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20.2 Detecting antigen-antibody complexes  (Page 8/12)

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Diagram of the complement fixation test. 1. Patient A’s serum contains antibodies to the suspected antigen. Patient B’s serum does not. Both patients have complement, but different amounts. 2. Heating the serum destroys all of the complement in the patient’s serum. Antibodies remain in Patient A’s serum. 3. An equal amount of complement is then added to the serum for both patients. Antigens are also added. In patient A’s serum, antibodies bind to antigens and complement fixation occurs. Patient B’s serum lacks antibodies, so complement fixation does not occur. 4. Sheep RBCs and antibodies to sheep RBCs are added to both samples. 5. In patient A, complement is already fixed and cannot lyse RBCs. The antibodies bind to RBCs and settle to the bottom. In patient B, antibodies bind to RBCs and complement lyses the RBCs. Serum turns pink.
The complement fixation test is used to determine whether a patient’s serum contains antibodies to a specific antigen. If it does, complement fixation will occur, and there will be no complement available to lyse the antibody-bound sheep red blood cells that are added to the solution in the next step. If the sample does not contain antibodies to the antigen, hemolysis of the sheep blood cells will be observed.
Link to learning graphic

View this video to see an outline of the steps of the complement fixation test.

  • In a complement fixation test, if the serum turns pink, does the patient have antibodies to the antigen or not? Explain.

[link] summarizes the various types of antibody-antigen assays discussed in this section.

Mechanisms of Select Antibody-Antigen Assays
Type of Assay Mechanism Examples
Precipitation Antibody binds to soluble antigen, forming a visible precipitin Precipitin ring test to visualize lattice formation in solution
Immunoelectrophoresis to examine distribution of antigens following electrophoresis
Ouchterlony assay to compare diverse antigens
Radial immunodiffusion assay to quantify antigens
Flocculation Antibody binds to insoluble molecules in suspension, forming visible aggregates VDRL test for syphilis
Neutralization Antibody binds to virus, blocking viral entry into target cells and preventing formation of plaques Plaque reduction assay for detecting presence of neutralizing antibodies in patient sera
Complement activation Antibody binds to antigen, inducing complement activation and leaving no complement to lyse red blood cells Complement fixation test for patient antibodies against hard-to-culture bacteria such as Chlamydia

Key concepts and summary

  • When present in the correct ratio, antibody and antigen will form a precipitin , or lattice that precipitates out of solution.
  • A precipitin ring test can be used to visualize lattice formation in solution. The Ouchterlony assay demonstrates lattice formation in a gel. The radial immunodiffusion assay is used to quantify antigen by measuring the size of a precipitation zone in a gel infused with antibodies.
  • Insoluble antigens in suspension will form flocculants when bound by antibodies. This is the basis of the VDRL test for syphilis in which anti-treponemal antibodies bind to cardiolipin in suspension.
  • Viral infections can be detected by quantifying virus-neutralizing antibodies in a patient’s serum.
  • Different antibody classes in plasma or serum are identified by using immunoelectrophoresis .
  • The presence of specific antigens (e.g., bacterial or viral proteins) in serum can be demonstrated by western blot assays, in which the proteins are transferred to a nitrocellulose membrane and identified using labeled antibodies.
  • In the complement fixation test, complement is used to detect antibodies against various pathogens.

Fill in the blank

When slowly adding antigen to an antiserum, the amount of precipitin would gradually increase until reaching the ________; addition of more antigen after this point would actually decrease the amount of precipitin.

equivalence zone or zone of equivalence

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The radial immunodiffusion test quantifies antigen by mixing ________ into a gel and then allowing antigen to diffuse out from a well cut in the gel.

antiserum

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Short answer

Explain why hemolysis in the complement fixation test is a negative test for infection.

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What is meant by the term “neutralizing antibodies,” and how can we quantify this effect using the viral neutralization assay?

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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