A Rapid Aflatoxin B 1 ELISA: Development and Validation with Reduced Matrix Effects for Peanuts, Corn, Pistachio, and Soybeans NANJU A. LEE,* ,† SHUO WANG, †,§ ROBIN D. ALLAN, ‡ AND IVAN R. KENNEDY † Faculty of Agriculture, Food and Natural Resources and Department of Pharmacology, University of Sydney, NSW, 2006 Australia Among the competitive ELISAs for aflatoxins that have been described, few have been adequately validated for reduced matrix effects. Using an aflatoxin B 1 (AFB 1 )-specific polyclonal antibody (produced from AFB 1 -oxime conjugated to bovine serum albumin (BSA)) and AFB 1 - and AFB 2 -enzyme conjugates, four direct competitive ELISAs based on 96-microwell plates (two standard assays and two rapid assays) were developed, paying special attention to producing a robust assay relatively free of interferences for a range of agricultural products. The antibody was AFB 1 -specific, detecting only AFB 1 in a mixture of four aflatoxins (AFB 1 , AFB 2 , AFG 1 , and AFG 2 ), but showed significant cross- reaction with AFG 1 (57-61%) when an individual compound was tested. Standard assays (long assays) exhibited higher sensitivities than rapid assays (short assays) with IC 50 values of 12 ( 1.5 and 9 ( 1.5 µg/kg in sample (with 1 in 5 dilution of sample extract) for AFB 1 and AFB 2 -enzyme conjugates, respectively. These assays have narrower detection ranges (7.1-55.5 µg/kg in sample) and required dilution of sample extracts to overcome solvent and matrix interferences, making these assays less ideal as analytical methods. Rapid assays exhibited IC 50 values of 21.6 ( 2.7 and 12 µg/kg in sample for AFB 1 - and AFB 2 -enzyme conjugates, respectively. These assays have ideally broader detection ranges (4.2-99.9 µg/kg in sample) and showed no methanol effects up to 80% with significantly reduced matrix interferences as a result of the shorter incubation times and increasing the amounts of enzyme conjugate used. Therefore, the rapid assays were formatted to perform without a need for extract dilution. The rapid assays can be completed within 15 min, potentially suitable for receival bays where quick decision-making to segregate low and high contamination is critical. Further validation using the rapid assay with AFB 1 -enzyme conjugate indicated relatively good recoveries of AFB 1 spiked in corn, peanuts, pistachio, and soybeans, which were free from significant matrix effects. It can be concluded that this rapid assay would be suitable for monitoring aflatoxin AFB 1 at current legal maximum residue limits of 10 µg/kg in food such as corn, peanuts, pistachio, and soybeans. KEYWORDS: Aflatoxin B1; ELISA; food; antibodies; carcinogen; monitoring; food safety INTRODUCTION Aflatoxins are toxic metabolites produced by fungi, mainly Aspergillus flaVus and A. parasiticus. They are listed as group I carcinogens by the International Agency for Research on Cancer (IARC), primarily affecting liver (1). The LD 50 of aflatoxins can be as low as 0.5 mg/kg body weight (1), which is significantly more toxic than most other known carcinogens. For these reasons, the presence of aflatoxins in food and animal feeds is potentially hazardous to the health of both humans and animals. Aflatoxin B 1 (AFB 1 ) has been shown to induce mutation at codon 249 in the tumor suppressor gene p53, which occurs in most heptatocarcinomas (2). Although some reports suggested that hepatocarcinogenesis in humans does not directly associate with aflatoxins (3, 4), the high incidence of liver cancer in South Africa (5), South-East Asia (5), Korea (6), Taiwan (7), and China (8) are still suspected to be linked to a combination of high dietary exposure to aflatoxins and hepatitis B viral infection. Furthermore, aflatoxin contamination affects the economic values of the crops as well as reduced efficiency of animal production, resulting in higher costs incurred by all sectors from production to consumption. The tolerance levels currently set by the regulatory bodies worldwide are typically 0.05 µg/kg for AFM 1 in milk, 10 µg/kg for AFB 1 and 20 µg/kg * To whom correspondence should be addressed. Tel.: +61 2 9351 8710. Fax: +61 2 9351 5108. E-mail: a.lee@acss.usyd.edu.au. † Faculty of Agriculture, Food and Natural Resources. ‡ Department of Pharmacology. § Current address: Faculty of Food Science and Bioengineering, Tianjin University of Science and Technology, Tianjin 300222, People’s Republic of China. 2746 J. Agric. Food Chem. 2004, 52, 2746-2755 10.1021/jf0354038 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/20/2004