Immunological techniques: Enzyme-linked Immunosorbent assay (ELISA)

Uses the highly specific binding reaction between antibodies and antigens.

Twp types of antibodies used:
1. Monoclonal
2. Polyclonal

Replace the detection or isolation stage on agar.

Positive result obtained is presumptive and must be confirmed using conventional tests.

Advantages:
- Easy to perform
- Can be applied to range of pathogens
- Can be semi-automated
- Give rapid result

ELISA formats:

- Sandwich ELISA:
Ø Uses two antibodies which trap the target antigens
Ø Test well is coated with antibody.
Ø Procedure:
1) Antibody is coated to the test well
2) Enriched sample added to the test well; presence of any target antigen will bind to the antibodies.
3) Washing procedure; remove food debris and unbound materials
4) Second antibodies added to the well with an enzyme label attached; presence of any target antigen will bind to the antibodies creating the ‘antibody sandwich.
5) Washing procedure
6) Addition of colourless substrate; convert by enzyme to coloured product.
7) Stop solution added; preventing further enzyme activity and any change in colour measured.

- Indirect Sandwich ELISA:
Ø Antibody does not carry enzyme label, instead it carry marker molecule which specifically bind to other molecule.
Ø Biotin is often used a s a marker with aviding as the protein binding site; resulting in a biotin-aviding, enzyme labeled antibody which catalyze conversion of colourless substrate to a coloured product.

- Competitive ELISA:
Ø Test well is coated with antigen
Ø Procedure:
1) Test well is coated with antigen
2) Sample and labeled antibodies are added to the test well simultaneously. Target antigen:
v Not present in sample; antibodies blind to the antigen coated on the wells.
v Present in sample; antibodies blind to the target antigen + antibodies coated on the wells.
3) Washing stage: antibody-antigen complexes in solution are removed; change in colour is due solely to the antibody-antigen complex on the test well.
4) Addition of colourless substrate; convert by enzyme to coloured product
v Coloured product: negative result
v Colourless (or weak colour): positive result.

Other Immunological techniques include:
1. Immunochromatography
2. Enzyme-linked immunofluorescent assays
3. Agglutination techniques.


References:

C.L. Baylis, Campden and Chorleywood Food Research Association, UK. 2003. Immunological techniques: Immunochromatography, Enzyme-linked immunofluorescent assays and Agglutination techniques. In: Thomas A. McMeekin. Detecting pathogens in food. North America: CRC Press LLC.217.

J. McCarthy, Unilever R&D Colworth, UK. 2003. Immunological techniques: ELISA. In: Thomas A. McMeekin. Detecting pathogens in food. North America: CRC Press LLC.241-245.