Polyacrylic polyhydrazides as novel reagents for detection of antibodies in immunoblotting assays

Abstract

By the glycoprotein specific staining method introduced recently (Heimgartner et al., 1989, Anal. Biochem. 181, 182–189) it is also possible to detect an antibody bound to its antigen on a membrane. The antibody is oxidized by periodate prior to incubation. Next, a polyacrylic polyhydrazide is coupled to the aldehyde groups generated in the sugar part of the antibody molecule. A periodate oxidized glycoenzyme, such as horseradish peroxidase, is then coupled to the remaining hydrazide groups of the polymer and incubation with a suitable enzyme substrate visualizes the glycoenzyme-polyhydrazide-antibody-antigen complex. The sensitivity of the detection is most critically dependent on the antibody class and the polyhydrazide reagent. Oxidation conditions are less important and the antibody does not need to be purified prior to periodate oxidation. Under standard conditions, the sensitivity obtained with IgG type antibodies was about ten times lower than with peroxidase-labeled secondary antibodies. However, a similar if not higher sensitivity can be expected with more glycosylated antibodies, such as IgM or IgE, or with chicken antibodies. This approach is advantageous because antibodies of all classes and from all species can be detected with the same reagent, the polyhydrazide, no foreign molecule is introduced in the antibody before its binding to the antigen and no conjugate needs to be prepared in advance.