An Antibody Mixing and Coating Method for Consistently Detecting Clinical Retinol Binding Protein 4 (RBP4)

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Purpose: The purpose of this study was to address the issue of inconsistent clinical results and poor clinical correlation between different manufacturers antibodies when using retinol-binding protein 4 (RBP4) assay kits (latex immunoturbidimetric assay) in clinical settings. By screening and combining different RBP4 monoclonal antibodies with polyclonal antibodies to enrich the recognition epitopes as much as possible and minimize the interference of other RBPs or related substances on the detection results, we aimed to develop an RBP4 assay kit (latex immunoturbidimetric assay) that improves the accuracy and consistency of clinical sample testing. Methods: Antibody screening: Different RBP4 monoclonal antibodies were screened to select two antibodies with high specificity and rich recognition epitopes. Preparation of the assay kit: The selected two RBP4 monoclonal antibodies were mixed with polyclonal antibodies and coated on latex particles to prepare the RBP4 assay kit (latex immunoturbidimetric assay). Comparative analysis: The reagent prepared with mixed antibodies was simultaneously used to detect the same clinical serum samples with an imported RBP4 polyclonal antibody-coated reagent and a widely recognized commercial kit, and the results were compared. Results: Clinical correlation analysis showed that the R² value between Reagent 3 (LUCI-Bio rabbit anti-human RBP4 polyclonal antibody coated reagent) and Reagent 2 (DAKO rabbit anti-human RBP4 polyclonal antibody coated reagent) was 0.9613. The R² value between the RBP4 concentration results from the reagent 1, a mixture of RBP4 monoclonal antibodies and polyclonal antibodies and the reagent 2, DAKO polyclonal antibodies coated latex particles was 0. 9756. When comparing the reagent (reagent 1) coated with a mixture of RBP4 monoclonal and polyclonal antibodies with the Beijing Strong Biotechnologies RBP4 kit, the R² value was 0.⁹⁵³⁵Conclusion: Measured RBP4 concentrations with the LUCI-Bio and DAKO rabbit anti-human RBP4 polyclonal antibody reagents showed good consistency (R²=0.9613) Measured RBP4 concentrations from the latex particles coated with a mixture of RBP4 monoclonal antibodies and polyclonal antibodies exhibited higher consistency and correlation (R²=0. 9756.) compared to those with DAKO polyclonal antibodies, indicating that the use of mixed antibodies enriched the antigen recognition epitopes and significantly improved the accuracy of clinical test results. Furthermore, the reagent coated with mixed antibodies also showed good consistency (R²=0.9535) with the Beijing Strong Biotechnologies RBP4 kit in clinical testing, further validating the effectiveness of the mixed antibody coating strategy. Therefore, the technique of combining RBP4 monoclonal antibodies with polyclonal antibodies for coating provides a new solution to enhance the consistency and accuracy of the RBP4 concentration measurements.