Dual-column immunoassays using protein G affinity chromatography

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Abstract
Tandem protein G affinity and reversed-phase chromatography (RPC) columns, coupled with a switching valve, were used for on-line immunoassays of antibodies and antigens. Columns with reversibly immobilized antibodies were prepared by adsorbing antibodies on the protein G column. Following antigen capture in the immunoaffinity column, antigen-antibody complexes were desorbed, dissociated, and transferred to the RPC column where they were separated and quantified. This system was used to determine the titer of a rabbit anti-human transferrin antibody sample with a precision of .+-. 2%. Quantitation of human transferrin in human serum had a precision of .+-. 6% and showed good agreement with rate nephelometry. The linear dynamic range for the transferrin, antigen immunoassay was 5 .times. 101 to 1 .times. 105 ng with a precision of .+-. 3.5%.