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 determinedby ultraviolet spectra(UV) andmatrix-assistedlaser desorptionionization time-of-flightmass 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).