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Methods. 2000 May;21(1):41-50.

Antibodies and immunoassays.

Author information

1
Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria.

Abstract

As a glycoprotein hormone, human chorionic gonadotropin (hCG) is not a single molecular entity. This term comprises not only the bioactive heterodimer hCG but also an array of molecular protein backbone and glycosylation variants, such as its free beta (hCGbeta) and alpha (hCGalpha) subunits and clipped, cleaved, terminally differently sialylated, and overglycosylated forms. This heterogeneity places great demands on selective detection systems for hCG-derived molecules. Measurement of hCG and/or its derivatives is highly dependent on the selection of target molecules and the natural variability of hCG in the specimens analyzed. Monoclonal antibody (mAb)-based immunoassays are still the state-of-the-art technique for both clinical and research applications but a major problem is the different extents of recognition of hCG variants by mAbs used in different immunoassays. On the whole, construction of sandwich-type assays obviously must take into consideration mAb characteristics, such as main and fine specificities, cross-reactivities, epitope locations and compatibilities, overlap and overhang in specificities (pairs of mAbs), and, finally, overspecificity. Consequences of overhang and overlap in antigen recognition of coating and detection mAb specificities are nondesirable assay cross-reactions and competitive interference by antigenic variants. The general agreement on the most favorable assay design is contrasted by the variety of isotopic and nonisotopic detection systems in current use. The immunoenzymometric assay (IEMA) technique is hampered by a relatively small measuring range and limited sensitivity. By measuring substrate absorption values off the absorption maximum, the measuring range of any IEMA can be extended significantly, as shown for 3,3',5, 5'-tetramethylbenzidine (TMB), without jeopardizing assay characteristics. Sensitivity of the IEMA can be enhanced by modifying the horseradish peroxidase (HRPO) labeling technique by using highly purified mAb preparations and higher-input HRPO/mAb ratios. We have also compared the assay characteristics of time-resolved fluoroimmunoassay (IFMA), IEMA, immunoradiometric assay (IRMA), and competitive radioimmunoassay (RIA) based on identical mAbs. Reasons for the observed superiority of the IFMA lie in its concept of signal detection and the high specific labeling of the detection mAb which on a molar basis can be up to 7-fold and 15-fold higher compared with (125)I and HRPO, respectively.

PMID:
10764605
DOI:
10.1006/meth.2000.0973
[Indexed for MEDLINE]

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