The development of energy transfer immunoassay methods
The use of fluorescein and rhodamine as donor and acceptor fluorescent labels in the development of energy transfer immunoassay (ETIA) methods was evaluated by developing an assay for human serum albumin. The sensitivity of the assay was found to depend on (i) the degrees of fluorophore labelling of antibody and antigen, (ii) concentrations of labelled antibody and antigen, (iii) the fluorimeter spectral bandwidth, and (iv) whether the donor (fluorescein) was conjugated to the antigen or the antibody. These results, including those relating to the stability of the labelled immune reactants on storage, lead to the conclusion that fluorescein and rhodamine are far from ideal as donor and acceptor. Nevertheless, the application of the assay to the analysis of test serum samples gave results that compared favourably with those obtained by electroimmunoassay. Other potential donor and acceptor fluorescent labels were also investigated, viz. Ca) fluorescamine and fluorescein, (b) MDPF and fluorescein, (c) dansyl chloride and rhodamine, (d) quinacrine and fluorescein, and (e) quinacrine and rhodamine. Of these, only (a) and (b) were found to be suitable donor-acceptor pairs, and they were applied to the development of a number of assays for both low and high molecular-weight analytes. Comparative studies of fluorescamine and MDPF as donor fluorescent labels together with fluorescein as the acceptor label were performed by developing immunoassay methods for the determination of human serum transferrin in four serum samples including a blood sample from the victim of a road traffic accident. Results obtained were generally in good agreement with those found by the radial immunodiffusion method. An ETIA developed for nortriptyline and related tricyclic antidepressants was capable of detecting nanomolar concentrations of the drugs in pure solution and in spiked sera. Other ETIA's developed include a sandwich assay for the quantitation of human immunoglobulin A, and a direct assay for the determination of human immunoglobulin G. The Fluram as well as the MDPF enhancement phenomena were also studied in detail and applied successfully to the development of fluorescence enhancement immunoassays for nortriptyline, human serum transferrin and immunoglobulin G. Finally, the automation of an energy transfer immunoassay was successfully performed by using the principles of stopped-flow injection analysis with merging zones.