Immune complex helps the host in various ways, such as activation of complement system, neutralization of toxins, enhancing production of phagocytes etc. But under certain conditions Ag-Ab complex induces pathological effects and results a number of diseases.
For example the precipitates or floccules formed are circulated and deposited on epithetical cells of various organs and initiates immune reactions. The immune cells cause damage to their own healthy cells to which the immune complex has been attached.
Diagnostic tests and Ag-Ab complex: Direct measurement of antibodies binding to antigens is the principle, generally used in majority of quantitative serological assays.
Since secondary reactions such as agglutination, complement fixation, precipitation etc. can be detected in a variety of ways, majority of diagnostic tests depend on secondary reactions followed by Ag, Ab binding. Some of the popular diagnostic tests are as follows.
1. ELISA or Enzyme linked immunosorbent assay:
ELISA is a widely-used method for measuring the concentration of a particular molecule (e.g, a hormone or drug) in a fluid such as serum or urine. It is also known as “enzyme immunoassay or EIA”. It is used as an initial screen for HIV detection.
Based on the principle of antigen – antibody interaction, this test allows for easy visualization of results and can be completed without the additional concern of radioactive materials use.
The test is performed in a 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.
In HIV test partially purified, antigens are pre-coated onto an ELISA plate. When patient serum with antibodies for pathogen/ HIV is added the antibodies bind to the antigens of pathogen on the plate.
2. Western Blot:
Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.
1) Proteins are separated by gel electrophoresis, usually sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
2) The proteins are transferred to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel.
3) The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time.
4) The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.
Immunofluorescence is a laboratory technique to identify specific antibodies or antigens. The exact technique may vary depending on the specific antibody that is being investigated and between different laboratories.
In general, cells, tissue, or some other substance is placed onto microscope slides. A small amount of sample containing antibodies (typically serum, a liquid portion of blood) is placed over the cells or tissue, allowing the antibodies that are specific for the particular tissue or cellular antigens to bind.
The serum is washed away, and a second antibody that binds to human antibodies (often made in another animal species such as rabbits or goats) is applied to the slide.
This second antibody has a fluorescent dye chemically linked to it. If serum of the person used for test has antibodies that bind to the tissue or cells, a bright fluorescence can be seen by use of a special microscope.