Antibodies and immunoassays

As a glycoprotein hormone, human chorionic gonadotropin (hCG) is not a sing le molecular entity. This term comprises not only the bioactive heterodimer hCG but also an array of molecular protein backbone and glycosylation vari ants, such as its free beta (hCG beta) and alpha (hCG alpha) subunits and c lipped, cleaved, terminally differently siatylated, and overglycosylated fo rms. This heterogeneity places great demands on selective detection systems for hCG-derived molecules. Measurement of hCG and/or its derivatives is hi ghly dependent on the selection of target molecules and the natural variabi lity of hCG in the specimens analyzed. Monoclonal antibody (mAb)-based immu noassays are still the state-of-the-art technique for both clinical and res earch applications but a major problem is the different extents of recognit ion of hCG variants by mAbs used in different immunoassays. On the whole, c onstruction of sandwich-type assays obviously must take into consideration mAb characteristics, such as main and fine specificities, crossreactivities , epitope locations and compatibilities, overlap and overhang in specificit ies (pairs of mAbs), and, finally, overspecificity. Consequences of overhan g and overlap in antigen recognition of coating and detection mAb specifici ties are nondesirable assay cross-reactions and competitive interference by antigenic variants,The general agreement on the most favorable assay desig n is contrasted by the variety of isotopic and nonisotopic detection system s in current use. The immunoenzymometric assay (IEMA) technique is hampered by a relatively small measuring range and limited sensitivity, fly measuri ng substrate absorption values off the absorption maximum, the measuring ra nge of any IEMA can be extended significantly, as shown tar 3,3',5,5'-tetra methylbenzidine (TMB), without jeopardizing assay characteristics. Sensitiv ity of the IEMA can be enhanced by modifying the horseradish peroxidase (HR PO) 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 detec tion and the high specific labeling of the detection mAb which on a molar b asis can be up to I-fold and 15-fold higher compared with I-125 and HRPO, r espectively, (C) 2000 Academic Press.