Complement Fixation Test

introduction image

Complement fixation test is a serological test which is used to detect the presence of antibody or antigen in the patient’s serum by using a complement. A complement participates in the immunological reaction and absorbs at the time of antigen and antibody binding. Complement particularly binds with the antibody, and in its absence, a complement lyse the erythrocytes.

A complement fixation test is a very complex process, which requires five reagents with separate standardization. It involves preparatory, complement fixation and indicator stage to complete the CF test. A complement fixation test requires an antigen, antibody, complement, sheep erythrocytes and amboceptor to perform the analysis.

Content: Complement Fixation Test

    1. Definition of Complement Fixation Test
    2. Principle
    3. Procedure
    4. Types

Definition of Complement Fixation Test

Complement fixation test can define as the immunological test, which makes the use of complement to detect the presence of antigen (about 0.04-0.06 mg) or antibodies (about 0.1 mg). A complement use as a test system which only fixes to the immunological complex or antigen-antibody complex. The complement causes immune cytolysis or the lysis of antibody-coated cells.

A complement remains free when there is no antibody in the test sample, and it will cause lysis of erythrocytes. If a complement fixes with the antibody, it does not cause hemolysis. CFT also makes the use of sheep erythrocytes as an indicator system which particularly binds with the immunoglobulin-G found in the patient’s serum.

Principle

Complement fixation test depends upon the principle of fixing complement to the antigen-antibody complex. Its principle is not based on the haemagglutinating activity and while performing CFT, the serum must be free of anticomplementary activity.

CFT depends upon two different systems, namely, test and indicator system. A test system includes antigen, antibody and a complement. The indicator system involves the addition of sheep erythrocytes to test for the presence of free complement.

If there will be specific binding between an antigen and antibody, a complement will fix to Ag-Ab complex and if no such binding is present, then a complement will remain free and will lyse the sheep RBCs.There are two ways to perform a complement fixation test, namely positive and negative complement fixation test.

In positive CF test, a complement fixes with the antigen-antibody complex and no hemolysis occurs.

positive complement fixation test
In contrast to this, complement does not fix due to the absence of antibody and hemolysis occurs in a negative CF test.

negative complement fixation test

Procedure

There are three steps or stages to perform a complement fixation test, which includes:

Preparatory Stage

It is the first stage of the complement fixation test, where all the reagents are standardized before performing the experiment. CFT requires, an antigen, inactivated patient’s serum, a complement and an indicator system (Sheep erythrocytes).

  • First, take a soluble or particulate antigen.
  • Then, take an antiserum and heat it to 56 degrees Celsius for half an hour before the complement fixation to remove anticomplementary agents.
  • Take a complement from the source of guinea pig serum and preserve it either in lyophilized or frozen state.
  • Before proceeding to the test, the reagents that have taken should be standardized. Titrate the guinea pig complement to the minimum haemolytic dose. The MHD of guinea pig serum is considered to be the highest dilution of guinea pig serum which can lyse about one unit volume of sheep erythrocytes in the presence of excess amboceptor within 30-60 minutes at 37 degrees temperature. One MHD of amboceptor is considered to be the highest dilution of inactivated amboceptor which can lyse about one unit volume of sheep erythrocytes in the presence of excess complement within 30-60 minutes at 37 degrees temperature. In CFT, saline added with calcium and magnesium ion is used for the titration of complement and amboceptor.

Complement Fixation Stage

After preparation of the reagents, there is a stage refers as “Complement fixation stage” where a complement fixes with the immunogenic complex (Ag-Ab complex).

  • In complement fixation stage incubate an antigen, inactivated patient’s serum and a standardized complement.

Positive result: If a serum contains a specific antibody which can bind with the specific antigen, can activate or fix the complement. Thus, a complement will attach to the antigen and antibody complex and gives a positive result for the complement fixation stage.

positive result for the complement fixation stage

Negative result: If there is no such binding between antigen and antibody, a complement will remain free in the solution and gives a negative result for the complement fixation stage.

negative result for the complement fixation stage

Indicator Stage

It is the third stage, which detects the presence of free complement.

  • Add Sheep erythrocyte to the above mixture containing antigen, patient’s serum and a complement.

Positive result: If lysis of sheep erythrocytes does not occur, then it indicates that the complement fixes with the antigen and antibody complex and gives a positive result for the CFT.

positive result for the indicator stage of CFT

Negative result: Lysis of sheep erythrocytes indicates the presence of free complement and gives a negative result for the CFT.

negative result for the indicator stage of CFT

Types

To perform a complement fixation test, there are the following types of methods which are explained below:

Wasserman Test

It is a routine method for the serological diagnosis of Syphilis. In Wassermann reaction, incubate a test serum of patient for one hour at 35-37 degrees Celsius. After incubation, mix a serum with the test antigen and two units of guinea pig complement.

If a serum contains syphilitic antibody, it will bind to the test antigen, and a complement will fix to the antigen-antibody complex. If a serum does not contain syphilitic antibody, then there will be no antigen-antibody formation, and the complement will remain free in the solution.

Take sensitized sheep erythrocytes and coat it with 4MHD hemolysin, to detect the presence of antibodies in the serum. Then the whole mixture is incubated at 35-37 degrees Celsius for 30 minutes.

Negative CFT: Lysis of sheep erythrocytes will indicate no complement fixation and the absence of antibodies.

Positive CFT: In this, the lysis of sheep erythrocytes does not occur. Absence of hemolysis will indicate the fixation of complement and the presence of antibodies.

Indirect Complement Fixation Test

This method is used to check the avian and mammalian serum which do not fix the complement of guinea pig. The indirect method involves two series of test. First, the standard antiserum fixes with the complement and the test antigen will get utilized.

The subsequent addition of antiserum will not fix the complement. Thus, in indirect complement fixation test, hemolysis of sheep erythrocytes indicates the positive result.

Conglutinating Complement Adsorption Test

It is an alternative method for the system which cannot fix the complement of guinea pig. In Conglutinating complement adsorption test, non-haemolytic horse compliment uses as an alternative of guinea pig. In this method, mix sensitized sheep erythrocytes with the bovine serum.

A bovine serum acts as an antibody which contains a beta globulin component and refers as “Conglutinin”. Thus, bovine serum agglutinates the sheep erythrocytes when it binds with the horse complement, and the process refers to as “Conglutination”.

If the formation of antigen and antibody complex occurs, then the horse compliment will get utilized, and agglutination of sheep erythrocytes will not occur.

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