Direct detection of idiotypic determinants on blotted monoclonal antibodies.
The protein-blotting technique has been tested as a mean to study the expression of idiotypic determinants. A monoclonal BALB/c antipoly (Glu60-Ala30-Tyr10) GAT antibody (G5) was caused to migrate on SDS gel and transferred to a nitrocellulose filter. To facilitate the renaturation of the idiotypic determinants, the blotted proteins were incubated in NP40 buffer, immediately after the transfer. The ability of two anti-idiotypic sera to detect two defined idiotypic specificities of the blotted G5 molecules was investigated. When G5 was electrophoresed on SDS gel under non-reducing conditions, a specific detection of two idiotypic specificities of the G5-blotted molecules was obtained. On the other hand, when G5 was migrated under reducing conditions, none of the two antiidiotypic sera gave a staining of the heavy and the light chains. This result indicates that molecules expressing conformational idiotypic determinants can be detected by protein-blotting technique after migration on SDS gel. Moreover, this suggests the possible interest of this technique to analyse non-antibody molecules bearing idiotypic determinants.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D007143:Immunoglobulin Heavy Chains / Q000032:analysis; D007130:Immunoglobulin Idiotypes / Q000032:analysis*; D007147:Immunoglobulin Light Chains / Q000032:analysis; D007158:Immunologic Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000276:immunology; D011108:Polymers; D011817:Rabbits
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007147:Immunoglobulin Light Chains; D010455:Peptides; D011108:Polymers; C020749:GAT