ABSTRACT
Protein A chromatography is an excellent technique for the purification of monoclonal antibodies. However, the presence of Protein A in therapeutic monoclonal antibody preparations due to leaching has been linked with toxicity in animals and humans. Two sandwich ELISAs were developed to monitor Protein A column leaching: (1) rabbit anti-Protein A for capture and anti-Protein A F(ab')2-HRP for detection; and (2) rabbit anti-Protein A for capture and anti-Protein A-biotin for detection. The biotin ELISA is sensitive to the subnanogram range. In addition, these assays were used to develop DEAE and gel filtration chromatography techniques that substantially reduce Protein A levels in monoclonal antibodies purified by Protein A chromatography.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Staphylococcal Protein A/analysis , Animals , Antibodies, Monoclonal/standards , Biotin , Chromatography, DEAE-Cellulose , Chromatography, Gel , Drug Contamination , Enzyme-Linked Immunosorbent Assay/standards , Horseradish Peroxidase , Immunoglobulin Fab Fragments , Rabbits , Staphylococcal Protein A/immunology , Staphylococcal Protein A/standardsABSTRACT
Serological standard curves are mostly sigmoidal in shape. Their transformation into straight lines by linear regression can be the source of serious error. Log/log or logit/log handling of the values can straighten the curve but only if their distribution is normal. A new way of calculating concentrations of antibody or antigen which leaves the standard curve unmanipulated is described. Computer programs for TI 59 (Texas Instruments) and--in BASIC--for a personal computer have been written and greatly facilitate routine work.
