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

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.