Preparation of compound-specific and group-specific antibodies to 7-methylguanine and related 7-alkylguanines and their use in immunoaffinity purification.
The preparation and characteristics of compound-specific and group-specific antibodies against 7-alkylguanines (7-alkGua) are described. A compound-specific antibody against 7-methylguanine was prepared using a hapten bound to carrier protein through the N2 position. In a competitive enzyme-linked immunosorbent assay (ELISA) 7-methylguanine (7-MeGua) showed 50% inhibition (I50%) at 10 pmol/well at room temperature, but the inhibition was found to be 40 times better at 4 degrees C (I50% at 250 fmol/well). When the antibody was bound to protein A-Sepharose CL4B 7-MeGua was retained in immunoaffinity columns. A group-specific antibody to 7-alkGua was prepared using 7-(2-carboxyethyl)guanine (7-CEGua) bound to carrier protein via the carboxyl group. In a competitive ELISA, this antibody cross-reacted well with 7-CEGua, 7-ethylguanine (7-EtGua), 7-(2-hydroxyethyl)guanine (7-HOEtGua) and 7-(2',3'-dihydroxy)-propylguanine (7-DHPGua) and some inhibition was seen with 7-MeGua. Immunoaffinity columns prepared from this antibody retained a number of 7-alkGua of diverse structure. 7-EtGua in calf thymus DNA treated with diethylsulphate and ethylnitrosourea was isolated by immunoaffinity purification and quantified by HPLC-fluorescence. These results illustrate the potential of immunoaffinity purification for both individual DNA adducts and groups of adducts.
Durand
M J
MJ
International Agency for Research on Cancer, Lyon, France.
Shuker
D E
DE
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation / Q000276:immunology; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004247:DNA / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D004563:Electrochemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D008401:Gas Chromatography-Mass Spectrometry; D006147:Guanine / Q000031:analogs & derivatives* / Q000276:immunology*; D006241:Haptens / Q000737:chemistry; D011817:Rabbits
D000477:Alkylating Agents; D000906:Antibodies; D006241:Haptens; C042808:7-(2-carboxyethyl)guanine; D006147:Guanine; C008450:7-methylguanine; D004247:DNA