K. Farrag*1,2, M. Rohlfs3, J. Ruppert3, F-P. Armbruster3, J. Stein1,2
1Interdisciplinary Crohn Colitis Centre Rhein-Main, Frankfurt/Main, Germany, 2DGD Clinics Sachsenhausen, Frankfurt/Main, Germany, 3Immundiagnostik AG, Bensheim, Germany
Ustekinumab is a monoclonal therapeutic anti-interleukin-12 and anti-interleukin-23 antibody approved for use in moderate to severe Crohn’s disease (CD). Analysis of data from the Phase 3 induction trials, UNITI-1 and UNITI-2, demonstrated a significant exposure–response relationship of ustekinumab in CD. Interindividual differences in response to ustekinumab treatment may be explained in part by interindividual variability in pharmacokinetics. The aim of this work was to develop and validate an enzyme-linked immunosorbent assay (ELISA) to measure ustekinumab drug concentrations.
Samples diluted at 1:200 were added to microtiter plates coated with recombinant human antibodies against ustekinumab for binding. Mouse anti-human immunoglobulin G1 (HRP-anti h IgG1) was used to detect bound ustekinumab. Assay performance characteristics were determined according to the European in vitro diagnostic devices directive 98/79/EC.
Both in serum and plasma, the method has been demonstrated to be linear from 1.10 to 37.35 ng/ml, showing a non-linear behaviour of less than ±20% in this interval. The limit of quantification (LoQ) for ustekinumab measurement in human serum samples was 0.953 ng/ml. Intra-assay variation (repeatability) was ≤9.5% (n = 23), while inter-assay variation (reproducibility) was ≤9.1% (n = 20). Linearity testing was performed by analysing three serially diluted samples spiked with ustekinumab; ustekinumab concentrations measured by the new assay were within 97%–117% of the expected concentrations. The assay detected no false-positive signals from the samples of untreated patients. The specificity of the antibody was tested by measuring the cross-reactivity against a range of compounds with structural similarity to ustekinumab. There was no cross-reactivity observed.
This newly developed ELISA offers a fast and accurate test with reproducible results. The specificity of the assay could be improved by the use of monoclonal antibodies to ustekinumab. This ELISA has potential utility in therapeutic drug monitoring of patients receiving ustekinumab, and additionally in pharmacokinetic/pharmacodynamic studies of the drug.