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Immunoassays are biochemical tests that use the binding of antibodies to antigens to detect and measure the concentration of specific molecules. They are relatively cheap, provide fast results and are highly specific due to the fact the tests are based on immune responses.
Food analysis looks at the chemical, microbiological, physical and sensory properties of food and drink. Testing on food needs to be done for safety and regulatory purposes, as well as to test for allergic reactions. Traditionally used for clinical studies, the use of immunoassays in the food industry has grown in popularity over recent years with the rise of fraudulent cases and food contamination.
Types of Immunoassay
There are five main types of immunoassay. These include:
1. Enzyme linked immunosorbant assays (ELISA)
ELISAs work by using enzymes linked to antibodies that give a color change when in the presence of certain antigens. The antigen is immobilized on a solid surface and is then complexed with the antibody that is linked to the enzyme. ELISAs can be direct, indirect, sandwich or competitive ELISAs.
Radioimmunoassays work by using radioactively labeled antigens or antibodies. Radioactivity emitted by bound antibody-antigen complexes is detected and recorded.
3. Fluoroimmnoassays (FIA)
Fluroimmunoassays work by using antibodies that have been labeled with fluorescent probes. Fluorescence from the antibody-antigen complex is detected and recorded.
4. Chemiluminescence immunoassays (CLIA)
Chemiluminescence immunoassays work in a similar way to ELISAs, but they measure light emitted from a chemical reaction.
5. Counting Immunoassays (CIA)
Counting Immunoassays use polystyrene beads that are coated with multiple antibodies complementary to the target antigen.
The main types of immunoassay involved in food analysis are enzyme-based immunoassays and radioimmunoassays.
Immunoassays are used in the food industry to test the raw materials as well as the final composition of food. They are also used to test for any contamination that may have occurred, as well as to test for allergens.
Potential contamination can occur during production and transportation. Contaminants tested for include toxins, micro-organisms and pesticides.
Allergens are tested for both known and unknown allergens. Known allergens are recorded on food labels, but sometimes food can be recalled due to accidental contamination with unexpected allergens.
Foodborne Pathogen Testing
Sandwich assays are commonly used for foodborne pathogen testing of E. coli and Listeria using polyclonal antibodies. A major problem with analyzing foodborne pathogens using polyclonal antibodies is that they have multiple epitopes. Due to this the specificity and sensitivity can be low and cause false positive results. Monoclonal antibodies are preferred and can be used to test for pathogens such as L. monocytogenes, L. innocua, S. Typhimurium and E. coli.
Bispecific antibodies that recognize red blood cells and L. monocytogenes have been specifically engineered. The bispecific antibodies caused agglutination of red blood cells only in the presence of L. monocytogenes, and the agglutination shows red colored clumps that are visible just by looking with the human eye.
Manmade single-stranded DNA molecules known as DNA enzymes are used to test for Candida albicans. A DNA RNA chimeric substrate at a single ribonucleotide junction is flanked by fluorophore and a quencher. When the fluorophore and quencher separate, there is an increase in fluorescence which can then be detected.
Mycotoxins are naturally occurring toxins produced by certain molds and they are hazardous to human health. The dangerous toxins include aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins. Mycotoxins can affect agricultural products such as corn, wheat, rice, oats, rye, soy, nuts, fruit and spices. Food and feed used can become contaminated with mycotoxins before being harvested, the time between harvesting and drying and when being stored. ELISA methods to detect mycotoxins have been available for over a decade. The most frequent way to test for mycotoxins by immunoassay is by using a direct competitive ELISA.
Allergen testing for food is crucial because allergic reactions caused by food can cause fatality. Food allergies cause abnormal responses to the proteins that certain foods contain and cause an immune response in specific individuals. Reactions occur from IgE antibody-mediated and cell-mediated reactions. Common allergens that are tested for include milk, eggs, nuts, sesame seeds, wheat and crustacea, and multiple testing kits are available for both home and industrial use.
Testing of final food products and swabs from equipment used in production can be used to verify any cross contamination and make sure companies are following regulations. Immunological testing methods can use human IgE from individuals allergic to specific foods, or animal IgG and IgY from polyclonal or monoclonal animal antisera. The polyclonal antisera is raised against specific proteins or mixtures of proteins, whereas the monoclonal antibodies are made specific to a particular protein or peptide.
Qualitative detection of food allergens can be done by using dot immunoblotting or SDS-PAGE, but Rocket immunoelectrophoresis and ELISAs are used for quantitation of hidden food allergens. ELISAs are used to detect proteins that are intact, but some ELISAS cannot detect protein hydrolysate. Lipophilic residues are difficult to detect, so improvement is needed for residual detection via ELISA.