Dye-protein interactions : protein staining and dye-IgY, dye-dextran-IgY complexes for antigen detection.
Loading...
Date
2004
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
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.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.
Keywords
Immunoglobulins., Proteins., Antigens., Theses--Biochemistry., Stains and staining (Microscopy)