An enzyme-linked immunosorbent assay for therapeutic drug monitoring of infliximab.

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.

Analysis of human CD4 T lymphocyte proliferation induced by porcine lymphoblastoid B cell lines.

BACKGROUND: This study was undertaken to characterize the two porcine lymphoblastoid cell lines L23 and L35, derived from a pig inoculated by the retrovirus Tsukuba-1, and to determine how they induce a strong human lymphocyte proliferation. METHODS: Phenotypic characterization was performed by flow cytometry and reverse transcriptase-polymerase chain reaction analyses. Xenogeneic mixed lymphocyte reactions (XMLR) were performed using unfractionated human peripheral blood mononuclear cells (huPBMC) and purified CD4+ T lymphocytes as responding cells, in the presence of blocking antibodies and fusion proteins. RESULTS: The immunoglobulin genes were demonstrated to be rearranged in L23 and L35 cell lines, in agreement with the expression of a B cell phenotype. Both induced a similar proliferation of huPBMCs and purified human CD4+ lymphocytes from adult or cord blood (naïve cells). Proliferation of CD4+ T lymphocytes was completely blocked by anti-SLA-DR plus anti-SLA-DQ mAbs, excluding human lymphocyte transformation by porcine viruses. The frequency of proliferative precursors was inconsistent with that induced by a retroviral superantigen but similar to classical direct xenoantigen presentation as observed with other porcine antigen-presenting cells. Extensive analysis of costimulatory signals led to the identification of the CD28 pathway, in agreement with membrane expression of B7 molecules on L23 and L35 cells, and of the CD2 pathway in L35 cells. CONCLUSION: These two porcine lymphoblastoid cell lines have been further characterized and clearly identified as belonging to the B cell lineage. By expressing major histocompatibility complex class II antigens and costimulatory molecules, they induce a vigorous proliferative response of human CD4+ lymphocytes through a direct presentation pathway.