Aflatoxins, antibodies

Plastic microdevices for high-throughput screening with MS detection were also prepared for detection of aflatoxins and barbiturates. These devices incorporated concentration techniques interfaced with electrospray ionization MS (ESI-MS) through capillaries [2], The microfluidic device for aflatoxin detection employed an affinity dialysis technique, in which a poly (vinylidene fluoride) (PVDF) membrane was incorporated in the microchip between two channels. Small molecules were dialyzed from the aflatoxin/antibody complexes, which were then analyzed by MS. A similar device was used for concentrating barbiturate/antibody complexes using an affinity ultrafiltration technique. A barbiturate solution was mixed with antibodies and then flowed into the device, where uncomplexed barbiturates were removed by filtration. The antibody complex was then dissociated and electrokinetically mobilized for MS analysis. In each case, the affinity preconcentration improved the sensitivity by at least one to two orders of magnitude over previously reported detection limits. 

Sophisticated and veiy sensitive methods have been developed in the food industry for detecting many other microbial toxins. For example, aflatoxin deteetion in seedstuffs and their oils is performed by solvent extraction, adsorption onto columns containing selective antibodies for them, and detected by exposure to ultraviolet light. 

Immunoaffinity chromatography is one of the most popular techniques of affinity derivatived method and it enables to produce ligands in case the ligand required is not available [7]. In this technique, stationary phase comprises of an antibody or antibody-related agent [1]. It is possible to isolate variable subtances using this technique due to high specifity of antibodies [1]. It is reported that immunoaffinity chromatography may be used for natural food contaminants such as aflatoxins, fumonisins and ochratoxins [11]. 

The assessment of DNA adducts may provide a sensitive indicatCH of previous exposure. The enzyme-linked immunosorbent assay (ELISA) has a lower limit of detection of about 0.08 femtomol per microgram of DNA (Perera et oL, 1982). This assay requires (1) the development of an antibody specific for a certain chemical metabolite bound covalently to DNA and (2) the isolation of DNA from some tissue sample, ag., skin biopsy, or lymphocytes of an exposed individual. It is anticipated that further refinement of such immunologic techniques may lower the threshold of sensitivity by one or two orders of magnitude. One such refined test is the ifitrasensitive ymatic radioimmunoassay (USE-RIA), purported to be about five times more sensitive than ELISA (Hsu et oL, 1981 Shamsuddin et oL, 1985 Harris et oL, 1985). Quantification by the development of monoclonal antibodies to aflatoxin Bj metabolites bound to DNA (Groopman et oL, 1982 Sizaret et oL, 1982) has now been reported. 

This methodology traditionally used by biologists is now applied in the areas of environment and food analysis (with pesticides, aflatoxins, anabolic steroids, PAHs). However, it is only possible to measure a compound if the adapted antibodies and enzyme conjugates are available. 

Enzyme-linked immunosorbent assay (ELISA) is a very useful technique for the specific and sensitive assay of certain compounds, in which suitable antibodies, monoclonal or polyclonal, to the compounds are available. The technique has found particular application m the monitoring of environmental contaminants and toxins, either studying the primarily contaminated materials, e.g., foodstuffs, or body fluids of potentially exposed humans. The technique has been increasingly applied to monitoring the carcinogenic mycotoxins, the aflatoxins. 

Aflatoxins are potent carcinogenic, mutagenic and teratogenic metabolites produced by molds. The major food affected with aflatoxins are corn, peanuts, rice, cottonseeds, dried fruit and milk from ingestion (103). The US action standards established by FDA are 20 pg/Kg for foods consumed by humans and 0.5 pg/kg for milk. In the case of animal feed, the levels are from 100 to 300 pg/kg. Therefore, assays capable of detecting at these levels have to be developed, (see Table 1 (104,105)). Detection of aflatoxins entails conjugation of these small molecules with carrier proteins like bovine serum albumin to produce antibodies (20). A number of commercial kits for aflatoxins are available (see sections on kits and immunoaflinity purification). 

Another common sample pre-concentration method is dialysis which serves to remove small molecules. For instance, affinity dialysis and pre-concentration of aflatoxins were achieved in a copolyester chip (see Figure 5.10). After affinity binding to the aflatoxin Bi antibody, various aflatoxins (Bj, B2, Gi> G2, G2J in a sample were retained, while the other small molecules passed through a PVDF dialysis membrane. Thereafter, the sample solution was exposed to a countercurrent flow of dry air, leading to water evaporation and analyte concentration. The concentrated and desalted sample was used in subsequent MS analysis [821], More details for MS analysis are described in Chapter 7, section 7.3. 

Zarba A, Wild CP, Hall AJ, Montesano R, Hudson GJ, Groopman JD (1992) Aflatoxin Mi in human breast milk from The Gambia, west Africa, quantified by combined monoclonal antibody immunoaffinity chromatography and HPLC. Carcinogenesis, 13(5) 891-894. 

