USE OF SYNTHETIC PEPTIDES AS TRACER ANTIGENS IN FLUORESCENCE POLARIZATION IMMUNOASSAYS OF HIGH-MOLECULAR-WEIGHT ANALYTES

This paper describes a homogeneous immunoassay based on fluorescence p olarization that enables subnanomolar detection of high molecular weig ht analytes. A monoclonal antibody (Mab) to human chorionic gonadotrop hin (hCG) was screened against a panel of 221 synthetic peptides using the method of Geysen et al. (Geysen, H. M.; et al. J. Immunol. Method s 1987, 102, 259-274. Geysen, H. M.; et al. J. Mol. Immunol. 1986, 23, 709-715). One of these peptides, which Was located near the C-terminu s of the hCG beta chain, bound to the Mab with high affinity. It was l abeled with tetramethylrhodamine (TMR) and used as the tracer antigen in a competitive fluorescence polarization immunoassay (FPIA) for hCG. The peptide-TMR conjugate binds specifically to the anti-hCG Mab with am antigen-binding affinity (K-a) of 1.5 x 10(7) M(-1) at 6 degrees C . Its fluorescence intensity was enhanced by similar to 20% upon bindi ng as a result of a prolonged excited-state lifetime. In a typical emb odiment, hCG was determined at a level of 1 x 10(-9) M (95% confidence limit)-a 100-fold improvement over similar systems reported in the li terature. This is mainly attributed to the large difference in hydrody namic volume between the tracer and the antibody, which resulted in la rge changes in polarization of the peptide tracer upon binding. Issues related to sensitivity, specificity, and reversibility were also inve stigated. This method is believed to be of significant importance to r apid and economical measurements of high molecular weight antigens of clinical interest.