An Enzyme-Linked Immunosorbent Assay for Therapeutic Drug Monitoring of Infliximab

Abstract

An enzyme-linked immunosorbent assay (ELISA) measuring serum infliximab concentrations in treated patients was developed. Microtiter plates were sensitized with tumor necrosis factor alpha (TNF-alpha) and saturated with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). Samples diluted 1:100 in PBS-1% BSA were added and bound infliximab was detected using peroxidase-conjugated goat anti-human immunoglobulin G specific for Fc fragment (HRP-anti hIgG). Reading was performed using an ELISA plate reader. The limit of detection, calculated by assaying 10 replicates of a drug-free serum sample or blank sample and defined as the lowest concentration distinguishable from zero at 2 standard deviations, was 0.014 microg/mL. Each quality control sample was tested on 7 occasions on 1 day and on 5 separate days. The intraday precision indices of the method were (percent coefficients of variation, CV%) 11.7%, 6.2%, and 6.9% for 0.04 microg/mL, 2 microg/mL, and 4.5 microg/mL, respectively. The corresponding bias measures (percent deviation) were -5.5%, -1.9%, and -7.9%, respectively. The between-days precision was 9.8%, 5.3%, and 5.3% for 0.04 microg/mL, 2 microg/mL, and 4.5 microg/mL, respectively. The corresponding bias were +0.3%, -0.3%, and -7.8%, respectively. Lower limit of quantitation and upper limit of quantitation were 0.04 microg/mL and 4.5 microg/mL, respectively. Trough serum concentrations of infliximab were measured in 6 adult patients with various diseases and in 5 pediatric patients with Crohn's disease. For the latter group, samples drawn 1 hour after the end of the infusion and repeated measurements also were available. Data were described using a 1-compartment population pharmacokinetic model. Terminal elimination half-life was 10.9 days. This method is rapid, accurate, and reproducible, and may be useful in therapeutic drug monitoring of infliximab.

The underlying inflammatory chronic disease influences infliximab pharmacokinetics

“…Trough serum infliximab concentrations were measured in the laboratory of Pharmacology-Toxicology of the University Hospital of Tours, France, using a validated enzyme-linked immunosorbent assay. 7 The limit of detection was 0.014 mg/L and the lower (LLOQ) and upper limits of quantification (ULOQ) were 0.04 mg/L (between-assay accuracy coefficient of variation: 9.8%) and 4.5 mg/L (between-assay accuracy coefficient of variation: 5.3%), respectively.…”

“…Despite the adjustment of dose based on patients' weight, large interindividual variability in infliximab serum concentrations is observed. [1][2][3] Infliximab pharmacokinetics has been analyzed in RA, 4 AS 5,6 and inflammatory bowel disease (IBD) [7][8][9][10][11][12] patients using population compartmental modeling. These studies showed that several individual characteristics such as body weight, [4][5][6]9,10,12,13 sex, 5,9,13 anti-drug antibodies, 14,15 cotreatment with methotrexate 4 or preinfusion C-reactive protein (CRP) 4 were related to infliximab pharmacokinetic parameters.…”

Detection of infliximab levels and anti‐infliximab antibodies: a comparison of three different assays

“…In Leuven, an in house developed direct ELISA was used to measure IFX based on a previously described method. 24 Briefly, high binding 96-well plates (Costari;…”

Tumor burden influences exposure and response to rituximab: pharmacokinetic-pharmacodynamic modeling using a syngeneic bioluminescent murine model expressing human CD20

“…Rituximab and infliximab plasma concentrations were measured using enzyme-linked immunosorbent assays adapted from methods described previously. 17,18 Briefly, microwell plates (Nalge Nunc International, Rochester, NY) were coated with an antirituximab idiotype monoclonal antibody (MCA 2260; Serotec, Cergy Pontoise, France) or recombinant human TNF-␣ (Tebu, Le Perray en Yvelines, France) at concentration of 1 g/mL and 0.75 g/mL, respectively. Mouse plasma, diluted 1/100 in PBS buffer, was added to the wells, and a goat antihuman IgG conjugated to horseradish peroxidase (A2290; SigmaAldrich, Lyon, France) was used for detection.…”