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A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of staphylococcal protein A (SpA) present as a trace contaminant of murine immunoglobulins purified on immobilized protein A

1992; Elsevier BV; Volume: 149; Issue: 1 Linguagem: Inglês

10.1016/s0022-1759(12)80044-5

1872-7905

Miguel A. J. Godfrey, Piotr Kwasowski, Roland Clift, V. Marks,

Advanced Biosensing Techniques and Applications

A specific non-competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detecting and quantifying protein A (SpA), present as a trace contaminant of therapeutic murine monoclonal antibodies (McAb) purified on immobilized SpA preparations. The assay employs a microtitration plate system, in which affinity-selected chicken anti-SpA antibodies from the egg yolks of immunized hens provide a specific capture antibody, followed by the addition of standards or sample with a McAb concentration of 1 mg/ml, in conditions unfavourable for Fc binding, and finally an affinity-selected rabbit anti-SpA peroxidase label. The working range of this assay is between 0.5 and 10.0 ng/ml (CV<5%), with a lower limit of detection of 0.2 ng/ml (CV<10%). This assay was used to evaluate SpA leakage when purifying a serum-free murine IgG1 cell culture supernatant using SpA immobilized on agarose (Protein A-Sepharose CL-4B) or controlled pore glass (Prosep A, high capacity). These gave average antibody SpA contamination levels of 6.7 ±1.6 and 2.4 ± 0.5 (mean ± SD) parts per million respectively.