Package 2- Immunoassays

Besides the conventional agar plate testing of foodborne pathogens, there are other methods such as Immunoassays, PCR, DNA hybridization etc.

Immunoassay refers to the qualitative or quantitative determination of antigen or antibody in a specimen by an immunological reaction. In addition, immunoassays are among the most commonly used techniques in clinical, food and environmental microbiology for rapidly detecting pathogenic bacteria.

There are 5 types of immunoassays techniques:

1) Immunofluorescence
This technique is the first type of immunoassay used to detect bacteria in biological specimens. Immunofluorescence kits for detecting Salmonella in food have been commercially available. In this method, bacteria from an enrichment culture are fixed to a microscope slide and the fixed cells are treated with fluoresin-conjugated somatic and flagellar antibodies specific for Salmonella. After excess reagent is removed, the slide is observed under fluorescence microscope for cells with fluorescent cell wall and/or flagella. Although immunofluorescence is a useful tool in the research laboratory, it is not used in most food microbiology laboratories because final evaluation of the reaction is performed by microscopic examination, which is tedious. Furthermore, trained and experienced personnel are needed to obtain reliable results.

2) Coagglutination (agglutination-enhancement)
This technique is popular in clinical microbiology because it is simple, rapid, moderately sensitive and does not require instrumentation. The most common type of antigen can be immobilized on inert latex particles by either passive adsorption or covalent bonding. Latex reagents prepared in this manner are stable for as long as 1 year when stored at 4°C. The test is performed as follows:

· After centrifugation of an enrichment culture, the pellet containing bacteria of interest is re-suspended in an appropriate buffer.
· A drop of the bacterial suspension is placed on a slide.
· A drop of the sensitized latex reagent is added and mixed thoroughly with the specimen.
· The slide is rocked for 1-5minutes and then visually examined under a high-intensity lamp for agglutination. The sensitivity of the latex agglutination test for bacteria is generally in the range 10^7-10^8 cells per ml.

It is important to perform appropriate controls each time to ensure that the senitized latex has retained reactivity and to detect false-positives caused by spontaneous agglutination of the senitized latex.

3) Immunoaffinity Chromatography
A monoclonal or polyclonal antibody to the target molecule- for example: aflatoxin B1, is immobilized on a solid inert support contained in a mini-column. A sample is suspected to contain the target antigen is allowed to flow through the column. The target molecule is retained while other materials are removed by extensive washing. The retained target molecule is eluted by changing the composition of the mobile phrase and quantitated by an appropriate method (for example: fluorometry).

4) Immunoimmobilization
Immunoimmobilization is a procedure that takes advantage of the immobilization of motile bacteria cells in a semisolid medium by a specific antibody directed against the flagella of the bacteria. Motile bacteria traverse the semi-solid medium until they meet the antiflagellar antibody diffusing from the opposite direction. A visible arc of immobilization forms at the antibody/bacterial interface; this arc indicated that target bacteria are present.

5) Enzyme Immunoassays
Enzyme immunoassays (EIA) also know as Enzyme-Linked-Immunosorbent Assay (ELISA), is the most commonly used format for the immunological detection of microorganisms and their metabolites (toxins). EIA depends on 3 principles:

Ø The exquisite specifity of antigen-antibody reactions
Ø Biological amplification of the antigen-antibody reaction by an enzyme
Ø The antibody’s ability to retain its immunoreactivity after conjugation with an enzyme.

Traditional immunoassays that used polyclonal antibodies often suffered from shortcoming caused by the presence of antibodies cross-reactive to bacteria other than the target organism and from batch-to-batch variations in antibody specificity.