Immunologic methods for the detection of benzo[a]pyrene metabolites in urine

Abstract

Two monoclonal antibodies (10C10 and 4D5) have been developed from the spleen cells of Balb/c mice immunized with 6-aminobenzo[a]pyrene covalently coupled to bovine serum albumin. These antibodies have been used in an immunoassay for the detection of benzo[a]pyrene and its metabolites in mouse urine. The antibodies were characterized in terms of sensitivity and specificity by competitive enzyme-linked immunosorbent assay (ELISA). With both antibodies, 50% inhibition of antibody binding is at 4 pmol of BP. The antibodies also cross-react with a number of BP metabolites as well as with several other polycyclic aromatic hydrocarbons (PAHs) including pyrene, 1-aminopyrene, and 7,12-dimethylbenz[a]anthracene but with different sensitivities. These results suggest that this assay will detect multiple PAH metabolites in urine. To test the assay on biological samples, mice were treated with [3H]BP, and urine was collected and digested with beta-glucuronidase and aryl sulfatase. Several methods were used to isolate BP and its metabolites from the urine, including ethyl acetate extraction, Sep-pak C18 cartridge chromatography, XAD2 resin chromatography, and immunoaffinity chromatography with antibody 4D5. Analysis of the urine extracts with antibody 4D5 gave 50% inhibition at 12-15 pmol of metabolites. Thus, quantitation of metabolites in this sample by competitive ELISA against a standard curve of BP would have underestimated actual metabolite levels by about 70%. This assay will be applied to the analysis of urines from individuals with environmental or occupational exposure. Since humans are usually exposed to BP in complex mixtures of PAHs, multiple metabolites may be present in the urine, making absolute quantitation difficult. This assay should thus serve as a general indicator of exposure to this class of chemicals.

Gold Electrode Modification with Thiolated Hapten for the Design of Amperometric and Piezoelectric Immunosensors

“…The relative frequency decrease increase with the increased concentration of the antibodies is indicative of the speci®c binding of the antibodies to the corresponding hapten portion immobilized on the gold surface. The observed slightly greater response of mAb4D5 to the pyrene modi®ed QCM is consistent with the better sensitivity of pyrene for mAb4D5 than for mAb10c10 reported by Santella et al [27] However, the possibility of nonspeci®c binding of mIgG under similar concentration levels can not be discounted.. The detection limit for mAb10c10 was about 400 ppb.…”

“…The attachment has been described as the formation of a stable thiolate complex on gold due to the cleavage of disul®de bonds. The monoclonal antibodies 10c10 and 4D5 showed high sensitivity for a number of benzo[a]pyrene (BaP) metabolites as well as with several other polycyclic aromatic hydrocarbons (PAHs) including pyrene [27]. In the present study, we investigated the application of gold-coated QCM modi®ed with a terminal pyrene group for piezoelectric immunosensing of the antibodies.…”

Detection of polycyclic aromatic hydrocarbon metabolites by high-pressure liquid chromatography after purification on immunoaffinity columns in urine from occupationally exposed workers

“…Besides, various immunoassays have been used to quantitate urinary PAH metabolites (Gomes and Santella 1990;Herikstad et al 1993). Thus, by the acquisition of greater knowledge of compound-antibody speci®city it should be feasible to quantitate several PAH metabolites after puri®cation on immunoanity columns.…”

Development of an Enzyme-Linked Immunosorbent Assay for 1-Nitropyrene

“…During the last years, environmental immunoassays were developed mainly for pesticides. Immunochemical determination of PAHs have been reported for benzo(a)pyrene (15)(16)(17)(18)(19), pyrene (20), and nitrofluoranthenes (21). Immunochemical test kits for screening of PAH contamination in soil and/or water are also available commercially (PAH-RIS Test/EnSys; D Tech PAH Test Kit/Merck; EnviroGard PAH Test Kit/Millipore; PAH Test/Quantix).…”