Inflammation is the main process of chronic inflammatory diseases where cytokine, tumor necrosis factor-α (TNF-α) plays an important role in pathogenesis. Anti-inflammatory biological drugs are mostly therapeutic monoclonal antibodies. This group includes TNF-α inhibitors that neutralize free cytokine and prevent its binding to the receptor. Infliximab (IFX) is an example of TNF-α inhibitor that is a chimeric, mouse/human monoclonal antibody. At first, biological drugs were believed to be very safe and effective, but over time, side effects were shown- for example primary and secondary response failure. It is important to monitor the levels of biological drug constantly, as this is the only way to adjust the therapy for the individual patient. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used technique for the detection of TNF-α inhibitors. The purpose of thesis is to develop and evaluate an in-house IFX-ELISA, using two different detection antibodies and to compare in-house ELISAs with the apDia IFX-ELISA kit. A well-established and evaluated method is the basis for further assessment of the results, so we first evaluated each method. In case of intra-assay and inter-assay precision the coefficient of variation (CV%) was lower than 20% for both variants of in-house IFX-ELISA. In case of anti-IFX secondary antibody conjugated with horseradish peroxidase used in in-house ELISA recovery was between 80 - 120%. When we used secondary antibody directed against human IgG conjugated with alkaline phosphatase recovery was 120,1% ± 11,2%. For linearity, the results we obtained were all within the limits of 80 - 120%. The variant of in-house IFX-ELISA in which we used anti-IFX secondary antibody conjugated with horseradish peroxidase is analytically specific. Comparison with apDia IFX-ELISA kit yielded very good repeatability (r between 0.90 and 0.99, p<0,001). In both versions of the in-house method, on average we obtained a slightly higher concentration than apDia IFX-ELISA. When using the anti-IFX secondary antibody conjugated with horseradish peroxidase 100% accuracy of classification of negative patients was achieved. In conclusion, both variants of an in-house IFX-ELISA give comparable results to apDia IFX-ELISA kit. Both are useful for detection of IFX, but in terms of analytical specificity and accuracy of negative results classification, the variant with specific anti-IFX secondary antibody conjugated with horseradish peroxidase is better.
|
