An analysis of experimental conditions influencing the anti-β2-glycoprotein I ELISA assay results

Five components of the anti-beta(2)-glycoprotein I (a beta(2)GPI) enzyme-linked immunosorbent assay (ELISA) (coating buffer, microplate brand, blocking buffer, dilution buffer, and conjugate) were analyzed to evaluate how they affect variability in test results. Thirty-two samples from patients with antiphospholipid syndrome (APS) positive for a beta(2)GPI IgG antibodies and three calibrators (a pool of a beta(2)GPI-positive patients, the monoclonal HCAL antibody, and a home-made calibrator) were tested. No differences with regard to the blocking step were noted. Differences were found between the neutral and basic coating buffer when HCAL was used. There were significant differences between Maxisorp and all the other brands of tested microplates. Differences were found between phosphate-buffered saline (PBS) and all the other dilution buffers examined, with exception of TRIS when HCAL or the homemade calibrator was used. There were differences between our routine conjugate and one of the other four conjugates tested when using two of the three calibrators. There were also significant differences between the routine and another conjugate analyzed when using the third calibrator. As variations in a beta(2)GPI ELISA conditions determine significant differences in the results, selecting the appropriate test variables is an important step toward a beta(2)GPI assay standardization.