActa Trop\u003c/em\u003e, \u003cem\u003e238\u003c/em\u003e, 106782. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.actatropica.2022.106782\u003c/span\u003e\u003cspan address=\"10.1016/j.actatropica.2022.106782\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurner, D., Ricciuto, A., Lewis, A., D'Amico, F., Dhaliwal, J., Griffiths, A. M., Bettenworth, D., Sandborn, W. J., Sands, B. E., Reinisch, W., Sch\u0026ouml;lmerich, J., Bemelman, W., Danese, S., Mary, J. Y., Rubin, D., Colombel, J. F., Peyrin-Biroulet, L., Dotan, I., Abreu, M. T., \u0026amp; Dignass, A. (2021). STRIDE-II: An Update on the Selecting Therapeutic Targets in Inflammatory Bowel Disease (STRIDE) Initiative of the International Organization for the Study of IBD (IOIBD): Determining Therapeutic Goals for Treat-to-Target strategies in IBD. \u003cem\u003eGastroenterology\u003c/em\u003e, \u003cem\u003e160\u003c/em\u003e(5), 1570\u0026ndash;1583. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1053/j.gastro.2020.12.031\u003c/span\u003e\u003cspan address=\"10.1053/j.gastro.2020.12.031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia, L. Y., Tang, Y. N., Zhang, J., Dong, T. Y., \u0026amp; Zhou, R. X. (2022). Advances in the DNA Nanotechnology for the Cancer Biomarkers Analysis: Attributes and Applications. \u003cem\u003eSemin Cancer Biol\u003c/em\u003e, \u003cem\u003e86\u003c/em\u003e(Pt 2), 1105\u0026ndash;1119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.semcancer.2021.12.012\u003c/span\u003e\u003cspan address=\"10.1016/j.semcancer.2021.12.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia, Y., Shen, H., Zhu, Y., Xu, H., Li, Z., \u0026amp; Si, J. (2017). A sensitive three monoclonal antibodies based automatic latex particle-enhanced turbidimetric immunoassay for Golgi protein 73 detection. \u003cem\u003eSci Rep\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e, 40090. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep40090\u003c/span\u003e\u003cspan address=\"10.1038/srep40090\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZabotti, A., Cabas, N., Cacioppo, S., Zoratti, C., Giovannini, I., Berretti, D., Luchetti, M. M., De Vita, S., Quartuccio, L., Terrosu, G., \u0026amp; Marino, M. (2024). The Challenge of IBD-Related Arthritis Screening Questionnaires in Early and Predominantly Entheseal Phenotypes. \u003cem\u003eRheumatol Ther\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(5), 1321\u0026ndash;1331. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s40744-024-00709-7\u003c/span\u003e\u003cspan address=\"10.1007/s40744-024-00709-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZygiel, E. M., \u0026amp; Nolan, E. M. (2018). Transition Metal Sequestration by the Host-Defense Protein Calprotectin. \u003cem\u003eAnnu Rev Biochem\u003c/em\u003e, \u003cem\u003e87\u003c/em\u003e, 621\u0026ndash;643. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-biochem-062917-012312\u003c/span\u003e\u003cspan address=\"10.1146/annurev-biochem-062917-012312\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 3 are available in the Supplementary Files section.\u003c/p\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":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"IBD, Calprotectin, Latex enhanced turbidimetric immunoassay","lastPublishedDoi":"10.21203/rs.3.rs-8411228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8411228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eFecal calprotectin (FCP) is a crucial non-invasive biomarker for diagnosing and monitoring inflammatory bowel disease (IBD). However, the standard enzyme-linked immunosorbent assay (ELISA) is time-consuming and labor-intensive. This study aimed to develop and evaluate a rapid, automated latex particle-enhanced turbidimetric immunoassay (LTIA) for FCP quantification.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRecombinant human S100A8 and S100A9 proteins (the subunits of calprotectin) were expressed in E. coli and purified. Polyclonal antibodies were generated in rabbits by immunization with these proteins. The antibodies were then covalently coupled to carboxylated latex particles (188 nm) to create the LTIA reagent. The linear repeatability, within-lab precision and interference substances of the reagent were determined, The performance of this method was evaluated using clinical fecal samples (n\u0026thinsp;=\u0026thinsp;104), including linearity and correlation with commercial ELISA Kit (B \u0026Uuml; Hlmann fCAl).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe developed LTIA demonstrated excellent linearity within the range of 50\u0026ndash;1500 \u0026micro;g/g (R\u0026sup2; = 0.9910). A strong correlation was observed between the new LTIA and the reference ELISA method, with a correlation coefficient (r) of 0.9830. The regression equation was y\u0026thinsp;=\u0026thinsp;1.0359x\u0026thinsp;+\u0026thinsp;8.8159. Bland-Altman analysis confirmed good agreement between the two methods, with 95.19% of data points within the 95% limits of agreement.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe LTIA showed strong correlation with the established ELISA for FCP quantification, offering a rapid, automated alternative potentially suitable for high-throughput labs. This study, completing development from antibody to reagent, supports its translational potential. However, this study has several limitations. Limitations include a single-center sample set, limiting generalizability, the need for multicenter validation, and unreported key analytical parameters such as LOD, LOQ, and inter-assay variation.\u003c/p\u003e","manuscriptTitle":"Development and validation of a latex particle-enhanced turbidimetric immunoassay for fecal calprotectin Bioprocessing and Bioengineering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 20:13:14","doi":"10.21203/rs.3.rs-8411228/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-04T22:16:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-22T11:16:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-22T11:15:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology Letters","date":"2025-12-20T09:36:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0677eb56-272d-4c93-be8c-01374847b0c5","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:01:19+00:00","versionOfRecord":{"articleIdentity":"rs-8411228","link":"https://doi.org/10.1007/s10529-026-03733-5","journal":{"identity":"biotechnology-letters","isVorOnly":false,"title":"Biotechnology Letters"},"publishedOn":"2026-04-24 15:57:28","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2026-02-04 20:13:14","video":"","vorDoi":"10.1007/s10529-026-03733-5","vorDoiUrl":"https://doi.org/10.1007/s10529-026-03733-5","workflowStages":[]},"version":"v1","identity":"rs-8411228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8411228","identity":"rs-8411228","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}