Background Infliximab (Remicade®) is a chimeric human-murine antihuman tumour necrosis factor (TNF) monoclonal antibody (75% human; 25% murine). It is composed of human constant and murine variable regions. It is produced by a recombinant cell line cultured by continuous perfusion. Infliximab binds to the soluble and transmembrane forms of tumour necrosis factor α (TNFα) and inhibits binding of TNFα with its receptors. It is approved for patients with rheumatoid arthritis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis and ulcerative colitis. People with certain diseases have too much TNFα that can cause the immune system to attack normal healthy parts of the body. Thus, Infliximab can block the damage caused by too much TNFα.
Purpose The purpose of this research has been to develop an ELISA assay for the quantification of Infliximab when reconstituted in sterile water for injection to give final concentrations of 10.0 mg/ml, 2.0 mg/ml and 0.5 mg/ml.
Materials and methods A direct and non-competitive ELISA test has been developed to determine Infliximab. This inmunometric test has been based the use of coated ELISA plates with Infiximab. The plates were incubated overnight at 4°C using samples of the reconstituted Infliximab diluted appropriately in buffer carbonate/bicarbonate pH 9.6 0.1 M to give final concentrations between 1.0 and 500.0 ng/mL. Blocker was then added and incubated for two h at 37°C. Then plates were incubated 30 min after the addition of antihuman IgG peroxidase conjugated to the sensitised Infliximab plates. At the end of the incubation period the substrate (OPD o-phenylenediamine dihydrochloride) was added to each well. The reaction terminated by addition of sulphuric acid (1.0 M). The absorbance was obtaining by subtracting the measurements at 450 and 620 nm using a 96-well plate reader and the results analysed using the XFluor4 software. Statistical data treatment for the validation of the method were performed using Stagraphics Plus 5.1 software.
Results The Infliximab ELISA method has been validated in terms of coating reproducibility, dynamic range of the assay including the estimation of the calibration function, accuracy (as recovery), intra e inter assay precision (as %CV), and sensitivity (as detection and quantification limits). Specificity was not included in the validation, since no cross-reactions are expected when analysing reconstituted Infliximab.
Conclusions The method here propose is ready to be used in further long term stability studies of Infliximab reconstituted in sterile water for injection to give the hospital conditions of use.
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