Mycotoxins ELISA

Enzyme-linked Imunosorbent Assay (ELISA) and Radioimmunoassay (RIA) are highly specific methods that have been used for the assay of a number of mycotoxins. ELISA is the preferred method of immunoassay because of the limited half life of the radioisotopes and the problems of disposal of radioactive waste. 

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. 

For mycotoxin analyses radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISAs) and affinity chromatography are the principal immunochemical methods in commercial application. Immunoaffinity columns or cartridges for specific mycotoxins are now being increasingly used in preliminary clean-up of extracts prior to final analysis by HPLC or GLC methods. 

Fluorescence may by induced using labeled mono- or polyclonal antibodies raised against the compound of interest. This technique has been used successfully to detect the presence of okadaic acid in cultures of Prorocentrum lima, and, further, to estimate quantities of the compound in individual cells.110 Immunofluoresence in combination with thin layer chromatography and ELISA techniques have also been used to detect multiple haptens in mycotoxin families.111112... 

Huang X, Domer JW, Chu FS Production of aflatoxin and cyclopiazonic acid by various aspergilli An ELISA analysis. Mycotoxin Res 1994 10 101-106. 

Enzyme - linked immnnosorbent analysis (ELISA) is a method known for decades. The technology is based on the ability of a specific antibody to distinguish the tri-dimensional structure of a given mycotoxin. The direct competitive... 

ELISA is usually used for the assay of mycotoxins. The commonly used ELISA needs equilibrium of the reaction antibody-antigen which requires an incubation period of approximately 1-2 h. Currently most ELISA tests for mycotoxins operate in the kinetic phase of linking antibody-antigen that reduces the incubation time to minutes. Although the reduction of the incubation time may lead to some loss of the sensitivity of the assay, the tests can produce accurate and reproducible results. 

The ELISA tests are preferred because they require low volume of the sample and fewer clean-up procedures of the extracted sample compared to the conventional methods like TLC (thin layer chromatography) and HPLC (high-performance liquid chromatography). ELISA tests are rapid, simple, specific, sensitive and portable for use in the field for the detection of mycotoxins in foods and fodder (Zheng et al. 2005). 

The ETA for mycotoxins has been used since the late 1980s. It is based on the principle of the direct competitive ELISA. Anti-mycotoxin antibody is coated on the surface of a membrane. 

To overcome the difficulties encountered with the bioassays and TLC methods, immunoassays using specific polyclonal and monoclonal antibodies have been developed for most of the major trichothecene mycotoxins and their metabolites.73 These antibodies have been used to produce simple, sensitive, and specific radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) for the mycotoxins. In the presence of the sample matrix, the lower detection limits for identification of trichothecene mycotoxins by RIA is about 2 to 5 ppb73 and by ELISA, 1 ppb.74 We conclude that immunoassays are useful tools for screening biomedical samples for evidence of a biological warfare attack with trichothecene mycotoxins. 

Although a number of Immunochemical methods have been used for the analysis of small molecular weight biological substances, only radioimmunoassay (RIA), enyzme-1Inked Immunosorbent assay (ELISA) and Immuno-affinity assay (lAA), have been developed for the analysis of mycotoxins. Recent developments have led to several quick screening tests and more than 10 types of commercial kits have become available In the last few years (8, 10, 13). In most cases, sample after extraction from the solid matrix and diluted In buffer can be directly used In the assay. Since the application of Immunoassay for several mycotoxins are covered by other speakers, I will only briefly highlight some of the recent progress on these methods. 

Competitive enzyme-linked immunosorbent assays (ELISA) Two types of ELISA have been used for the analysis of mycotoxins and both types are heterogenous competitive assays. One type, i.e. direct ELISA, involves the use of a mycotoxin-enzyme conjugate and the other system, i.e. indirect ELISA, involves the use of a protein-mycotoxin conjugate and a secondary antibody to which an enzyme has been conjugated. Although horseradish peroxidase (HRP) is most commonly used as the enzyme for conjugation, other enzymes such as alkaline phosphatase and beta-galactosidase, also have been used (5, 9, 13). 

