TESTS FOR AUTOANTIBODIES ASSOCIATED WITH AUTOIMMUNE DISEASE

Agglutination Assays
Red cells are incubated with purified specific antigen (eg, thyroglobulin), which is adsorbed to the cell surface. The antigen-coated cells are suspended in the patient's serum, and antibody is detected by red cell agglutination. Antigen-coated latex particles are substituted for red cells in latex fixation tests.
Enzyme-Linked Immunosorbent Assay
Antibodies to various tissue antigens can be readily detected by these tests. Extracted and purified antigens are fixed to a plastic microtiter well or beads. The patient's serum is added, and excess proteins are removed by washing and centrifugation. Adherent immunoglobulin is then detected when a second antibody coupled to an enzyme (eg, alkaline phosphatase) is added. Finally, the enzyme's substrate is added; color forms and is measured in a spectrophotometer. This test can also be adapted for antigen detection by placing the antibody on the plastic surface. ELISA is very sensitive and less cumbersome than radioimmunoassay techniques.
Immunofluorescence Microscopy
This technique is most frequently used for detection of antinuclear antibody (ANA). Frozen sections of mouse liver or other substrates are cut and placed on glass slides or, alternatively, monolayers of cultured cell lines may be used. A patient's serum is placed over the sections and incubated. Fluorescein-conjugated rabbit anti-human immunoglobulin is then applied and washed. ANA specifically binds to the nucleus, and the fluorescein conjugate binds to the human antibody. Fluorescence of the cell nucleus on microscopy indicates a positive test.
Complement Fixation
Specific antigen, unknown serum, and complement are combined. Sheep red blood cells coated with anti-sheep cell antibody are added for 30 minutes at 37 °C. If antigen-specific antibody is present in the patient's serum, complement is bound and consumed, preventing lysis of sheep red cells.

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