Abstract
The double antigen bridging immunoassay has been used extensively for detection of immunogenicity responses to therapeutic monoclonal antibodies. We have analyzed parameters affecting performance of this type of immunoassay including microtiter plate antigen coating concentration, enzyme-labeled antigen conjugate dilution and assay format (one-step versus two-step). We present results demonstrating that the format of the assay has a significant impact on the optimal parameters to maximize assay performance. A one-step assay format achieves maximal sensitivity across a broad range of coating concentrations and at a lower concentration of conjugate than that in a two-step format. In contrast, a two-step format requires very low coating concentrations and higher conjugate concentrations to achieve maximal sensitivity and suffers from significantly reduced sensitivity at higher coating concentrations. Together, these findings indicate that a one-step assay format can greatly reduce the effect of coating concentration variation on assay performance.
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