Method Comparison Between ELISAs Suggests Therapeutic Ranges for Infliximab Should Be Assay-Specific

Infliximab is an anti-tumor necrosis factor (TNF)1 monoclonal antibody therapy used in the treatment of inflammatory bowel disease (IBD). Patient response to infliximab varies from complete clinical, endoscopic, and biochemical remission to no improvement in symptoms, either after infliximab infusions have commenced [primary nonresponse (PNR)] or after an initially positive response to infliximab [loss of response (LOR)]. The etiology of PNR and LOR is not well-understood, although genetics and immunogenicity are likely to play a role. Clinical management in suspected PNR or LOR is complex—options include dose escalation or stopping infliximab, switching treatment, or addition of an immunomodulator. Currently, management is based on clinical assessment, but, increasingly, concentration of trough infliximab is augmenting treatment decisions. For example, trough concentration of infliximab is associated with mucosal healing (1). In the US, the AGA (American Gastroenterological Association) have made recommendations on therapeutic drug monitoring in IBD (2); however, an international consensus therapeutic concentration of infliximab has not yet been determined. In the UK, the National Institute for Health and Care Excellence (NICE) provides guidance and advice to improve health and social care. NICE have undertaken a Diagnostics Assessment program to determine the cost and clinical effectiveness of measuring infliximab drug levels in management of IBD patients in the National Health Service setting, and conclude that there was insufficient data to comment (3). NICE states that further research into the analytical and clinical validity of the ELISA kits for measuring infliximab drug levels is recommended (3). Two major studies [Trough Concentration Adapted Infliximab Treatment (TAXIT) and Personalized Anti-TNF Therapy in Crohn disease (PANTS)] both aimed to address the diagnostic utility of anti-TNF drug monitoring in the management of IBD have used different ELISA methods for determining infliximab drug levels. Currently, ELISA methods for measuring infliximab concentration are not standardized, making comparability and generalizability of results between the studies difficult (4). The TAXIT randomized controlled trial showed that targeting patients' infliximab trough concentrations in a 3–7-mg/L window resulted in a more efficient use of the drug in patients with IBD (5). As a result of this, a target therapeutic range of 3–7 mg/L is widely quoted for infliximab-treated patients. The assay used in the TAXIT trial is an in-house method utilizing a polyclonal antibody to detect infliximab. This assay was subsequently replaced by an ELISA using a specific monoclonal antibody (MA-IFX6B7), which is now commercially available as a CE-marked ELISA kit manufactured by R-Biopharm. The monoclonal assay was benchmarked to the clinically validated original polyclonal antibody assay (6). The PANTS study is a prospective, observational study that aims to investigate the clinical, serological, and genetic factors that determine PNR, LOR, and adverse drug reactions in patients treated with infliximab with active luminal Crohn disease. The study is recruiting from over 110 UK hospitals currently participating in the UK Inflammatory Bowel Disease Genetics Consortium pharmacogenetics program. The trough concentration of infliximab in patient serum is determined at multiple time points throughout the course of the study, at the Exeter Blood Sciences Central Laboratory. It is anticipated that the PANTS study will further inform about relevant therapeutic ranges for infliximab, in both the induction and maintenance phases of treatment. The ELISA assay used in the PANTS study is manufactured by Immundiagnostik, and the correlation of this assay with the R-Biopharm ELISA is not known. The aim of this study is to compare the R-Biopharm and Immundiagnostik ELISA assays. The results will be of importance to NICE and the IBD and Clinical Biochemistry communities. The 126 patient samples received for infliximab analysis through the Exeter Blood Sciences routine clinical service were analyzed using both the R-Biopharm (RIDASCREEN® IFX Monitoring, G09041) and Immundiagnostik AG (IDKmonitor® Infliximab drug level, K 9655) methods. There is broad agreement between the 2 methods (Fig. 1, linear regression), however the Immundiagnostik assay is consistently lower than that obtained in the R-Biopharm assay across all concentrations (paired t-test, P < 0.01). The Bland–Altman plot shows that there is a proportional bias. In the R-Biopharm assay, the wells are coated with monoclonal antibodies against TNFα. The secondary antibody is a monoclonal antibody against infliximab (MA-IFX6B7, isolated and characterized at KU Leuven) that is conjugated with horseradish peroxidase. In the Immundiagnostik assay, wells are coated with an antiinfliximab fragment, and after binding of infliximab, the Fc domain of an antihuman IgG1 Fc, coupled to peroxidase, is added. The correlation between the assays was determined up to an infliximab concentration of 12 mg/L in the R-Biopharm method. Both methods had acceptable analytical performance (intraassay and interassay precision, %CV ≤ 10%). For both assays, no change in [infliximab] was observed up to a hemolysis index of 550, nor for samples spiked with adalimumab (10 mg/L) or vedolizumab (250 mg/L), nor for samples exposed to 3 freeze–thaw cycles. Accuracy was determined by recovery experiments for samples spiked with Remicade (R-Biopharm, 108%–111%; Immundiagnostik, 120%–152%) and CT-P13 (RemsimaTM and InflectraTM, R-Biopharm, 82%–124%; Immundiagnostik, 91%–139%). These data highlight that it would be inappropriate to draw direct comparison between infliximab concentrations reported in the TAXIT trial and PANTS study. Healthcare professionals must be aware of these differences when interpreting infliximab concentrations and recognize that therapeutic ranges are not transferable between kits. Greater concordance between infliximab assays should be observed after alignment with the infliximab standard material, produced by the National Institute of Biological Standards and Controls, UK. tumor necrosis factor inflammatory bowel disease primary non-response loss of response National Institute for Health and Care Excellence Trough Concentration Adapted Infliximab Treatment randomized controlled trial Personalized Anti-TNF Therapy in Crohn's disease study National Institute for Health Research.