Question

how would you set up and execute an ELISA test to detect a toxic fungal component? What components would you need?

Answer 1

Fungal toxins also called Mycotoxins which are secondary fungal metabolites with structural and toxicological properties that induce a variety of toxic and carcinogenic effects when food contaminated with these compounds is ingested. In order to test fungal components in a sample ELISA( Enzyme-linked Immunosorbent assay ) test can be used.

In the competition, ELISA a microtitre plate is coated with a known amount of antibody against the mycotoxin looked for. After being washed the test solution, containing an unknown quantity of the mycotoxin, is added together with a known amount of enzyme-labelled mycotoxin. Labelled and non-labelled mycotoxin compete for the active sites of the bound antibody. After incubation, the plate is washed again and the captured enzyme is determined by adding chromogenic substrate. The resulting colour or the intensity of the resulting colour can be measured visually or, more precisely, instrumentally with an ELISA reader. In both cases, the determination of the amount of mycotoxin in the test solution is made by using a series of standards in various concentrations, which have undergone the same procedure. Often, the test solutions are applied in various dilutions if no idea exists about the order of magnitude of the mycotoxin concentration in the test portion. The lower the product concentration of the enzyme reaction, the lower the amount of bound enzyme and the higher the mycotoxin concentration in the test portion.

For example, we set up an experiment to test Aflatoxin B1 which is one of the mycotoxins

Aflatoxin B1 is conjugated to an enzyme. This conjugate is used as the known antigen (enzyme-conjugate). Antibodies specific to aflatoxin B. are coated onto plastic microtitre wells. Aflatoxin B. is extracted from the sample with a solvent. The extract is mixed equally (v/v) with enzyme-conjugated aflatoxin B, and the mixture is placed in the antibody-coated microtitre wells. The contaminating aflatoxin B.. from the sample, if present, and the enzyme-conjugated aflatoxin B. compete for the binding sites on the antibody. "Unbound aflatoxin B1 from sample and enzyme-conjugated is "washed" away. A solubilized enzyme substrate is added to each well, and catalyzed by the bound enzyme, if present, from a colourless solution to a blue solution. The intensity of colour decreases as the amount of aflatoxin B, in the sample increases. After a few minutes, the change in colour can be evaluated visually.

Procedure

Preparation of the test sample: Grind the laboratory sample so that it completely passes through a sieve with a 1 mm mesh.

Extraction: Weigh out 5 g test sample into a test tube with screw cap. Add 25 ml methanol solution, shake vigorously for at least 1 minute. Filter through a filter paper into a 50 ml test tube or equivalent. All samples to be tested should be prepared during the same time period. Preparation of the enzyme-conjugate working solution: Add 2 ml enzyme-conjugate hydration solution to the aflatoxin B. enzyme-conjugate. Mix well to completely dissolve. Do not shake hard enough to cause foaming in the bottle.

CAUTION: The working solution must be at room temperature before use and used within 30 minutes after preparation.

Preparation of the enzyme-substrate working solution: Transfer 1 ml enzyme-substrate and 1 ml hydrogen peroxide to a mixing tube. Close the tube and shake vigorously.

CAUTION: The working solution must be used within one hour after preparation.

Enzyme Immuno Assay:

NOTE: A different pipette/tip must be used with each of the following steps.

Always pipette into the wells in the same order. Set up the mixing wells. Do not use the antibody-coated microtitre wells. Pipette 0.1 ml aflatoxin B, the enzyme-conjugate working solution into each well. Use one well for each sample to be tested and one well for each control. Up to six aflatoxin B1 control wells can be used with each strip. Add 0.1 ml of aflatoxin B. standard solutions and sample filtrate into the separate wells and mix thoroughly by drawing the fluid back into the pipette/tip. Change the pipette/tip between each sample and control. Set up the antibody-coated wells. Transfer 0.1 ml of the solution from each mixing well to the corresponding antibody-coated well. Change the pipettes/tips between each transfer. Let stand at ambient temperature (22 - 25° C) for 10 minutes. 78 Shake the solution out of the antibody-coated wells into the waste receptacle containing decontaminating solution (See Safety Notes). Do not get decontaminating solution into the wells. Wash the wells 10 times by filling with distilled water and shaking out the wash solution. After the final wash, invert the wells on absorbent paper and tap to remove excess water.

NOTE: Only one pipette tip need to be used for each of the following steps:

Immediately add 0.1 ml enzyme-substrate working solution to each antibody-coated well and let stand at ambient temperature for 5 minutes. Gently agitate the well several times during the assay by holding on the countertop and tapping on the side. Add 0.1 ml colour stopping solution to each antibody well and compare the colour development of the control wells with the sample wells. Hold the strips against a white background for comparison.