Analytical and clinical evaluation of a new immunoassay for therapeutic drug monitoring of infliximab and adalimumab

In the last decade, biological agents have revolutionised rheumatology. Tumour necrosis factorα (TNF α ) neutralising antibody constructs as infl iximab and adalimumab are widely used as treatment for rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and psoriasis. Infl iximab is a chimeric (mouse-human) monoclonal antibody against TNF α . It has been shown to induce the formation of human antichimeric antibodies. Adalimumab is a fully human antibody against TNF α and therefore thought to be less immunogenic than chimeric antibodies. However, even fully human antibodies may lead to the production of human antihuman antibodies. Unfortunately, despite the overall effectiveness of these biological agents, a signifi cant proportion of patients do not respond or lose response over time. An explanation may be that antibodies are formed against these therapeutic agents. Another reason could be the necessity of achieving trough anti-TNF α drug concentrations in a target range (1, 2) . In addition, the incidence of anti-drug antibodies could be associated with adverse events. Higher concentrations of anti-infl iximab antibodies were associated with a higher risk of infusion reactions (3) , and severe arterial and/or venous thromboembolic events have been related with the presence of anti-adalimumab antibodies (4) . Until now, in the absence of direct measurement of drug levels and anti-drug antibodies, decision-making in the case of failure to anti-TNF α treatment was based on clinical outcome alone. Recently, a new enzyme linked immunosorbent assay (ELISA) has been developed to detect soluble drug levels and antibody formation among patients receiving infl iximab or adalimumab (Promonitor ® , Proteomika, Derio, Vizcaya, Spain, distributed by Menarini Diagn o stics S.A. ® , Badalona, Barcelona, Spain). The aim of this work was to evaluate the analytical performance and the clinical validation of these four assays with the purpose of testing the hypothesis that they are suitable for therapeutic drug monitoring of infl iximab and adalimumab in treated patients. Following the recommendations for the optimisation of immunoassays (5) , we performed our evaluation of these assays during a period of 6 months and two different reagent lots were used. Intraand inter-assay variability were evaluated on three different days with two replicates of the same sample in each assay. The linearity was performed by diluting different proportions of a standard from the upper and lower end of the dynamic range for each analyte to be evaluated. For the clinical validation study, serum was collected from 69 patients with rheumatic diseases treated with infl iximab (Remicade ® , Schering-Plough) or adalimumab (Humira ® , Abbott Laboratories). Informed consent of the patients was required. Before each infusion of infl iximab or subcutaneous injection of adalimumab, 10 mL of serum was collected and stored at – 20 ° C. All sera were tested under standardised conditions specifi ed by the fabricant. Six dilutions of serum samples and 10 for each standard curve were made. All the analytical development was carried out without knowledge of clinical data. Briefl y, in the anti-TNF α drug assay, recombinant human TNF α was coated overnight at room temperature on the solid phase and recognised by infl iximab or adalimumab in each case. The therapeutic monoclonal antibody was detected by an anti-human inmunoglobulin specifi c antibody conjugated to biotine. Serum concentrations of antibodies against these drugs were measured using a double-antigen ELISA based on their capture by drug-coated microplates and their detection by biotine-coupled anti-TNF α drug. This ELISA has a detection limit of 2 ng/mL for infl i ximab and 0.4 ng/mL for adalimumab. And the cut-off value for the drug is 0.053 mg/L for infl iximab and 0.002 mg/L for adalimumab. And for antibodies, the cut-off value is 37 UA/mL for infl iximab and 8 UA/mL for adalimumab. Descriptive statistics were provided using median (range) or percentages. Statistical analysis was performed using the Sigma Plot ® program. Differences between independent groups were traced with the use of Student t-test for normally distributed values. p-Values < 0.05 were considered statistically signifi cant. *Corresponding author: Francisca Llinares-Tello c/o Col o n 120 2 ° A, 03570, Villajoyosa, Alicante, Spain E-mail: paquillinares@eresmas.com Received January 25, 2012; accepted March 9, 2012 ; previously published online March 26, 2012