LIQUID-PHASE BINDING ASSAY OF ALPHA-FETOPROTEIN USING A SULFATED ANTIBODY FOR BOUND FREE SEPARATION/

A rapid immunoassay using a sulfated antibody for bound/free separatio n in a liquid-phase binding assay is described, A first anti-alpha-fet oprotein monoclonal antibody was labeled with peroxidase (Fab'-POD) an d a second monoclonal antibody was conjugated with polysulfated tyrosi ne peptide (Fab'-YS), The monoclonal antibodies and alpha-fetoprotein (AFP) were mixed, incubated, and analyzed directly by anion-exchange c olumn chromatography, The amount of POD activity in the column effluen t was determined fluorophotometrically. The bound (Fab'-POD + AFP + Fa b'-YS) and free (Fab'-POD) forms of the conjugate were clearly and eas ily separated by ionic charge, and the free sulfated antibody (Fab'-YS ) was not detectable fluorophotometrically. The elution position of th e bound conjugate was adjusted by varying the length of the polysulfat ed tyrosine peptide, This method is convenient for antigen measurement because (1) only two modified antibodies are used in a buffer solutio n, (2) the concentration of antibodies and other assay conditions are easily set, (3) no solid phase is required, and (4) no washing is nece ssary.