Complement fixation reaction

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The complement fixation reaction (KBR) is a test for the detection of antibodies (Ab) in the blood serum of human and animal patients. The KBR has been replaced by the ELISA .

principle

  1. Antigen - antibody complexes activate the complement system , also called complement, and lead to the consumption of complement factors .
  2. The consumption of these complement factors is the measure of the amount of antibodies in the blood serum.
  3. The consumption of complement factors is made visible via antibody-laden erythrocytes ( hemolytic system ).

This results in:

  • Serum with spec. Ak → formation of Ak-Ag complexes → complement is used up → complement cannot trigger hemolysis → serum is Ak-positive
  • Serum without spec. Ak → no Ak-Ag complexes → complement still available, leads to → hemolysis → serum is Ak-negative

application

In human medicine

Proof of Ak against:

In veterinary medicine

Proof of Ak against:

Preliminary tests

Complement preliminary experiment

target
Determination of the optimal complement concentration.
Overdose
Too much complement causes a false negative result in a positive patient serum. This is due to the fact that the complement is not completely used up in the test system. This leads to partial or complete hemolysis.
Underdosing
Too little complement causes a false positive result in a negative patient serum. That is, the complement is insufficient to partially or completely hemolyze the hemolyzing system.
execution
In the preliminary complement experiment, the patient's serum is replaced by veronal buffer. The antigen is used to include the possible anti-complementary effect of the antigen.
evaluation
The highest dilution that shows complete hemolysis corresponds to one unit of complement.

Amboceptor preliminary test

target
Determination of the optimal amboceptor concentration. (The amboceptor are antibodies (Ab) against e.g. sheep erythrocytes, which in the experiment attach to the added sheep erythrocytes and in the negative case attract the complement factors, which causes hemolysis of these erythrocytes).
Overdose
If there is too much amboceptor in the main experiment, agglutination occurs. This is due to the fact that agglutinins are also present.
Underdosing
In the main test, a negative patient serum is false positive. This is due to the fact that the HBK are not completely resolved by the lack of an amboceptor.
execution
In the preliminary amboceptor experiment, patient serum and antigen are replaced by veronal buffer. This is necessary to get the same volume. evaluation
The highest dilution that shows complete hemolysis corresponds to one amboceptor unit.

Antigen preliminary test

target
Determination of the optimal antigen concentration.

Reactions taking place

  1. The patient's serum is inactivated, that is, the complement is withdrawn from it.

Antigen (Ag) of the pathogen against which antibodies (Ab) are to be detected in the serum and complement (which is obtained from guinea pig serum) is added to it.

  1. If the patient is infected, i.e. if there are Ab against the pathogen in the serum, they bind to the added Ag and form a complex. The complement attaches itself to this complex to strengthen the Ag-Ak reaction. If there are no Ak, Ag and complement are free in the solution.
  2. Addition of the hemolytic system. (The haemolytic system consists of sheep erythrocytes and Ak (amboceptor) against them: They are obtained by injecting sheep erythrocytes into rabbits, for example → The erythrocytes are viewed as foreign by the rabbit organism and Ak against them are formed → these are isolated and puts them in the experiment together with other sheep erythrocytes as a "hemolytic system").

A complex is formed from the sheep erythrocytes and the anti-sheep erythrocytes (the sheep erythrocytes act as Ag). If the patient is infected, the complement has already been used up and the sheep erythrocytes are not dissolved (i.e. no hemolysis) because no complement is attached to the anti-sheep erythrocyte-Ak-sheep erythrocyte complex → the test is positive because Ak were present. In the negative case, complement is still available, which attaches to the complex with the sheep erythrocytes and ensures that the Ak can do its job and dissolve the erythrocytes (→ hemolysis).

Individual evidence

  1. ^ A b Anton Mayr, Michael Rolle: Medical Microbiology, Infection and Disease Theory. Georg Thieme Verlag, 2007. ISBN 9783830410607 . P. 127.