Antibody quantitation using an immunoadsorbent and the unlabeled antibody enzyme method.
Measurement of specific immunoadsorbent-bound antibodies has been accomplished by the unlabeled antibody enzyme method (Sternberger et al., 1970). Sepharose-4B containing specific antigen (or ligand) is treated with diluted specific immune serum (primary serum), such as 1 ml of serum diluted 6000-20,000 fold. followed by antiserum against immunoglobulin G (IgG) of the primary serum and then by peroxidase-antiperoxidase (antigen-antibody) complex (PAP) derived from the same species as the primary serum. Radiolabeled primary antibody and anti-IgG have confirmed the stoichiometry of the reaction. The immunoabsorbent binds the antibody of interest quantitatively and to equal extent after 15 min or 48 h. The enzymatic activity of the PAP complex followed a direct linear relationship to its concentration indicating the stability of binding in the PAP complex. A direct relation between the enzymatic activity measured when both the primary antiserum and the anti-IgG are used allows for quantitation of the antibody level of the primary serum.
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D007124:Immunoenzyme Techniques; D007163:Immunosorbent Techniques; D011817:Rabbits; D012709:Serum Albumin / Q000276:immunology; D012710:Serum Albumin, Bovine / Q000032:analysis
D000906:Antibodies; D007106:Immune Sera; D012709:Serum Albumin; D012710:Serum Albumin, Bovine