Dual-column immunoassays using protein G affinity chromatography

• Published in: Analytical chemistry (Washington) Vol. 61; no. 17; pp. 1901 - 1906
• Main Authors: Janis, Linda J, Regnier, Fred E
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.09.1989
• Subjects: Biological and medical sciences; Biotechnology; Chromatography, Affinity; Fundamental and applied biological sciences. Psychology; Immunoassay - instrumentation; Methods. Procedures. Technologies; Nerve Tissue Proteins - analysis; Others; Various methods and equipments
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac00192a024

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 x 10(1) to 1 x 10(5) ng with a precision of +/- 3.5%.