Analysis of Antigen-Antibody Reactions by High-Performance Liquid Chromatography

Abstract

A simple and convenient method for the analysis of antigen-antibody reactions using high-performance liquid chromatography is described. A mixture of human serum albumin and anti-albumin monoclonal antibody in a buffer was analyzed on a gel filtration column. After the free albumin and antibody were rapidly separated from the antibodyantigen immune complex, the three amounts in the mixture were directly calculated from the peak heights. The concentration of the antigen and antibody as well as other reaction conditions were easily varied by this method. The effects of the reaction time, the concentrations of albumin or antibodies, and the pH were examined. The reactivities of the antigen binding sites of immunoglobulin G, pepsin digested fragment F(ab')2 and reduced monomeric form Fab' were studied; the results suggest that the Fc region of IgG may participate in the antigen-antibody interaction at those binding sites. Epitope typing of monoclonal antibodies was also carried out with a combination of two antibodies. This represents a new immunochemical technique for characterizing antibodies and for the quantitative assay of protein antigens.

“Electrokinetic Analyte Transport Assay” for α‐fetoprotein immunoassay integrates mixing, reaction and separation on‐chip

“…In our previous report, we demonstrated the "liquid-phase binding assay" (LBA) that separates bound and free forms by using polysulfated tyrosine peptide-modified antibody as a separation improvement molecule in HPLC [9][10][11]. It has the advantage that optimization of assay conditions such as antibody concentration can be done easily and precisely because of stoichiometric reaction in the liquid phase, resulting in good reproducibility and linear dose-response curve with wide assay range [12].…”

Analysis of Antibody–Antigen Interactions in Mixtures Containing Reactive and Nonreactive Components Using Size-Exclusion High-Performance (Pressure) Liquid Chromatography

“…Size-exclusion high-performance (pressure) liquid chromatography (SE-HPLC) 1 provides a rapid means of separating stable antibody-antigen complexes (CPLX) from unbound antibody (Ab) and antigen (Ag).…”

“…SE-HPLC elution profile analysis has been used to determine binding parameters of monoclonal Ab and Ag interactions based on equations derived from the law of mass action (1)(2)(3)(4). Complex mixtures of antibodies and antigens such as polyvalent antisera and venom may contain both reactive and nonreactive (NR) components.…”

Automatic and Simultaneous Analysis of Lens culinaris Agglutinin-Reactive α-Fetoprotein Ratio and Total α-Fetoprotein Concentration

“…This antibody recognizes AFP obtained from benign and malignant liver disease similarly; however, the antibody is inhibited in its binding to LCA-reactive AFP by LCA. During this search, we developed a new automatic immunoassay method for human chorionic gonadotropin, thyroxine, and α-fetoprotein , based on a liquid-phase binding assay (LBA) system. In LBA, an antibody reacts with antigen without a solid phase.…”