Immunochemical methods are procedures with reactions between antigens (proteins with additional groups such as carbohydrates, lipides, etc) and antibodies (proteins produced by an animal's immunosystem in response to the introduction of a foreign protein which specifically binds with it) and can detect specific microorganisms or their metabolits.
Some early methods consisted of adding a radioactive label (eg. 125I) to a soluble antigen followed by reaction of the antigen with its homologous antibody - Radio Immunoassays, but food laboratories were reluctant in using these kind of methods because of the use of radioactivity. A nonradioactive version was developed creating the so-called Enzyme Immunoassays (EIA) which are now much more used to detect microbial toxins in foods. These methods have a colored end point which is acceptable to the food industry and allow either visual or spectrophotometric reading of the results.
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Most commercially avaliable EIAs use an antibody sandwich technique for the capture and detection of target bacteria or their toxins. Figure shows how this technique works. The antibody is immobilized on an inert solid such as polystyrene (some kits are already supplied with the antibody attached to the solid surface). A suitable enriched sample is added to the antibody-coated surface under conditions in which the target antigen (such as a specific Salmonella protein) attaches to the antibody. The resulting antigen-antibody complex is further reacted with a second specific antibody to which an easily assayed enzyme has been covalently linked. Excess antibody-antigen is washed away leaving an antibody-antigen- antibody sandwich. |
A colored substrate, chosen for the particular enzyme, is added and is converted to a colored product in the presence of the enzyme and is detected either visually or using a microtiter plate reader.
The assay takes between 2 and 2½ hours and results indicate the presence or absence of the target bacterial cells or any other substance, such as proteins. Results indicating the presence of target cells are presumptive and need to be confirmed biochemically and by serology. EIA tests for Listeria and Salmonella in food are done on suitably enriched samples allowing a product giving negative results to be released two days after sampling, compared to four or five days for conventional tests.
Choice of enzymes
The choice of enzymes depends largely on whether the assay format is heterogeneous or homogeneous. In heterogeneous EIAs, separation of antibody bound enzyme conjugate from free enzyme-labeled material is critical to achieve the desired relationship between the final enzymatic activity and the antigen concentration. This assay is normally referenced to as Enzyme Linked Immuno Sorbant Assay - ELISA. In homogeneous assays, separation of free from bound enzyme conjugate is not required. This assay is referenced to as Enzyme Multiplied Immunoassay Technique - EMIT.
In both cases, however, the choice of enzymes is limited. For heterogeneous assays, the enzymes used include:
Whereas for homogeneous assays they include:
Choice of enzyme substractes
Enzyme immunoassays that are used for food analysis are largely based on colorimetric end-points. However other forms of substrates are now available which can be used to generate amplified end-point signals. The next table shows some of the substrates used in EIAs for food analysis.
| Enzyme | Substrate | Application |
| Peroxidase | o-phenylenediamine (colorimetric) | Staphylococcal enterotoxin B |
| Tetramethylbenzidine (colorimetric) | Salmonella | |
| Pyrogallol/H2O2 | Staphylococcal enterotoxin B | |
| Alkaline phosphatase | Ethanol, NAD, Iodo-nitrotetrazolium (colorimetric) | Botulinal neurotoxins |
| p-Nitrophenyl phosphate (colorimetric) | Campylobacter | |
| 4-Methylumbelliferyl phosphate (fluorogenic) | Quinidine | |
| AMPPD (chemiluminescent) | Salmonella | |
| b-galactosidase | o-nitrophenyl-b-D-galactopyranoside (colorimetric) | Proteinase |
| 4-Methylumbelliferyl-b-D-galactopyranoside (fluorogenic) | Proteinase |
Enhanced Enzyme Immunoassays
The sensitivity of the enzyme immunoassays can be enhanced considerably by determining the enzymatic activity using sensitive end-point detections, for example, fluorescence, chemiluminescence and enhanced colorimetric reactions.