Characterization of Hapten-Protein Conjugates for Immunoassay of Polycyclic Aromatic Hydrocarbons(PAHs)

Abstract

Preparation and characterization of the hapten-protein conjugates are fundamental to developing environmental immunoassays. As a hapten, 1-pyrenebutyric acid(PBA) was conjugated to the carrier protein of bovine serum albumin(BSA) or ovalbumin(OVA) by active ester method. Infrared spectra(IR) showed that PBA-BSA and PBA-OVA conjugates were successfully prepared. The number of the haptens conjugated to the carrier protein was determined by ultraviolet spectra(UV) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS). The calculated average binding ratios of PBA/BSA and PBA/OVA were 18:1 and 10:1 by UV, and 31:1 and 22:1 by MALDI-TOF-MS, respectively. Although there was a discrepancy between the results determined by the two methods, both of them were useful for the characterization of the hapten-protein conjugates. The antibody was produced against the antigen of PBA-BSA, and the affinity was tested by the double agar diffusion method. The conjugates and the antibody could be used for developing a sensitive and selective immunoassay of polycyclic aromatic hydrocarbons(PAHs).