Fig. 5 NRL Array Biosensor mixed format immunoassays (modified from [119]). Schematic of (a) the sandwich and (b) the competitive immunoassay fonnats used in the detection of the Campylobacter jejuni and aflatoxin Bi (AFBi), respectively, (a) Sandwich format antigen captured by the immobilized antibody then quantified by passing a second, fluorescently labeled, antibody over the surface, (b) Competitive format competition for binding sites on the fluorescently labeled antibody occurs between the unlabeled antigen in solution and the surface-bound antigen analog, (c) Final charge-coupled devices image taken with the NRL Array Biosensor of a waveguide exposed simultaneously to the C. jejuni (5 x 10" cfu/mL) sandwich assay (SAND) and the aflatoxin Bj (AFBi-1 ng/mL) competitive assay (COMP) in various combinations...

In addition, at least six foreign companies are marketing similar kits. In most cases these kits are dependent on generation of antibodies from the same hapten, i.e. aflatoxin B,. Consequently, there is usually considerable cross reactivity toward other aflatoxin derivatives (B, G, G,). It is important for proper evaluation of results obtained using the kits that this cross reactivity be known. 

Our initial studies using the kits were disappointing, invariably resulting in modifications of the kits themselves and changes in protocols for use of the kits. Following this initial phase of work, we decided one kit was ready for collaborative study. This first collaborative study involved use of the Neogen Agri-Screen kit. The antibodies have specific ability to bind aflatoxin B, and very low cross reactivity to aflatoxins B, G, and G,. This kit contains Antibody-coated microtiter wells Aflatoxin standard solution Dilution buffer (Tris)... 

In this collaborative study fourteen laboratories analyzed six different commodities containing aflatoxin B, at two concentration levels as blind duplicates. Two standards (15 and 50 ng B,/g) were provided, and collaborators were asked to report their results as <15 or >15 ppb. Laboratories with microtiter well readers were asked to determine aflatoxin concentrations both visually and spectrophotometrically. In this kit aflatoxin B,-antibodies are coated onto plastic microtiter wells. The aflatoxin-containing sample is extracted with Me0H-H,0 (55+45). The extract is defatted with hexane, and the MeOH extract mixed with the aflatoxin-enzyme conjugate and added to the well of the antibody-coated microtiter plate. The aflatoxin in the extract and the aflatoxin-enzyme complex compete for the antibody binding sites. The enzyme substrate(ABTS) and HjOj solution are then added, the reaction leading to a colored product in the presence of enzyme. The intensity of color is determined visually or spectrophotometrically at 580nm. 

A third collaborative study (10) was conducted examining the Immuno Dot Screen Cup (Afla-20, IDS Cup), a test kit containing antibodies with considerable cross reactivity with aflatoxins B2 and G,. The manufacturer s procedure was also modified to increase the reliability of detection at 20 ng/g total aflatoxins, and to broaden the applicability to include peanut butter samples. 

In the analysis, antibodies specific to the aflatoxin are coated on the base of a plastie eompartment or microtiter j well in an airay on a plate such as that shown in Figure I... 

Takahashi, N., C.L. Miranda, M.C. Henderson, D.R. Buhler, D.E. Williams and G.S. Bailey. Inhibition of in vitro aflatoxin B1 DNA binding in rainbow trout by CYP1A inhibitors - alpha-naphthoflavone, beta-naphthoflavone and trout CYP1A1 peptide antibody. Comp. Biochem. Physiol. 110C 273 -280, 1995. 

The objectives of this our study were to investigate the properties of immobilized horseradish peroxidase on magnetic particle coated with copolymer of acrylamide and acrylonitrile, used furder in fibre optic smart biosensor constructions with simultaneously immobilized different antibodies for aflatoxines (Fig. 4). 

This classic biological methodology is widely used in research, diagnosis, and testing because it is affordable and sensitive to tiny amounts of material. It is applied to domains such as the environment and agriculture (pesticides, anabolic steroids, aflatoxins, HPA) for which numerous assay kits exist. However only compounds for which there exist adapted antibodies and the conjugated enzymes can be measured. 

Groopman, J. D and Donahue, K. F. (1988). Aflatoxin, a human carcinogen Determination in foods and biological samples by monoclonal antibody affinity chromatography. J. Axsoc. Off-Anal. Chem. 71. 861-867. 

Several research groups have reported antibodies for aflatoxins and other mycotoxins (27). Commercial kits for aflatoxin detection in various substrates have been announced. The introduction of such kits will permit on-site detection of aflatoxins to be confirmed immediately rather than having to wait for analytical results from a remote laboratory following detection of fluorescing materials in a commodity. Since aflatoxins and other microbial toxins have a number of structural variations, the antibodies used in their analysis must be carefully selected to assure that the proper compounds are being detected and accurately measured. 

---