Coupling dyes to chicken IgY antibodies for the development of immunodiagnostic tests.

Abstract

The aim of this study was to develop a highly simplified, sensitive and specific malarial diagnostic test at the lowest possible cost. Initial work and optimisation of procedures was achieved with chicken antibodies by covalently attaching commercially available dye to them. Chicken antibodies were easily isolated from egg yolk and dye is cheap, easily visible and requires no equipment for identification of results. A dipstick dye-immunoassay was developed with nitrocellulose as the capture phase. The dye-immunoassay is an alternative to the traditional enzyme linked immunosorbent assay (ELISA) technique, which employs the use of an enzyme-substrate reaction. Numerous dyes were investigated and included Reactive black 5, trypan blue, Cibacron Blue, Congo red, Acid-black 2, dianix blue, dianix red, para-nitroaniline and primulin. Most of these dyes have dark colours which are essential for good contrast on nitrocellulose and in a microtitre plate. Some dyes contain amino (NH2) groups, which are targeted in a covalent linking step and attached to the lysine residues on antibody molecules or to the carbohydrate groups on antibody molecules. Attachment of dye molecules to antibodies with glutaraldehyde was the chief coupling method explored and conditions were optimized in this study. Unbound dye was removed by dialysis. Reactive black 5 is sensitive down to 50 nanograms of antigen on nitrocellulose. A second covalent coupling method was investigated by means of attaching dye to the carbohydrate moieties on the antibody. Reactive black 5 was sensitive down to 50 nanograms of antigen. The carbohydrate method appears to be more sensitive than the glutaraldehyde method at lower antibody concentrations. Primulin, a yellow dye, was similarly investigated. This dye does not have a dark colour initially, but can be diazotized to change its colour to orange or purple. It also fluoresces under ultra-violet light. This dye was sensitive down to 500 nanograms of antigen with both the glutaraldehyde and carbohydrate coupling techniques. A dye-linked immunosorbent assay (D-LlSA) protocol for direct antigen detection has been developed whereby the dye-antibody solution (dianix blue dye) acts as the primary antibody and substrate respectively. Sensitivity levels compare with traditional ELISAs. Dianix blue is sensitive down to 25 nanograms of antigen in a microtitre plate. Unique protein staining abilities of the dyes used in this study were indicated by staining IgY in electrophoretic gels. Acid-black 2 indicated better protein staining abilities than that of Coomassie brilliant blue. Evidence shows that dye was successfully covalently attached to antibodies and that antigen detection is possible by visualising the dye developed spots. Although malarial antibodies were not used, all procedures with chicken antibodies were optimised. Highly simplified, sensitive and specific diagnostic tests were developed.