Mycotoxins, antibodies

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. 

SPR systems also showed encouraging results with their ability to detect mycotoxins. The BIACORE was used to detect a mycotoxin, DON, produced by Fusarium species, from spiked wheat sample in a competitive inhibition assay (Schnerr et ah, 2002). Biotinylated DON was immobilized on the sensor chip which was previously coated with strep-tavidin. Mycotoxin extracts from wheat samples were first allowed to react with the antibody and then injected into the BIACORE. The detection range was established to be 0.13-10 pg/ml. In a slightly modified format, DON was also detected by SPR at a range of 2.5-30 ng/ml (Tudos et ah, 2003). 

The semi-lethal dose of T2 mycotoxin is considered to be 5 mg/kg, although at a dose of 0.1 mg/kg vital changes in a number of biochemical indexes are observed. The maximal permitted concentration of this toxin in com is 100 pg/kg [32, 34]. Taking into account this value we chose appropriate T2 mycotoxin concentrations for the experiments. Initially it was dissolved in ethanol and then diluted with 0.05 M Tris-HCl buffer (pH 7.3) to make a series of solutions with different concentrations. T2 mycotoxin was kindly provided by Dr. Kotik from the Institute of Poultry Farming (Ukraine). Specific antibodies (Ab) against T2 were purchased from Sigma (USA). 

The sNPS photosensitivity increased after the immobilization of antibodies but it rose sharply following the addition of T2 mycotoxin at the concentration of 100 ng/mL (Fig. 9.7). 

Fig. 9.7 The photocurrent values on the sPNS surface 1 - bare 2 - with antibodies against T2 3 - with Ab and mycotoxin T2 (antigen)...

The deposition of the specific Ab on sNPS increases the photoluminescence level, but upon formation of the specific immune complex it decreases. The level of photoluminescence decrease depends on the concentration of T2 mycotoxin in the solution (Fig. 9.8). If a non-specific antibody is used or serum bovine albumin is used as an antigen the photoluminescence level does not change. 

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. 

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... 

The mode of action of mushroom-produced mycotoxins varies considerably. Alpha amanitin, amatoxin produced by some species of Amanita, is a class A poison that acts by inhibiting a critical nuclear polymerase that enables the cell to make protein. Once the function of this RNA polymerase is curtailed, basic life processes cease. Attempts to kill alpha amanitin with antibodies have proven to be even more harmful to patients than the poison itself. Most forms of mushroom poisoning can be treated with rapid lavage (induced vomiting) or medically approved ingestion of charcoal to absorb the toxin before it is absorbed into the stomach. 

Studies that have investigated inhalation of mold and mold products found that inhalation produces more potent effects than ingestion. These effects are as potent as intravenous administration. Mycotoxins upon inhalation may produce immunosuppression, carcinogenesis, cytotoxicity, neurotoxicity (including acute or chronic central nervous system damage), mucous membrane irritation, skin rash, nausea, acute or chronic liver damage, and endocrine effects. These effects may be independent of infection or stimulation of antibodies (in contrast to the Mycobacterium mycotoxins). 

The aim of the present review is to present development of new biosensors for mycotoxines determination in foods, utilizing new polymeric membranes with immobilized enzymes and antibodies. 

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 antibodies are specific and sensitive. Their target compounds are mycotoxins, and not antigens, compounds with similar chemical groups that can react with the antibodies. This is the so-called matrix effect (Zheng and Richard 2006). 

The control zone will always be visible regardless of the presence or absence of mycotoxin because the second antibody always binds to a second antibody gold particle complex thus showing the validity of the test. The benefits of the immunochromatographic test are that they are, very rapid, have long-term stability and are particularly suitable for testing for mycotoxins. 

The angle of incidence in this process changes as mass concentration of the sensor surface changes. The variations in the mass concentration measured by the sensor are due to the binding and dissociation of interacting molecules. In this case those molecules are between the immobilised agent (the mycotoxin) and a specific antibody added to the sample. 

In this method the fixed concentration of the mycotoxin specific antibodies are mixed with a sample containing some concentration of the mycotoxin. Then the mixture is passed over the sensor surface on which the mycotoxin is immobilised. The surface plasmon resonance has a number of benefits. 

Choi G H, Lee D H, Min W K, et al. (2004). Cloning, expression, and characterization of single-chain variable fragment antibody against mycotoxin deoxynivalenol in recombinant Escherichia coli. Protein Expr. Purif. 35 84-92. 

Detection and Quantification of Naturally Occurring Compounds. Antibodies can be prepared for naturally occurring compounds as well as for pesticides and drugs. This opens the way for developing rapid immunoassays for plant and microbial products such as mycotoxins, plant hormones, and high value plant components such as flavor and fragrance compounds and pharmaceutical precursors. 

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. 

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. 

The mycotoxins are low-molecular-weight compounds that must be conjugated to a carrier protein to produce an effective antigen.73 When T-2 toxin is conjugated to a protein, it develops relatively low antibody titers and is still a marked skin irritant.91 This would preclude mycotoxins use as immunogens in the production of protective immunity. To circumvent such problems, a deoxy-... 

Chanh TC, Hewetson JF. Structure/function studies of T-2 mycotoxin with a monoclonal antibody. Immuno-pharmacology. 1991 21 (2) 83—90. 

Immunoassays offer a sensitive, specific, cost-effective means of screening many samples for trace residues of toxic chemicals, their metabolites, and adducts. Antibodies can be used both as detectors to quantify the amount of a chemical present and in immunoaffmity chromatography to purify and concentrate material for subsequent analysis. Applications of these assays include detection of pesticide residues, mycotoxins, biomarkers of toxicity, and industrial chemicals. 

Using different mycotoxin protein conjugates as Immunogens, specific antibodies, both polyclonal and monoclonal antibodies, against most mycotoxins have been obtained (1-13). Table II summarizes the different antibodies which have been made against various mycotoxins and mycotoxin metabolites. 

The specificity, generally expressed as the cross-reactivity, of an antibody Is determined primarily by the type of mycotoxin or Its metabolites that has been used In the antibody production as well... 

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