ایمونواسی ترکیبی برای تجزیه و تحلیل حساسیت بالای آفلاتوکسین M1 در شیر

An ultra-sensitive sandwich ELISA was developed for detection of AFM1 in milk. The assay involved the immobilization of rat monoclonal antibody of AFM1 in 384 microtiter plate to capture AFM1 antigen. This was detected by tracer secondary rabbit poly-clonal antibody labelled with horseradish peroxidase upon addition of a luminol-based substrate. Milk samples with different fat percentage were analyzed after pre-treatment. Linear range of AFM1 detection 250–6.25 pg/mL was achieved in 3% fat milk. The miniaturised assay (10 μL) enabled ultra trace analysis of AFM1 in milk with much improved lower limit of detection at 0.005 pg/mL. A sensitive magnetic nanoparticles (MNPs) based ELISA was also developed and coupled with micro plate ELISA for analysis in milk. The hybrid-assay, by coupling the 1°Ab immobilized MNPs column with microwell plate assay enabled simultaneous measurement of low (0.5 pg/mL) and high AFM1 contamination (200 pg/mL). The most promising feature of this MNPs-ELISA is the small column size, high capture efficiency and lower cost over other reported materials. The proposed assay can be deployed for simultaneous analysis and monitoring of AFM1 in milk.

