Diagnostic immunology

1. Diagnostic immunology

2. There are many practical applications that one can use in the diagnostic immunology laboratory with respect to disease detection and disease monitoring. • Many of the same processes that occur in the human body, such as Ag-Ab interactions, can be utilized in a laboratory setting to augment disease conditions.

3. The following 3 laboratory based detection methods are examples of very common diagnostic tools used in clinical settings, public health labs, and in research settings

4. 1.) Agglutination Reactions • Agglutination reactions occur when an Ab and Ag are specific for each other and a "lock and key" lattice of Ab-Ag forms to allow for an observable agglutination reaction.

5. An example is the Rapid Plasma Reagin (RPR) kit used for the detection/screening of syphilis caused by the bacterium, Treponema pallidum. • The IgM antibody is often referred to as early Ab or the 1st Ab to appear in an infection.

6. It is also known as the Ab of agglutination because of it's molecular make-up. IgM is typically arranged as a pentamer (5 Ab's bound together which allows for 10 sites of Ag attachment). • An IgM pentamer (see figure in text) allows for many Ag's to bind and form a complex lattice that will become insoluble in solution and be visible to the naked eye

7. Thus, one can use specific Ab for syphilis to bind to specific Treponema pallidum Ag's and form a complex (this is typically called a "reactive"). • The test is easy and low cost....because of these features, it makes an RPR test a useful and cheap screening test for this disease.

8. 2.)Fluorescent Antibody (FA) assays Fluorescent Antibody (FA) assays are one of the most common kits available to diagnostic labs for the detection of a variety of microbes. A kit will simply have an Ab that has been "labeled" with a fluorochrome such at FITC. The FITC will fluoresce when exposed to a uv light source on a microscope and "light up" as apple green fluorescence.

9. A kit will simply have an Ab that has been "labeled" with a fluorochrome such at FITC. The FITC will fluoresce when exposed to a uv light source on a microscope and "light up" as apple green fluorescence. • If the reaction on a slide is not specific (ie. the Ag doesn't match the labeled Ab) then there is no fluorescence.

10. Enzyme Linked Immunoabsorbent • Enzyme Linked Immunoabsorbent Assays (ELISA) are also a very common kit available for diagnostic labs. • The HIV screening test is an ELISA based test. • The kit will have a specific Ab for HIV in a plastic coated well. • A technician will then add serum from a patient. If the serum has HIV Ag, it will bind to the Ab in the plastic coated well.

11. This step is followed by another Ab that is labeled with an enzyme. Then a substrate is added to the "sandwich" ELISA. • If the labeled enzyme has attached in the previous step, the substrate will fit into the enzymes active site and a colorimetric reaction will take place. • So, a typical color like yellow will be produced with a positive test. See the text for illustrations of this test.