Suchergebnisse

Abstract

Objective
PAHs are produced during the incomplete burning of organic materials. PAH sources include vehicle exhaust, tobacco smoke and waste incineration. Cancer is a significant endpoint of PAH exposure. Pyrene is a common component of PAH exposures and metabolism of pyrene leads to the excretion of 1-OHPyrG in urine. Measurement of 1-OHPyrG has been used to assess occupational and environmental exposure to PAHs. The production of Mabs to 1-OHPyrG will allow the development of novel PAH biomonitoring tests, facilitating the introduction of routine screening and safeguarding human health.


Methods
1-Pyrenebutyric acid was coupled to a carrier protein and a sheep Mab raised to 1-OHPyrG. A second Mab which binds the Mab-1-OHPyrG immune complex was also produced and a novel urinary non-competitive ELISA developed. Careful formulation of assay buffers has overcome the heterogeneous nature of urine. Assay performance was confirmed through binding studies with structurally similar metabolites, reproducibility testing and a comparative method study.


Results
A sensitive and specific ELISA (measuring range 20-500pg/ml) was used to detect elevated levels of 1-OHPyrG in urine. Occupational samples determined by ELISA and HPLC-fluorescence were in good agreement (R2=0.91, N=20). A test kit is now being transferred to manufacture.


Conclusions
The development of a novel ELISA for 1-OHPyrG has allowed the production of a sensitive and specific PAH biomonitoring test kit. Kits increase the accessibility of biomonitoring and facilitate the introduction of routine screening. Alternate assay formats are being investigated. A lateral flow "point of care" test will enable on-site testing and significantly increase the utility of biomonitoring.

Abstract

Objective
Anti 1-OHPyrG sheep Mabs have been raised, characterised and employed in a sensitive and specific ELISA. The development of a LF test will enable the manufacture of a simple, rapid and cost-effective point of use test. On-site screening will allow employers to immediately confirm safe working practice and significantly increase the efficacy of biomonitoring.


Methods
A quantitative LF test has been developed which employs an anti-1-OHPyrG sMab to capture the PAH metabolite. The captured metabolite is visualised through a second Mab raised against the sMab-1-OHPyrG immune complex. This sandwich assay provides a positive correlation between the assay signal and biomarker concentration. The LF test can determine biomarker concentrations in non-occupationally exposed individuals and performance has been determined in a comparative study.


Results
A specific LF immunoassay, with a measuring range of 20-500pg/ml of 1-OHPyrG, can detect increasing levels of PAH metabolite in urine due to lifestyle factors (e.g. smoking and consumption of chargrilled food) in samples from non-occupationally exposed individuals. The LF determined concentrations compared well to literature values. Urines determined by LF and ELISA were in excellent agreement (R2=97, N=8).


Conclusions
Antibodies enable the development of quantitative LF tests, providing "real-time" analysis to occupational health professionals. On-site testing allows the immediate confirmation of safe working practice and provides immediate reassurance to those involved in potentially hazardous activities. An 'app' to support LF testing is now under development and a survey of potential industry end-users has highlighted the desired features.