The use of ELISA for detection of the antibody-induced conformational change in a viral protein and its intermolecular spread.
Antibody-induced conformational changes of proteins have been recently frequently suggested to explain a variety of observations. In spite of the fundamental importance of this phenomenon for both in vivo and in vitro antigen-antibody interactions, it is not generally accepted because of the lack of conclusive evidence. This report utilizes a novel approach to the study of antibody-induced antigenic conformational changes. Pairs of monoclonal antibodies (mAb) were used to induce and to assess conformational changes in potato virus X (PVX) protein. Blocking ELISA with native and glutaraldehyde treated virus was used to detect conformational changes. Double antibody sandwich (DAS) ELISA was designed to investigate possible inter-molecular spread of conformational changes. Detection of one way blocking in a blocking ELISA, with a pair of mAbs reacting to non-overlapping epitopes, suggested conformational change as the mechanism of blocking. The putative conformational change was confirmed when the one way blocking was prevented using conformationally restrained virus. Inter-molecular spread of the conformational change among the molecules of PVX protein was demonstrated in DAS-ELISA, when capture mAb inhibited binding of detecting mAb in the absence of steric hindrance. Unlike X-ray crystallography, the methodology utilized in this study indicates directly the significance of a changed conformation to antibody binding.
Cepica
A
A
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Canada.
Yason
C
C
Ralling
G
G
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000502:physiology*; D000956:Antigens, Viral; D001667:Binding, Competitive; D015153:Blotting, Western; D002213:Capsid / Q000276:immunology; D036022:Capsid Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005557:Formaldehyde / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D010942:Plant Viruses / Q000276:immunology*; D011487:Protein Conformation; D011198:Solanum tuberosum
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; C058750:coat protein, Potato virus X; D005557:Formaldehyde; D005976:Glutaral