An automated analytical method for analyzing alpha-fetoprotein (AFP) carbohydrate chain microheterogeneity based on competitive reaction between lectin and anti-AFP monoclonal antibody in liquid phase is described. The antibody used binds to all species of AFP molecule without Lens culinaris agglutinin (LCA); however, its binding reaction to LCA-reactive AFP was inhibited by LCA. Sulfated tyrosine octamer was conjugated to the antibody, and sulfated tyrosine pentamer and peroxidase were conjugated to other monoclonal antibodies, respectively. Serum reacted with three anti-AFP monoclonal antibodies and LCA in liquid phases, and two types of immune complex were observed. The two types were separated directly by the liquid-phase binding assay system equipped with an anion-exchange column. Peroxidase activity of immune complex was determined fluorophotometrically. Total AFP concentration and the ratio of LCA-reactive AFP in samples were calculated simultaneously, using the sum of the two peaks and the ratio of peaks obtained by LCA inhibition to sum of two peaks. The results correlated well with conventional methods. The method is simple and convenient for routine clinical assays.

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A rapid immunoassay using a sulfated antibody for bound/free separation in a liquid-phase binding assay is described. A first anti-alpha-fetoprotein monoclonal antibody was labeled with peroxidase (Fab'-POD) and a second monoclonal antibody was conjugated with polysulfated tyrosine peptide (Fab'-YS). The monoclonal antibodies and alpha-fetoprotein (AFP) were mixed, incubated, and analyzed directly by anion-exchange column chromatography. The amount of POD activity in the column effluent was determined fluorophotometrically. The bound (Fab'-POD + AFP + Fab'-YS) and free (Fab'-POD) forms of the conjugate were clearly and easily separated by ionic charge, and the free sulfated antibody (Fab'-YS) was not detectable fluorophotometrically. The elution position of the bound conjugate was adjusted by varying the length of the polysulfated tyrosine peptide. This method is convenient for antigen measurement because (1) only two modified antibodies are used in a buffer solution, (2) the concentration of antibodies and other assay conditions are easily set, (3) no solid phase is required, and (4) no washing is necessary.

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.

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