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.
HirschfeldT. Optical microscopic observation of single small molecules. Appl Opt1976; 15: 296–7.
2.
EkekeGIExleyD.The assay of steroids by Fluoroimmunoassay. In: PalSB, ed. Enzyme-Labelled Immunoassay of Hormones and Drugs. Berlin: Walter de Gruyter, 1978.
3.
DiamandisEPMortonRCRichsteinEKhosraviM. Multiple fluorescence labelling with europium chelators. Application to time-resolved fluoroimmunoassays. Anal Chem1989; 61: 48–53.
4.
CanfiABaileyMPRocksBF. Multiple labelling of immunoglobulin G, albumin and testosterone with a fluorescent terbium chelate for fluorescence immunoassays. Analyst1989; 114: 1407–11.
5.
HassanMLandonJSmithDS. Multi-fluorescein-substituted polymers as potential labels in fluoroimmunoassay. FEBS Lett1979; 103: 339–41.
6.
RocksBFBertramVMRBaileyMPRileyCThomBT. Detection of antibodies to the human immunodeficiency virus by a rapid fluorescence-labelled immunosorbent assay. Ann Clin Biochem1988; 25: 522–9.
7.
PourfarzannehMWhiteGWLandonJSmithDS. Cortisol directly determined in serum by fluoroimmunoassay with magnetisable solid phase. Clin Chem1980; 26: 730–3.
8.
CarlssonJDrevinHAxenR. Protein thiolation and reversible protein-protein conjugation. Biochem J1978; 173: 723–37.
9.
CarlssonJDrevinHAxenR.Reagens zur Verwendung in immunochemischen Untersuchungsmethoden. German patent DE 28 08 476 A1. 1978.
