DOP055. Development of a new immunoassay for the accurate determination of anti-infliximab antibodies in inflammatory bowel disease
J.M. Semmler1, A. Pilch1, F.P. Armbruster1, A. Dignass2, W. Kruis3, J. Stein4, 1Immundiagnostik, AG, Bensheim, Germany, 2Agaplesion Markus Hospital, Department of Medicine I, Frankfurt, Germany, 3Ev. Krankenhaus, Med. Klinik, Köln, Germany, 4Crohn Colitis Center, DGD Krankenhaus Sachsenhausen, Frankfurt, Germany
The formation of antibodies to infliximab (ATIs) is closely associated with loss of response to infliximab in patients with inflammatory bowel disease (IBD). Therapeutic drug monitoring strategies have been based mainly on assessment of serum ATI levels by immunoassays based on the double-antigen format, using immobilized infliximab or protein A to capture ATIs on a carrier. In these assays, however, the presence of infliximab can interfere with the binding of labeled infliximab to the captured ATIs, leading to false-negative results. In this study, we developed a novel immunoassay for ATIs to enable their measurement in the presence of infliximab.
The new assay is based on the dissociation of immune complexes between infliximab and ATI at low pH-values and the use of biotinylated and peroxidase-labeled infliximab to prevent re-formation of immune complexes. Western blot analysis confirmed the presence of ATIs in the samples found positive by the new method but negative by the conventional method. Measurement of ATI levels was performed in 29 patients with Crohn's disease (CD) on infliximab maintenance therapy using both the novel immunoassay and the conventional method. Enzyme-linked immunosorbent assay was used to determine serum infliximab trough levels.
Whereas ATIs were detected in 7 out of 29 patients (24.1%) using the new method, they were detected by the conventional method in only 1 patient (3.4%), for whom the two highest ATI titers were determined by the new assay. The addition of infliximab to the samples dose-dependently inhibited the detection of ATIs in the conventional method but not in the new method. Patients positive for ATIs had significantly lower serum trough levels of infliximab (P < 0.01) and significantly higher clinical activity scores (p < 0.001) in comparison to patients negative for ATIs.
The novel assay allows the measurement of serum ATI levels independent of the presence of infliximab and anti-adalimumab antibodies (data not shown). The new method is therefore a useful tool in strategy selection for the optimal management of IBD patients showing loss of response to TNFalpha-antibodies .
1. Bendtzen et al., (2009), Monitoring of bioavailability, pharmacokinetics and, Scan. J. Gastroenterol, 44: 774–781.