Quantitative analysis of aflatoxin-albumin adducts

Abstract

Quantitative exposure assessments made using biologically relevant markers will facilitate epidemiological studies of risk from environmental carcinogens. Blood proteins are readily accessible macromolecules that have been shown to be targets for activated chemical carcinogens. Serum albumin is quantitatively the most abundant target for aflatoxin B1 and the measurement of aflatoxin-serum albumin adducts has been used to detect exposed individuals. The goal of these experiments was to devise an analytical procedure that would increase the overall recovery of aflatoxin adducts in serum albumin, and thereby improve the accuracy of exposure monitoring. The method developed consisted of the following procedures. Proteins were precipitated from serum (< or = 100 microliters) with 80% ammonium sulfate, with incubation at 4 degrees C for 2 h. Following dialysis against phosphate-buffered saline (pH 7.0 for 3 h at 4 degrees C), the proteins were digested with protease (Pronase) (1:4.1 w/w enzyme:protein) for 15 h at 37 degrees C with shaking. Enzyme and other undigested proteins were precipitated with acetone (1:2 v/v, 40 min, 4 degrees C). After evaporation of the acetone under vacuum, levels of aflatoxin B1-albumin adducts were determined by radioimmunoassay carried out on 300 microliters fractions. This procedure obviated the isolation of albumin prior to analysis and reduced interference in the radioimmunoassay. High recoveries of aflatoxin B1 adducts were achieved together with a low limit of detection. The applicability of the procedure in epidemiological studies of human aflatoxin exposure was illustrated by results of analysis of aflatoxin-albumin adducts in serum samples from residents of Chongming Island, People's Republic of China.

Human Aflatoxin Albumin Adducts Quantitatively Compared by ELISA, HPLC with Fluorescence Detection, and HPLC with Isotope Dilution Mass Spectrometry

“…[5][6][7][8][9]. Each of these methods has inherent strengths and weaknesses that affect sensitivity and sample throughput.…”

Quantitative Comparison of Aflatoxin B1 Serum Albumin Adducts in Humans by Isotope Dilution Mass Spectrometry and ELISA

“…This adduct has been measured by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection and more recently by isotope dilution mass spectrometry (IDMS; refs. 10,[21][22][23][24]. To date, the high-throughput, relatively inexpensive equipment needs and robust performance of the ELISA have made it the method of choice.…”

Development of Aflatoxin B 1 -Lysine Adduct Monoclonal Antibody for Human Exposure Studies

“…To our knowledge, this is the first report of the development of a specific monoclonal antibody for AFB-lysine adduct. Previous studies either used a polyclonal antiserum-based ELISA (5, 33) or a monoclonal antibody 2B11-based RIA (8,26,30) to detect AFB-albumin adducts in large quantities of human serum samples collected in epidemiological studies. The availability of IIA4B3 will improve the specificity and sensitivity for AFBlysine adduct measurements in future human studies.…”

“…Its ability to recognize AFB-lysine adduct prompted the earlier development of an RIA-based method to measure total AFB-albumin adducts in human serum samples (8,26,30). In this study, we compared the affinities and specificities of monoclonal antibodies IIA4B3 and 2B11 for recognizing AFB and its metabolites and adducts.…”

“…Data from human exposure studies have also demonstrated that the excretion of urinary AFB-N 7 -Gua and the formation of AFB-albumin adducts are highly correlated (13). Three major analytical techniques are currently available for measuring AFB-albumin adduct levels in human blood: enzyme-linked immunosorbent assay (ELISA) (5, 33, 35), radioimmunoassay (RIA) (8,26,30), and immunoaffinity chromatography followed by high-performance liquid chromatography (HPLC) with fluorescence detection (24,30,33). All of these methods are antibody-based assays; therefore, the results of each assay are influenced by the specificity and the sensitivity of the antibodies used.…”