Use of synthetic peptides as tracer antigens in fluorescence polarization immunoassays of high molecular weight analytes

Abstract

This paper describes a homogeneous immunoassay based on fluorescence polarization that enables subnanomolar detection of high molecular weight analytes. A monoclonal antibody (Mab) to human chorionic gonadotrophin (hCG) was screened against a panel of 221 synthetic peptides using the method of Geysen et al. (Geysen, H. M.; et al. J. Immunol. Methods 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-terminus of the hCG beta chain, bound to the Mab with high affinity. It was labeled 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 an antigen-binding affinity (Ka) of 1.5 x 10(7) M-1 at 6 degrees C. Its fluorescence intensity was enhanced by approximately 20% upon binding as a result of a prolonged excited-state lifetime. In a typical embodiment, 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 literature. This is mainly attributed to the large difference in hydrodynamic volume between the tracer and the antibody, which resulted in large changes in polarization of the peptide tracer upon binding. Issues related to sensitivity, specificity, and reversibility were also investigated. This method is believed to be of significant importance to rapid and economical measurements of high molecular weight antigens of clinical interest.