No 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 x 10(1) to 1 x 10(5) ng with a precision of +/- 3.5%.

A new type of chromatographic immunoassay based on sequential addition is described. On a protein A column, the antibody, the sample containing the antigen, and then a known amount of antigen are sequentially injected. This assay is designed to shorten analysis times and reduce complexity of dual-column chromatographic immunoassays, circumvent desorption buffer interferences common to affinity chromatography, and eliminate the need for tagged molecules. This new technique is named kinetic immunochromatography sequential addition (KICQA). Because of its kinetic nature, flow rate will have a large effect on KICQA, and the impact of changing flow rate is studied extensively. By use of various amounts of antibody, the dynamic range of KICQA is shown to be selectable over 2.5 orders of magnitude. Finally, KICQA was used to determine transferrin and albumin in human serum. Both analytes show good agreement with their respective reference methods, and an albumin assay was performed in under 1 min.

No abstract

No abstract