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Title: Development of an immunoassay to detect benzene adducts in hemoglobin

Abstract

The purpose of this project was to develop an immunoassay to detect the adducts formed in hemoglobin after exposure to benzene, which is known to cause bone marrow degeneration and acute myelogenous leukemia. The use of benzene-adduct detection as a biological monitoring method would permit measurement of low exposures and exposures sustained weeks earlier. The reactivity of hydroquinone, an important benzene metabolite, with blood proteins and amino acids was investigated in order to decide which antigens and analytes were likely to be suitable for immunoassay development. The second section determined the combination of benzene-metabolite and antigen need to produce an immunoassay with the requisite low detection limit and specificity. The immunoassays with the best performance were tested on hemoglobin from benzene-exposed mice. In vitro studies showed that hydroquinone efficiently formed adducts with erythrocyte membranes and hemoglobin but not with albumin. Adduction efficiency was greater in incubations using purified hemoglobin than whole blood. Cysteine accounted for 15 to 27% of the adducts formed by hydroquinone. The site of the other adducts were not identified although there was evidence that the hemoglobin heme was adducted. Adducts were found on only 1 of the 2 globin chains. Tryptic digestion of the globin failedmore » to associate the adducts with a specific peptide. Antigens made from hydroquinone-adducted hemoglobin but not hydroquinone-adducted cysteines coupled to carrier proteins effectively elicited adduct-specific antibodies. Interference due to reactivity to hemoglobin was controlled by using uniform quantities of hemoglobin in all wells. The mid-range of the best assays were approximately 12 pmoles HQ per well. Antibodies directed toward hemoglobin adducted with the benzene metabolites phenol, catechol and 1,2,4-trihydroxybenzene were also made. The performance of the anti-1,2,4-trihydroxybenzene were suitable for quantitative immunoassays.« less

Authors:
Publication Date:
Research Org.:
California Univ., Berkeley, CA (United States)
OSTI Identifier:
7026118
Alternate Identifier(s):
OSTI ID: 7026118
Resource Type:
Miscellaneous
Resource Relation:
Other Information: Thesis (Ph.D.)
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 59 BASIC BIOLOGICAL SCIENCES; ADDUCTS; DETECTION; BENZENE; BIOLOGICAL INDICATORS; ENZYME IMMUNOASSAY; FEASIBILITY STUDIES; HEMOGLOBIN; ANTIGEN-ANTIBODY REACTIONS; BONE MARROW; CYTOCHEMISTRY; LEUKEMIA; ANIMAL TISSUES; AROMATICS; BIOASSAY; BIOCHEMISTRY; BODY; CARBOXYLIC ACIDS; CHEMISTRY; DISEASES; GLOBINS; HEMATOPOIETIC SYSTEM; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; HYDROCARBONS; IMMUNE SYSTEM DISEASES; IMMUNOASSAY; NEOPLASMS; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; PIGMENTS; PORPHYRINS; PROTEINS; TISSUES 560300* -- Chemicals Metabolism & Toxicology; 550200 -- Biochemistry

Citation Formats

Grassman, J.A. Development of an immunoassay to detect benzene adducts in hemoglobin. United States: N. p., 1993. Web.
Grassman, J.A. Development of an immunoassay to detect benzene adducts in hemoglobin. United States.
Grassman, J.A. Fri . "Development of an immunoassay to detect benzene adducts in hemoglobin". United States. doi:.
@article{osti_7026118,
title = {Development of an immunoassay to detect benzene adducts in hemoglobin},
author = {Grassman, J.A.},
abstractNote = {The purpose of this project was to develop an immunoassay to detect the adducts formed in hemoglobin after exposure to benzene, which is known to cause bone marrow degeneration and acute myelogenous leukemia. The use of benzene-adduct detection as a biological monitoring method would permit measurement of low exposures and exposures sustained weeks earlier. The reactivity of hydroquinone, an important benzene metabolite, with blood proteins and amino acids was investigated in order to decide which antigens and analytes were likely to be suitable for immunoassay development. The second section determined the combination of benzene-metabolite and antigen need to produce an immunoassay with the requisite low detection limit and specificity. The immunoassays with the best performance were tested on hemoglobin from benzene-exposed mice. In vitro studies showed that hydroquinone efficiently formed adducts with erythrocyte membranes and hemoglobin but not with albumin. Adduction efficiency was greater in incubations using purified hemoglobin than whole blood. Cysteine accounted for 15 to 27% of the adducts formed by hydroquinone. The site of the other adducts were not identified although there was evidence that the hemoglobin heme was adducted. Adducts were found on only 1 of the 2 globin chains. Tryptic digestion of the globin failed to associate the adducts with a specific peptide. Antigens made from hydroquinone-adducted hemoglobin but not hydroquinone-adducted cysteines coupled to carrier proteins effectively elicited adduct-specific antibodies. Interference due to reactivity to hemoglobin was controlled by using uniform quantities of hemoglobin in all wells. The mid-range of the best assays were approximately 12 pmoles HQ per well. Antibodies directed toward hemoglobin adducted with the benzene metabolites phenol, catechol and 1,2,4-trihydroxybenzene were also made. The performance of the anti-1,2,4-trihydroxybenzene were suitable for quantitative immunoassays.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Fri Jan 01 00:00:00 EST 1993},
month = {Fri Jan 01 00:00:00 EST 1993}
}

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