Case Study: Immunological Methods as Screening Tools

Immunological methods have become increasingly popular as front line screening methods for the detection and monitoring of allergens, chemical contaminants and pathogens due to their ease of use and relatively inexpensive design.

Diagnostic immunology is a collective term for a variety of techniques that rely in the specificity of the bond between antibodies and antigens. The method is well suited for the detection of even the smallest amounts of bio(chemical) substances. Antibodies specific for a desired antigen can be conjugated with a radio label, fluorescent label or colour-forming enzyme that are used as a “probe” to detect it.

Well known applications of immunological methods include pregnancy tests, immunoblotting and ELISA. The speed, accuracy and simplicity of such tests has led to the development of rapid techniques for the diagnosis of disease, microbes and contaminants in food. The following steps outlines the key principles of an immunological technique.

ELISA Technique

In order to develop an immunological method we need antibodies. Antibodies are used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, called an antigen. Each antibody contains a paratope (a structure similar to a lock) that is specific for one particular epitope (a structure analogous to a key) on the antigen. This allows an antibody and antigen to bind together with precision.

In food, low molecular weight chemicals do not produce an immune response. They act on the receptor or enzyme causing either a toxic or chemical response. Therefore, the method requires conjugating the toxin to a protein through a protein conjugation the protein then becomes a carrier to the chemical or toxin to which there is an immune response. The toxin conjugated to the protein is known as the immunogen. The immunogen is immunised into an animal where the antibodies are raised. Typically an imminisation program or plan is put in place.

The enzyme-linked immunsorbent assay (ELISA) is based on this selective and specific antibody-antigen recognition. Performing an ELISA test requires at least one specific antibody for a specific antigen. The sample with an unknown amount of antigen is immobilised on a solid support either non-specifically or specifically. After the antigen is immobilised, the detection antibody is added and interacts with the antigen in the antibody-antigen lock and key type system.

This interaction can be visualized by enzymes, linked to secondary antibodies or antigens, and indicate if a antigen-binding has occurred. The plate is typically washed with a mild detergent solution between each step to ensure any proteins or antibodies that are not specifically bound are removed. After the final wash step, a final enzymatic substrate is added to produce a visible colour which can be measured by a spectophotometer.

Characteristics of an ELISA

ELISA can be classified as a labelled method. Labelled methods attach a “tag” to either the antigen or antibody. This “tag” can be detected and measured. Other labelled methods can include: direct flourescnce, indirect flouresence, chemiluminescence and lateral flows.

ELISA would be classified as a screening test used to detect a disease or contaminant. They are highly sensitive but are not as specific as confirmatory tests. A food sample is first tested with a screening method such as an ELISA. If the sample is compliant, i.e. no analyte (antigen) has been detected the food sample is compliant and no further action is required. However, if an analyte is present the sample will then undergo confirmatory analysis by physiochemical methods, typically Liquid Chromatography-Mass Spectrometry (LC-MS).

What we would like you to do

Please consider the following questions and add your thoughts to the discussion area below:

• Have you any experience with screening or confirmatory methods for food analysis? If so can you share your thoughts on their advantages and disadvantages to your fellow learners

• Why do you think screening and confirmatory methods are used to analyse a food sample?