A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25–50？pg/mL. AFM1 as low as 1？pg/mL was detected by this method with assay volume 40？μL. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling. 1. Introduction Aflatoxins are highly toxic, mutagenic, carcinogenic, and teratogenic compounds contaminating a wide range of food commodities [1, 2]. The major naturally occurring aflatoxins, namely, AFB1, AFB2, AFG1, and AFG2, constitute a class of structurally related toxic fungal metabolites. They are extremely potent carcinogens and can have significant economic impacts, making them important targets for detection and quantification [3]. Commodities frequently contaminated by aflatoxins include cereals, nuts, dried fruits, spices, and pulses [4, 5]. When animals consume AFB1 contaminated foodstuffs, the toxin is metabolized in the liver and excreted as AFM1 via milk and urination [1]. Humans are exposed to the deleterious effects of aflatoxins either directly by eating contaminated grains or indirectly via animal products [6]. Evidence of hazardous human exposure to aflatoxins through various foods including dairy products has been shown by several investigators [1–3, 7]. Many analytical methods have been developed for estimation of aflatoxins in agricultural commodities. Among them optical and electrochemical transducers are most widely used for detection of aflatoxins. The optical detection method is regarded as one of the sensitive techniques for aflatoxin analysis. The optical signal is not influenced by electrical, magnetic, or ionic fields. Table 1 gives an account of some of the reported biosensors for analysis of AFM1 and AFB1. Table 1: Reported biosensing techniques for analysis of AFM1
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