 Determination of Aflatoxin B 1 –DNA Adduct in Rat Liver by Enzyme Immunoassay T. Vidyasagar, N. Sujatha and R. B. Sashidhar* Department of Biochemistry, University College of Science, Osmania University, Hyderabad-500 007, India A simple, rapid and highly sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B 1 (AFB 1 )–DNA adducts is reported. Polyclonal antibodies specific to the aflatoxin B 1 –N 7 –guanine adduct were produced using a novel synthetic antigen, bovine serum albumin (BSA)–guanine– AFB 1 . The antibodies were characterized by the Ouchterlony double diffusion technique and by antibody capture assay. The working range of the indirect competitive assay developed was between 0.45 and 330 ng of the analyte [calf thymus (CT)–DNA–AFB 1 ]. A 50% inhibition was attained at 15 ng of the analyte (CT–DNA–AFB 1 ). The antibody capture assay indicated that the antibody produced cross-reacted 100, 92 and 110% with BSA–guanine–AFB 1 , CT-DNA–AFB 1 and CT-DNA–formamidopyrimidine–AFB 1 , respectively. When free AFB 1 and guanine were used as competing analytes, the antibodies showed @5% and zero cross-reactivity at the 50% inhibition level. Spiking studies indicated a recovery in the range 96–97 and 74–78% when standard CT-DNA–AFB 1 was added to 10 mm phosphate buffer (pH 7.2) and control rat liver tissue, respectively. Rats exposed to a single oral dose of 1 mg kg 21 body mass of pure AFB 1 were used to validate the method. The AFB 1 –DNA adduct formed in the liver tissue after 48 h of exposure was determined using the ELISA method developed. The liver AFB 1 –DNA adduct ranged between 6.06 and 7.94 mg mg 21 DNA. The proposed method may find application in the biological monitoring of aflatoxin B 1 in molecular epidemiological studies to assess the dietary exposure of aflatoxins. Keywords: Aflatoxin B 1 ; calf thymus DNA–aflatoxin B 1 ; aflatoxin B 1 –N 7 –guanine; calf thymus DNA–formamidopyrimidine–aflatoxin B 1 ; aflatoxin B 1 –DNA; enzyme-linked immunosorbent assay Aflatoxins are potent hepatotoxic and hepatocarcinogenic compounds produced by Aspergillus flavus and Aspergillus parasiticus species. A variety of human foods such as cereals, millets and oil seeds are susceptible to these ubiquitous fungi, which infect and produce aflatoxins during growth, harvest, transport and storage. 1,2 Aflatoxin B 1 (AFB 1 ) is one of the most potent carcinogens and is classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC, Lyon, France). 2 Exposure to AFB 1 has been associated with an increased incidence of primary hepatocellular carcinoma (PHCC), which is the seventh most frequent cancer in the world and particularly in South-East Asia, China and Sub-Saharan Africa. 3–7 After gaining entry into the systemic system through the diet, AFB 1 is metabolically activated by liver microsomal enzymes (cytochrome P 450 -dependent enzymes) into a highly reactive electrophilic species, an 8,9-epoxide, which efficiently binds to nucleophilic sites of cellular macromolecules. 8–10 The carcino- genic 8,9-epoxide specifically binds at the N 7 -position of guanine in DNA or attacks the e-amino group of lysine in proteins. 8,9,11,12 In the recent past, biological monitoring of the AFB 1 –N 7 - guanine adduct has been used as a molecular dosimeter of exposure to AFB 1 in molecular epidemiological studies. 13–16 Various analytical methods have been developed for the detection and determination of the guanine–AFB 1 adduct from human and animal tissues, including chromatographic, im- munological and immunocytochemical methods. 15–18 How- ever, all these methods were based on antibodies which were produced to the AFB 1 , moiety. An AFB 1 -specific monoclonal antibody affinity chromatographic method coupled with high- performance liquid chromatography(HPLC) was successfully used to detect AFB 1 –N 7 -guanine from acid-hydrolysed DNA samples in human tissues after acute poisoning with AFB 1 . 17 Similarly, a multiple monoclonal antibody, specific to aflatoxin B 1 , has been used in affinity chromatography along with HPLC for the detection of AFB 1 –N 7 -guanine in rat urine. 16 An immunoanalytical method was also developed in which mono- clonal antibodies raised against AFB 1 –formamidopyrimidine was used to detect AFB 1 –formamidopyrimidine adducts in DNA samples from rat liver tissues. 15 A sensitive im- munocytochemical method for the quantification of AFB 1 bound to DNA in various rat tissues using AFB 1 -specific monoclonal antibodies has also been reported. 18 This paper reports an in vitro method to determine AFB 1 –N 7 - guanine in intact DNA by using AFB 1 –N 7 -guanine-specific polyclonal antibodies. An indirect competitive ELISA was developed to determine the AFB 1 –N 7 -guanine adduct in DNA extracted from liver tissues of rats exposed to a single oral dose of AFB 1 . Experimental Apparatus A DU-50 recording spectrophotometer (Beckman, Fullerton, CA, USA) was used for spectral analysis. An SLT-Spectra II microplate reader (Gr¨ odig, Salzburg, Austria) was used to measure the optical density. Polystyrene microtitre ELISA plates were purchased from Greiner (N¨ urtingen, Germany). Reagents AFB 1 , bovine serum albumin (BSA) (fatty acid free, RIA grade), dimethyl suberimidate, guanine, calf thymus (CT) DNA, Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), anti-rabbit immunoglobulin G (IgG) labelled with alkaline phosphatase raised in goat (whole molecule), gelatin, trinitrobenzenesulfonic acid (TNBS) and polyester silica gel G TLC plates (20 cm 3 20 cm; particle size 2–25 mm) were purchased from Sigma (St. Louis, MO, USA). m- Chloroperbenzoic acid (MCPBA) was purchased from Merck (Darmstadt, Germany). All other chemicals were of analytical- reagent grade. Analyst, June 1997, Vol. 122 (609–613) 609 Published on 01 January 1997. Downloaded on 10/06/2016 16:53:04. View Article Online / Journal Homepage / Table of Contents for this issue