A developmental study of an in-house ELISA kit for the detection serum ANA

Abstract

Background: The production of ANA (Anti-Nuclear Antibody) occurs through an autoimmune mechanism that targets the DNA, RNA, and histone proteins of one’s own cells. The detection of ANA in serum is a diagnostic marker in autoimmune diseases, and the development of an in-house ELISA has clinical and economic value. Aim: To develop an in-house ELISA kit for serum ANA detection. Materials and Methods: The HEp-2 cell line, rich in diverse cellular antigens, served as the antigen source. Two methodologies were explored: utilizing whole cells and isolated cell-derived proteins. In the former, 20,000 cells per well were employed, while the latter utilized proteins isolated from cells at a concentration of 5μg/ml per well. Serum samples categorized as positive or negative for ANA were obtained as primary antibodies from the Center for Rheumatology of the First Central Hospital of Mongolia. The ELISA procedure involved measuring Optical Density (OD) values using primary and secondary antibodies. Results: The developed in-house ELISA kits using both methodologies were influenced by various factors including antigen binding, non-specific binding inhibition, and antibody exposure duration. A refined protocol was devised, augmenting the disparity in OD values between positive and negative controls. The difference between the positive and negative control values of the model using protein was close and there was no statistically significant difference (p>0.05). The difference in OD values between the positive and negative controls using HEp-2 cell protein increased in experiments 3 and 5. Optimal protocol conditions were developed that maximized the difference between the OD values of the positive and negative controls including fix the cells attached to the reaction plate with 1:1 methanol-acetone for 20 minutes at room temperature. Cell endogenous peroxidase was inhibited with 0.1% quenching buffer, and nonspecific binding was inhibited with 10% calf serum albumin. Primary antibody at 1:300 for 30 minutes and secondary antibody at 1:20,000 for 1 hour. There was a statistically significant difference (p˂0.0001) between the positive and negative control values using whole HEp-2 cells. Conclusion: We successfully developed an in-house ELISA kit technology for the detection of serum ANA by HEp-2 cell-based ELISA methodology.