Complement Fixation

When complement binds to an antigen-antibody complex, it becomes fixed and used

up. Complement fixation tests are very sensitive and can be used to detect extremely small
amounts of an antibody for a suspect microorganism in an individuals serum. A known antigen
is mixed with test serum lacking complement (figure 1a).

Fig: 1a. Test serum is added to one test tube. A fixed amount of antigen is then added to both
tubes. If antibody is present in the test serum, immune complexes form.
When immune complexes have had time to form, complement is added (figure 1b) to the
mixture. If immune complexes are present, they will fix and consume complement.

Fig: (b) When complement is added, if complexes are present, they fix complement and
consume it.
Afterward, sensitized indicator cells, usually sheep red blood cells previously coated with
complement-fixing antibodies, are added to the mixture. Lysis of the indicator cells (figure 1c)
results if immune complexes do not form in part a of the test because the antibodies are not
present in the test serum.
In the absence of antibodies, complement remains and lyses the indicator cells. On the
other hand, if specific antibodies are present in the test serum and complement is consumed by
the immune complexes, insufficient amounts of complement will be available to lyse the
indicator cells. Absence of lysis shows that specific anti-bodies are present in the test serum.
Complement fixation was once used in the diagnosis of syphilis (the Wassermann test) and is still
used as a rapid, inexpensive screening method in the diagnosis of certain viral, fungal, rickettsial,
chlamydial, and protozoan diseases.

Fig: (c) Indicator cells and a small amount of anti-erythrocyte antibody are added to the two
tubes. If there is complement present, the indicator cells will lyse (a negative test): if the
complement is consumed, no lysis will occur (a positive test).
Immunoblotting (Western Blot)
Another immunologic technique used in the clinical microbiology laboratory is
immunoblotting. Immunoblotting involves polyacrylamide gel electrophoresis of a protein
specimen fol-lowed by transfer of the separated proteins to nitrocellulose sheets. Protein bands
are then visualized by treating the nitrocellulose sheets with solutions of enzyme-tagged
antibodies. This procedure demonstrates the presence of common and specific proteins among
different strains of microorganisms.
Immunoblotting also can be used to show strain-specific immune responses to
microorganisms, to serve as an important diagnostic indicator of a recent infection with a
particular strain of microorganism, and to allow for prognostic implications with severe
infectious diseases.
Immunoprecipitation
The immunoprecipitation technique detects soluble antigens that react with antibodies
called precipitins. The precipitin reaction occurs when bivalent or multivalent antibodies and
antigens are mixed in the proper proportions. The antibodies link the antigen to form a large
antibody-antigen network or lattice that settles out of solution when it becomes sufficiently large
3

(figure 2a). Immunoprecipitation reactions occur only at the equivalence zone when there is an
optimal ratio of antigen to antibody so that an in-soluble lattice forms. If the precipitin reaction
takes place in a test tube (figure 2b), a precipitation ring forms in the area in which the optimal
ratio or equivalence zone develops.

Fig:2 Immunoprecipitation. (a) Graph showing that a precipitation curve is based on

the ratio of antigen to antibody. The zone of equivalence represents the optimal ratio for
precipitation.

Fig: 2(b) A precipitation ring test. Antibodies and antigens diffuse toward each other in a test
tube. A precipitation ring is formed at the zone of equivalence.