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A sandwich ELISA for measuring benzo[a]pyrene-albumin adducts in human plasma.

ABSTRACT: Exposure to polycyclic aromatic hydrocarbons has often been quantified via DNA or human serum albumin (HSA) adducts of the carcinogenic metabolite benzo[a]pyrene diol epoxide (BPDE). We previously reported a sandwich ELISA, using 8E11 as capture antibody and anti-HSA as detection antibody, that detected intact BPDE adducts in HSA isolated from plasma. After confirming that BPDE binds to HSA at His146 and Lys195, we modified the ELISA to measure intact BPDE-HSA directly in human plasma. To adjust for interference due to nonspecifically bound HSA on well surfaces and to cross-reactivity of the antibodies, the ELISA employs paired wells with and without addition of BPDE tetrols to deactivate 8E11. By performing assays in quadruplicate, a series of sample-specific adjustments and screening steps are used to reduce measurement errors that are a consequence of detecting low BPDE-HSA concentrations in the general population. ELISA measurements of BPDE-HSA in plasma from smoking and nonsmoking subjects (range 0.280-2.88 ng BPDE-HSA/mg HSA) and from highway workers with and without exposure to asphalt emissions (range 0.346-13.9 ng BPDE-HSA/mg HSA) detected differences in BPDE-HSA levels in the a priori expected directions.

SUBMITTER: Chung MK

PROVIDER: S-EPMC6354764 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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A sandwich ELISA for measuring benzo[a]pyrene-albumin adducts in human plasma.

Chung Ming Kei MK Regazzoni Luca L McClean Michael M Herrick Robert R Rappaport Stephen M SM

Analytical biochemistry 20130116 2

Exposure to polycyclic aromatic hydrocarbons has often been quantified via DNA or human serum albumin (HSA) adducts of the carcinogenic metabolite benzo[a]pyrene diol epoxide (BPDE). We previously reported a sandwich ELISA, using 8E11 as capture antibody and anti-HSA as detection antibody, that detected intact BPDE adducts in HSA isolated from plasma. After confirming that BPDE binds to HSA at His146 and Lys195, we modified the ELISA to measure intact BPDE-HSA directly in human plasma. To adjust ...[more]

PMID: 23333225

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Project description:Electrophilic diol epoxide metabolites are involved in the carcinogenicity of benzo[a]pyrene, one of the widely studied polycyclic aromatic hydrocarbons (PAHs). The exposure of humans to this PAH can be assessed by measuring stable blood protein adducts, such as to histidine and lysine in serum albumin, from their reactive metabolites. In this respect, measurement of the adducts originating from the genotoxic (+)-anti-benzo[a]pyrene diol epoxide is of interest. However, these are difficult to measure at such low levels as are expected in humans generally exposed to benzo[a]pyrene from air pollution and the diet. The analytical methods detecting PAH-biomarkers still suffer from low selectivity and/or detectability to enable generation of data for calculation of in vivo doses of specific stereoisomers, for evaluation of risk factors and assessing risk from exposures to PAH. Here, we suggest an analytical methodology based on high-pressure liquid chromatography (HPLC) coupled to high-resolution tandem mass spectrometry (MS) to lower the detection limits as well as to increase the selectivity with improvements in both chromatographic separation and mass determination. Method development was performed using serum albumin alkylated in vitro by benzo[a]pyrene diol epoxide isomers. The (+)-anti-benzo[a]pyrene diol epoxide adducts could be chromatographically resolved by using an HPLC column with a pentafluorophenyl stationary phase. Interferences were further diminished by the high mass accuracy and resolving power of Orbitrap MS. The achieved method detection limit for the (+)-anti-benzo[a]pyrene diol epoxide adduct to histidine was approximately 4 amol/mg serum albumin. This adduct as well as the adducts to histidine from (-)-anti- and (+/-)-syn-benzo[a]pyrene diol epoxide were quantified in the samples from benzo[a]pyrene-exposed mice. Corresponding adducts to lysine were also quantified. In human serum albumin, the anti-benzo[a]pyrene diol epoxide adducts to histidine were detected in only two out of twelve samples and at a level of approximately 0.1 fmol/mg.

Project description:BackgroundWe describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H alpha2beta2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-alpha subunit antibody magnetic beads and an anti-beta subunit antibody binds to the PDI/beta subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.ResultsWe applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.ConclusionsHere we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.

Dataset Information

A sandwich ELISA for measuring benzo[a]pyrene-albumin adducts in human plasma.

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A sandwich ELISA for measuring benzo[a]pyrene-albumin adducts in human plasma.

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