Multiple releasable fluorescein labels for immunoassay: the principle illustrated by an immunoassay for antibodies to the human immunodeficiency virus.
Attempts to increase the sensitivity of fluorescein-based fluorescence immunoassays by using multiple labelling have generally been unsuccessful because of concentration quenching. We have labelled antibodies to human immunoglobulin G with multiple fluorescein fluorophores attached by means of a disulphide linkage: this linkage can be rapidly and easily broken by treatment with dithiothreitol, allowing fluorescein to be released from the antibody and measured in free solution. Application of this technique to a fluorescence labelled immunosorbent assay for antibodies to the human immunodeficiency virus gave an approximately 20-fold increase in signal compared with an equivalent assay using fluorescein isothiocyanate.
Bertram
V M
VM
Biochemistry Department, Royal Sussex County Hospital, Brighton, UK.
Bailey
M P
MP
Rocks
B F
BF
D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis*; D005452:Fluoresceins; D015200:Fluoroimmunoassay; D015483:HIV Antibodies / Q000032:analysis*; D015488:HIV Antigens / Q000032:analysis; D006801:Humans; D007074:Immunoglobulin G
D005452:Fluoresceins; D015483:HIV Antibodies; D015488:HIV Antigens; D007074:Immunoglobulin G; C027101:fluorescein thiocarbamylethylenediamine