Indirect competitive ELISA (or double antibody ELISA) In the indirect ELISA, Instead of using a mycotoxin-enzyme conjugate, a mycotoxin-protein (or polypeptide) conjugate is first prepared and then coated to the microplate (1-13). The plate is then Incubated with specific antibody against the mycotoxin in the presence or absence of the homologous mycotoxin. The amount of antibody bound to the plate coated with mycotoxin-protein conjugate is then determined by reaction with a second antibody-enzyme complex such... 

Morgan, M. R. A. Mycotoxin immunoassays (with special reference to ELISAs). Tetrahei on, 45 223 49. 1989. 

Ueno, Y. Ohtani, K. Kawamura, O. Nagayama, S. ELISA of mycotoxins with monoclonal antibodies. Tanpakushitsu Kt usan Koso, Bessatsu, (31), 80-9. 1987. Japanese. 

Enzyme-linked immunosorbent assay (ELISA) methods are widely used in medical and food analysis laboratories and it seems appropriate that such methods could be used for the detection of mycotoxins in foods. In the direct ELISA method the antibody is bound to a solid surface, such as a microtiter plate, and it is necessary to prepare a conjugate of the mycotoxin to be analyzed with the enzyme, such as phosphatase or peroxidase, to be used for color development. The sample or standard is mixed with the enzyme-linked mycotoxin and the two allowed to compete for the antibody bound to the surface. After washing away soluble material, the... 

ELiSA Enterotoxins, mycotoxins, 1 h for toxins 0.01-0.1 ng Staph, enterotoxinr,... 

Determination test kits are based on direct but more often on indirect-type ELISA techniques. They use 96-well microtitration plates or beads or sticks. Analysis for mycotoxins can be completed within 3 h. This kind of test needs a photometric reader for the quantitation by plotting sample value against standard-curve derived values. Nowadays, a number of companies are developing such tests for aflatoxins, OTA, patulin, zearalenone, T-2 toxin, fumonisins, DON, etc. with limits of quantification as low as 1 pgperkg or even less. 

The main advantages of the ELISA techniques are mainly the rapidity of analysis, the high sensitivity of detection (ultrasensitive tests), and the cost per unit sample, but the main drawback is the production of false positives (results being positive when no mycotoxins are present in the extract) and sometimes false negatives (results being negative when mycotoxins occur in the extract). Especially for food or feed complex matrices, such a limitation is very important and explains why ELISA procedures are seldom selected as official methods. 

Zeranol and zearalenone and their metabolites have been recently analyzed by Hsieh et al. by dual UV-vis and ED in rice, corn flakes, and soybean [ 148]. The method provided quick and reliable semiconfirmative and quantitative information on the occurrence of these analytes and supplement to ELISA screening method for total mycotoxins. Other HPLC-ED schemes for these mycotoxins have been applied to cattle [149], cheese [150], and edible animal tissue [151], In the last work, these mycotoxins were enzymatically hydrolyzed, in order to be easily oxidized on GCE (Table 4.7). 

The most common assay to determine mycotoxins is based on a quick solvent extraction (methanol-water, ethanol, chloroform, etc.) of the mycotoxin. The extract is then filtered and quantified via ELISA (enzyme-linked immunoassay) columns. The use of minicolumns is the most common because it is fast, repeatable, and reliable, and requires little expertise to run the assay. The test usually takes 5 minutes, so it allows processors to make important decisions about the acquisition and economic value of the grain. Other more time-consuming and complicated tests are thin-layer chromatography or other chromatographic techniques based on fluorescence or UV detectors. The main advantage of these tests is the identification of specific types of mycotoxins. 

The utilization of ELISA columns to selectively isolate mycotoxins followed by Iheir cpiantification via fluorescence allows for cpiick and rehable detmnination of these important metabohtes, which are r ulated by most r tdatory agencies. Test can be pmTimned in less than lOmin. 

The utilization of ELISA colunms to selectively isolate mycotoxins followed by their quantification via fluorescence allows a quick and reliable determination of these important metabolites, which are regulated by most regulatory agencies. Tests can be performed in less than 10 min. 

ELISA Enzyme-linked immunosorbent assay. A sensitive biotechnology analytical technique in which an enzyme is complexed to an antigen or antibody. The analyte is bound and complexed, and then removed for quantification via color development or other instrumental analysis techniques (i.e., fluorescence). ELISA is used as a rapid and accurate technique to quantify most mycotoxins. 

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