5.1.5 Immunological methods of detection of Microbial pathogens

5.1.5 Immunological methods of detection of Microbial pathogens

Serotyping

This method is widely applied to Gram negative enteric bacterial pathogens such as Salmonella and E.coli. It employs the use of specific antibodies to indentify homologous antigens. In case of food borne pathogens, the antigens are particulate and agglutination methods are employed. For soluble antigens like toxins gel diffusion assays may be used.

Radio immunoassay

This technique consists of adding a radioactive label to an antigen, allowing the labeled antigen to react with specific antibody and measuring the amount of antigen – antibody complex by the use of a radio activity counter. 125 I is commonly used. Toxins and other metabolites can also be detected by RIA.

Immunofluorescence

ELISA – Enzyme Linked Immuno-Sorbant Assy

Chemical conjugation of an enzyme to either antigen or antibody allows detection of immune complexes formed on a solid phase. Enzyme linked immunosorbant assay is a simple, versatile and highly sensitive test for detection of antigen (eg. Microbial pathogen or toxin) or antibody (Immunoglobulin produced against a bacteria / toxins).

ELISA and related assays depend on four major principles

1. Most antigens bind spontaneously to plastic surfaces such as the wells of polystyrene microtitre plates. Antibodies being protein also attach while retaining their antigen binding activity. Once they bind to solid surface, they become resistant to washing. Thus antigen or antibody coated plates can be prepared in the initial step.

2. In subsequent steps immune complexes are formed. Unbound reactants can be washed away.

3. A variety of enzymes (horse radish peroxidase – HRP, Alkaline phosphatase) can be chemically coupled to either antibody or antigen without affecting their biological properties.

4. The enzyme component of the immune complex reacts with the substrate resulting in colour change. The reaction is stopped at an appropriate stage and interpreted by visual comparison or by optical density measurement.

There are many ways of performing ELISA

1. Simple ELISA (Simple 2 layer assays)

ELISA plates are prepared by coating with either antigen or antibody and are then exposed to potentially binding enzyme labeled complementary reactant (antibody or antigen). The plates are then washed and the substrate added to detect the binding of the enzyme labeled components.

This simple ELISA can be made quantitative in 2 ways: i. Antigen inhibition assay and ii. Competitive assay.

2. Sandwich ELISA (Two site assay)

In this assay, antibody coated on a solid phase is exposed to test samples and after washing, the complex is exposed to diluted reference antibody-conjugate to the same antigen. Then, the substrate is added for colour reaction to reveal the formation of immune complex.

3. Indirect ELISA

This assay is very popular for detecting presence of specific antibodies and has been a valuable tool in epidemiological studies. Antigen (usually viral, bacterial or parasite extracts) is used to coat the ELISA wells. Serum is then applied and bound specific antibody is revealed by the use of an antiglobulin enzyme conjugate, followed by colour reaction.

A major advantage of this assay is that it can utilize a “Universal” antispecies immunoglobulin (anti immunoglobulin).

ELISA and related assays depend on four major principles

Last modified: Tuesday, 27 December 2011, 2:13 PM