Generation of a Highly Specific Monoclonal Anti-Infliximab Antibody for Harmonization of TNF-Coated Infliximab Assays.

Background: Determination of infliximab (IFX) serum concentrations has been used for treatment optimization of patients with inflammatory bowel disease. A wide range of enzyme-linked immunosorbent assays (ELISA) exists to quantitate IFX. Most of these assays lack specificity and cross-react with other anti-tumor necrosis factor (TNF) agents. The ability of these IFX assays to detect IFX in complex with antidrug antibodies is not known. The objective of our study was to develop an IFX-specific immunoassay to monitor IFX serum concentrations and to evaluate the impact of antidrug antibodies on the assay performance.

Methods: A panel of monoclonal antibodies toward IFX (MA-IFX) was generated by hybridoma technology and evaluated to replace the polyclonal antibody in a TNF-coated IFX assay. The selected monoclonal antibody-based (MA-based) IFX ELISA was benchmarked to a clinically validated, reference polyclonal antibody-based (pAb-based) IFX ELISA using 209 inflammatory bowel disease serum samples.

Results: Fifty-five MA-IFX were generated and grouped into 9 clusters. Of the 22 monoclonal antibodies tested, MA-IFX6B7 was selected for use in the IFX ELISA and the assay was further optimized. MA-IFX6B7 is a high-affinity (KD = 1.40E-09 mol/L), noninhibitory IgG1 antibody that binds to the Fab fragment of IFX and exhibits no cross-reactivity with other anti-TNF drugs. The linearity of an IFX dose-response curve was demonstrated in the range of 1.2-37.5 ng/mL (R = 0.988). The MA-based assay showed a good Pearson correlation (R = 0.986) and agreement (intraclass correlation coefficient = 0.985) with the pAb-based assay. The MA-based assay detects IFX in complex with nonneutralizing anti-IFX antibodies but not when complexed with neutralizing anti-IFX antibodies.

Conclusions: In this study, a highly specific MA-IFX was developed as detection antibody in an ELISA to quantify IFX serum concentrations. The assay was benchmarked to the clinically validated reference pAb-based IFX ELISA.