Dye-protein interactions : protein staining and dye-IgY, dye-dextran-IgY complexes for antigen detection.

Abstract

In order to develop a cheaper alternative to the conventional enzyme-linked immunosorbent assay system, application of dye molecules as labels in immunoassay was investigated in this study. This chromogenic dye-antibody conjugate could be used in colourimetric immunodetection diagnostic assays that could be used in a rural African setting. The chemistry of the interaction between twenty-six dyes of anionic, cationic and ligand dye classes with IgY and other proteins were studied for protein detection and conjugation to antibodies. Out of the twenty-six dyes studied, Direct Red 81 proved to be a good protein stain on nitrocellulose and polyacrylamide gels with comparable sensitivity to Coomassie Blue R 250. Direct Red stained proteins faster (< 5 min) than Coomassie Blue R 250 in polyacrylamide gels. Aurintricarboxylic Acid, Ethyl Red and Gallocyanine with carboxylic acid and/or hydroxyl functional groups were selected, activated with carbonyldiimidazole (CDI) to form amine reactive-imidazole intermediates and conjugated to anti-rabbit albumin IgY. Gallocyanine gave the best molar coupling ratio with IgY (76:1 dye:IgY). The dye-antibody conjugates were used to detect rabbit albumin on nitrocellulose. Aurintricarboxylic Acid-IgY and Gallocyanine-IgY detected 50 ng of rabbit albumin on nitrocellulose, which was 10 fold less sensitive than HRPO-IgY conjugate. Cross-linking of the antibodies by the dyes compromised the immunoreactivity of the Aurintricarboxylic Acid-IgY and Gallocyanine-IgY conjugates. The immunoreactivity of Ethyl Red-IgY was not compromised. Anti-rabbit albumin IgY was conjugated to derivatized dextran as an alternative immunoassay reagent and used to detect rabbit albumin on nitrocellulose by staining the polysaccharide (dextran) in the immune complex with PAS reagent. IgY-dextran complex was able to detect 25 ng of rabbit albumin on nitrocellulose, but PAS staining resulted in high background staining of the nitrocellulose membrane. Dextran-antibody conjugates may have better potential as immunodetecting reagent than dye-IgY conjugates, if a more sensitive and specific method of detecting the dextran in the Ag:Ab-dextran immune complex is developed.