Aflatoxins are highly toxic mycotoxins produced by Aspergillus species in a wide range of food and animal feedstuffs stored under temperature and humidity conditions favorable to mold growth. When aflatoxin B1 contaminated feed is ingested by cattle, it is transformed into its hydroxylated product, AFM1, which is then secreted in the milk. AFM1 is known for its hepatotoxic and carcinogenic effects. The toxic and carcinogenic effects of AFM1 recently lead WHO-IARC to change its classification from group 2 to group 1 ( IARC, vol. 82, 2002). AFM1 is relatively stable during milk pasteurization, storage as well as during the preparation of various dairy products ( Codex Committee, 2001 and Badea et al., 2004). To date, aflatoxins are regulated in many countries worldwide. Due to the fact that milk intake in infants is high and when young they are vulnerable to toxins, the European Community legislation imposes maximum permissible levels AFM1 of 50 ng/L in milk and 25 ng/L for infant formulae ( Henry et al., 2001). To minimize the occurrence of AFM1, it is essential to trace the sources of contamination using rapid, selective, sensitive and cost effective assays. Several methods for AFM1 determination have been developed. High-performance liquid chromatography (HPLC) ( AOAC Official Method, 2000.08), thin layer chromatography (TLC) ( Kamkar, 2006) and enzyme-linked immunosorbent assays (ELISA) ( Rastogi et al., 2004) are mainly used in routine analysis. For an effective screening and monitoring of AFM1 in foodstuffs such as milk at ultra low level, analytical methods combining simplicity with high detectability and analytical throughput are required. This can be achieved by means of immunological methods in conjunction with a highly sensitive detection of the label ( Magliulo et al., 2005). HPLC, TLC techniques require extensive sample preparation steps and well-trained personnel. Moreover, the reagents and instrumentation used are expensive ( Thirumala-Devi et al., 2002). Immunochemical techniques are becoming very popular for mycotoxins analysis with many literatures reporting the use of a commercially developed ELISA ( Thirumala-Devi et al., 2002, Lopez et al., 2003, Rodriguez et al., 2003 and Rastogi et al., 2004). ELISA is not only suitable tool for quick and sensitive analysis with high sample throughput, but also cost-effective and requires only a small sample volume for analysis ( Pei et al., 2009 and Parker and Tothill, 2009). Among the established ELISA techniques, sandwich-type immunoassay is an effective bioassay due to the high specificity and sensitivity ( Knopp, 2006). Enzyme labels have experienced widespread popularity since their first use in 1971 in an ELISA (Van Weeman and Schuurs, 1971). Enzyme labels are not consumed, and their reactions can be initiated and stopped. Furthermore, enzymes amplify the signal because an enzyme can produce many detectable molecules, up to 107 molecules of substrate per minute per enzyme molecule, by its catalysis of a substrate product reaction. They can be used in both homogeneous and heterogeneous immunoassays. Enzymes are the most commonly used labels as they can produce colored, fluorescent, luminescent, and electroactive compounds enabling detection by a variety of techniques (Gracia et al., 2005). Enzyme labels detected by chemiluminescent (CL) substrates, such as the luminol (5-aminophthalyhydrazide)/peroxide/enhancer system for horseradish peroxidase (HRP) or dioxetane-based substrates for alkaline phosphatase represent the most sensitive detection system in immunoassay development. CL compounds produce light in response to chemical reactions and as labels in immunoassay, they can be more sensitive than radio labelled and fluorescent forms (Krick and Wild, 2001). In addition, the CL signal detection can be performed immediately after substrate addition, thus shortening the overall analytical procedure when compared with conventional colorimetric assays (Magliulo et al., 2005). Gracia et al. (2005) have reported that luminol could be used as an enzyme substrate for HRP that yield high sensitivity. Owing to their high surface area, nano-materials can facilitate miniaturization and thereby provide enhanced number of binding sites. MNPs have special relevance in bio-analytical chemistry because of their larger surface to volume ratio, enhanced antibody–antigen kinetics, lower mass transfer resistance and easy separation of immobilized bio-molecules from reaction mixture using magnetic field (Yu, 1998). Recently, widespread use of MNPs in biosensors has been witnessed through a flurry of literature (Cheng et al., 2005, Nikitin et al., 2007 and Ravindranath et al., 2009). Various antibody coupling strategies using nanoparticles have also been reported (Wang et al., 2006, Radoi et al., 2008, Wang and Gan, 2009 and Ahirwal and Mitra, 2010). The covalent binding of antibody through self assembled monolayer is more suitable than the other conventional methods like physical adsorption and polymer entrapment (Liu et al., 2006 and Shankaran and Miura, 2007). The advantages of using covalent binding over physical adsorption to anchor antibodies and other proteins to a substrate surface are well documented in the literature (Ivanova et al., 2006). Despite MNPs prevalent applicability in biosensor field, reports on MNPs being deployed for analysis of AFM1 have been scarce. In one such testimony, reported by Radoi et al. (2008), AFM1 detection was done using MNPs coated with protein G by physical adsorption. In the present work, we report a novel approach where a highly sensitive microplate sandwich ELISA was developed and integrated with MNPs which could detect ultra trace amount of AFM1 in milk. The functionalized MNPs were used as an affinity capture column wherein antibodies immobilized on their surface could capture AFM1 from milk sample. To circumvent interference attributed to whey proteins and fat, the effect of pre-treatment of milk sample with trichloro acetic acid (TCA) followed by centrifugation and filtration was investigated to assess the performance of integrated ELISA. The miniaturised assay was used for high throughput analysis of AFM1 which showed an astoundingly low detection limit 0.005 pg/mL as against other reported ELISA (Thirumala-Devi et al., 2002, Rastogi et al., 2004 and Magliulo et al., 2005). Moreover the assay was validated by performing recovery studies in certified reference material for AFM1 (ERM-BD 282). The proposed assay further demonstrated the use of functionalized MNPs as affinity capture material through a miniaturised column for AFM1. This gave better efficiency of capture and cost effectiveness when compared to materials like protein A and protein G (Fuentes et al., 2005 and Radoi et al., 2008) in affinity column.

In the present work, a fast, reliable and ultra sensitive sandwich ELISA was developed for detection of AFM1 in milk. The miniaturised assay (assay volume 10 μL) enabled ultra trace analysis of AFM1 in milk with much improved lower limit of detection at 0.005 pg/mL. The total assay time to detect AFM1 by sandwich ELISA was approximately 4–5 h. Moreover, a sensitive MNPs-ELISA was also developed and coupled with micro plate ELISA for analysis in milk. The hybrid system by coupling the 1°Ab immobilized MNPs column with microwell plate assay enabled simultaneous measurement of low (0.5 pg/mL) and high AFM1 contamination (200 pg/mL). The most promising feature of this MNPs-ELISA is the small size, high capture efficiency and lower cost over other reported materials. It can be further extended for separation of AFM1 from the contaminated milk.