Immunological Methods

Microorganisms have specific antigen properties, and many of these antigens can elicit the production of antibodies in animals. With the appropriate antibodies, one can then use the antibodies to identify the unknown by antigen-antibody reactions. Historically, polyclonal antibodies have been used for detection and characterization of bacteria. More recently, monoclonal antibodies have been used for the reactions. Both types of antibodies are useful for anaerobic microbiology. The reactions can be agglutination of cells directly with antibodies or indirect agglutination of antigens reacting with antibodies fixed on a variety of support systems, including blood cells, latex beads, plastic steel balls, etc. The most popular format of antigen-antibody reactions used is ELISA. In this test, the antibody is fixed on a support surface and the antigen is captured. A second antibody then reacts with another site of the antigen. The second antibody is conjugated with an enzyme. A substrate is then applied, and if the enzyme is present a color reaction will occur. The intensity of the color reaction will indicate the presence of the antigen. A cutoff color intensity is established to indicate a positive or negative test of the presence of the antigen. At first, this test was done manually, but recently, it has been completely automated. The analyst presents the instrument with a suspect sample, and the instrument will automatically complete the test with a computer printout.

An exciting new development in immunological analysis is the field of immunomagnetic capture technology. In this technology, beads are magnetized and then antibodies or other capture particles are attached on the surface of the beads. Antibodies against whole cells, antigens, and other target particles are fixed on the beads. The charged beads are then introduced to a broth or food sample to interact with target bacteria or other molecules. In this phase, the sample is mixed thoroughly with the charged beads tumbling in the liquid food to interact with target organisms. After the interaction, a powerful magnet is applied to the side of the reaction tube. The magnet holds all the beads with or without captured organisms. The rest of the debris is discarded, and the beads are released and washed. The captured organisms can be subject to growth in a special broth or agar, or these may be used for further immunological reaction, PCR, DNA/RNA reactions, or other tests.

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