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 less than 5%), with a lower limit of detection of 0.2 ng/ml (CV less than 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.
Godfrey
M A
MA
School of Biological Sciences, University of Surrey, Guildford, UK.
Kwasowski
P
P
Clift
R
R
Marks
V
V
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D016477:Artifacts; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G; D007136:Immunoglobulins / Q000302:isolation & purification; D012016:Reference Values; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000032:analysis*
D000911:Antibodies, Monoclonal; C016512:IgY; D007074:Immunoglobulin G; D007136:Immunoglobulins; D013205:Staphylococcal Protein A