Antigen itself antibody: an alternative one‐step immunoassay for measuring the anti‐idiotypic antibody titer

Abstract

Immunoassay designs rely on the great specificity of antibodies and a suitable marker that facilitates generation of a quantitative signal. Currently, there is no reliable method for measuring the titers of an anti-idiotypic antibody. Our initial attempt to measure titers of mouse anti-idiotypic antibody after idiotypic vaccination with HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT) failed. Because the injected antigen, nmAb-KT, is a mouse IgG, using a commercial antibody to measure the antibody titer always gave a false positive signal against control mouse serum antibody in parallel with the antigen-treated immunized serum antibodies. To get a reliable and clearly differentiable signal by ELISA, idiotypic antigen was labeled with HRP and HRP-conjugated-nmAb-KT used to measure the antibody titers in the antigen-treated mice. Compared with control mice, signals were found in high anti-nmAb-KT IgG responses in test mice; however, untreated control mice had a significant amount of purified non-specific IgG. This method is amenable to long read lengths and will likely enable anti-idiotypic antibody titer measurement in a more specific and cost effective way without requiring commercial antibody.Key words anti-idiotypic antibody, killer toxin neutralizing monoclonal antibody, one-step immunoassay, titer measurement.Killer toxins are protein molecules that disrupt cell functions in a number of ways (1, 2). Among the yeast killer toxins, HM-1 is highly stable against heat treatment and a wide range of pH values (pH 2-11); it also exhibits a wide spectrum of anti-fungal activity (3, 4). These unique properties have made this toxin attractive for therapeutic applications, but not for clinical use both because of its instability in host physiological conditions and its antigenicity (2, 4, 5). To overcome the intrinsic toxicity and chemical instability of HM-1 and simultaneously take advantage of its immunogenicity, nmAb-KT has been produced (6). With the aim of producing a new anti-fungal drug, we used nmAb-KT as an immunogen in mice to produce an antigenspecific anti-idiotypic antibody that shares structural and/or functional similarities with the active site of HM-1 (7-15).Antibodies are highly specific, naturally evolved molecules that recognize specific antigens and can be classified according to their mode of action as they react to and set about defending the body against foreign invaders (16). Anti-idiotypic antibodies are a special set