Immunochemical detection of adducts of sulfur mustard to DNA of calf thymus and human white blood cells
| Autor: | A. G. Scheffer, Robert A. Baan, Roos H. Mars-Groenendijk, G. P. Van Der Schans, Hendrik P. Benschop, A. Fidder |
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| Rok vydání: | 1994 |
| Předmět: |
Guanine
Thio Enzyme-Linked Immunosorbent Assay Thymus Gland In Vitro Techniques Toxicology Adduct chemistry.chemical_compound Mice Antibody Specificity Mustard Gas Leukocytes Animals Humans Antiserum Mice Inbred BALB C biology Immunochemistry Antibodies Monoclonal Sulfur mustard General Medicine DNA chemistry Biochemistry Hemocyanins biology.protein Cattle Female Hapten Haptens Keyhole limpet hemocyanin |
| Zdroj: | Chemical research in toxicology. 7(3) |
| ISSN: | 0893-228X |
| Popis: | As part of a program to develop methods for dosimetry of exposure to sulfur mustard, we developed immunochemical methods for the detection of the major adduct, N7-[2-[(hydroxyethyl)thio]ethyl]guanine (N7-HETE-Gua), formed after alkylation of DNA with sulfur mustard. After immunization of rabbits with calf thymus DNA treated with sulfur mustard, we obtained the antiserum W7/10 with a high specificity for DNA adducts of sulfur mustard. With this serum, a competitive enzyme-linked immunosorbent assay was developed in which sulfur mustard adducts to DNA could be detected with a minimum detectable amount of 1-5 fmol per well and a selectivity that allows detection of one N7-HETE-Gua among 5 x 10(6) unmodified nucleotides in single-stranded DNA. The complications that arise to isolate double-stranded DNA from biological samples and to make the DNA single-stranded without destruction of the sulfur mustard adducts result in about a 20-fold higher limit for adduct detection in DNA from human blood than in single-stranded DNA. Presently, adducts in white blood cells can be detected after exposure of human blood to sulfur mustard concentrations > or = 2 microM. We synthesized N7-HETE-GMP for use as a hapten to generate monoclonal antibodies against this adduct. After immunization of mice with this adduct coupled to the carrier protein keyhole limpet hemocyanin we obtained several hybridomas producing monoclonal antibodies that recognize N7-HETE-Gua, containing an intact imidazolium ring. The sensitivity of the competitive ELISA with the monoclonal antibodies was comparable to that of the assays performed with the rabbit antiserum.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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