Complement fixation test (CFT)



Purpose

It is a serological test used to detect unknown Ag or Abs.

Complement

It is a protein present in the serum and has a role in humoral immunity (B and T cells immunity). There are many types of complement proteins e.g., C1, C2, C3,... and C11).

Principle

    "Complement protein has the ability to bind with Fc site of the antibody. It reacts with the Ag-Ab complex present on the cell surface. This complex degrades protein into C1a and C2a (larger and smaller molecules respectively)". C1b acts as a signaling agent. It gives signals to macrophages and these macrophages engulf that Ab-Ab complex and degrade it.

Why it is called CFT

We mix Abs, Ag, and complement protein. The complement will fix with Abs so-called CFT.

Stages of test

CFT comprise of 2 stages
  1. Complement stage
  2. Indicator stage

Complement stage requirements

In this stage, we require Ag (unknown), Abs (known), and complement protein (taken from guinea pig).

1. Procedure for the complement protein collection

  1. Take the blood of guinea pig by cardiac puncture.
  2. Incubate blood at room temp. for some time.
  3. Centrifuge blood at 3000 rpm for 15 minutes.
  4. Collect supernatant (contain serum) and discard sediment (RBCs).
  5. Again centrifuge supernatant at 3000 rpm for 15 minutes. Collect supernatant and discard sediment.

2. Indicator stage requirements

This stage is for the detection of the complement stage. For this stage, we require sheep RBCs and amboceptors.

I. Collection of sheep RBCs

  • Collect blood from the jugular vein.
  • Centrifuge blood.
  • Collect sediment (contains RBCs).
  • Wash RBCs.
  • Store RBCs at 4oC.

II. Amboceptors

These are anti-RBCs. We inject 1-2 % solution of sheep RBCs in-ear vein of the rabbit. Abs will be produced by rabbit against sheep RBCs.

The procedure of injecting RBCs

  • On day 1st: We inject 0.2 ml of 1-2 % RBCs in the ear vein of the rabbit.
  • On day 2nd: Leave this day.
  • On day 3rd: Increase dose up to 0.4 ml and inject.
  • One day 4th: Leave
  • On day 5th: Increase dose up to 0..6 ml and inject.
Continue this procedure until the dose rate becomes 1 ml (9th day).
  • After the last injection, on day 14th, we slaughter the rabbit and collect blood. We collect serum from the blood.
  • This serum contains amboceptors and complement protein.
  • Heat serum at 56oC. Complement protein will degrade at this temperature. The serum contains only amboceptors.

Standardization

We standardize complement and amboceptors before performing the test.

1. Titration of complement

  • Take microtitration plate.
  • Pour 50 ul of normal saline in microtitration well.
  • Pour 50 ul of complement and make 2 folds serial dilution.
  • Add 50 ul mixture of amboceptor and 1 % RBCs in all the wells.
  • Incubate plate at 37oC for 30 minutes.

Result

  1. If the reaction starts, hemolysis will occur and a reddish solution will be formed (hemolysis is due to C1b).
  2. If the reaction does not start, there is no hemolysis, and button formation will occur.
Note: Divide the conc. of last hemolysed well with 8. Use the complement equal to the conc. of result for the performance of the fixation test.

2. Titration of amboceptor

  1. The procedure is the same as described above except that in this titration, divide the last hemolysed well with 4 and use amboceptor equal to resulted in conc. for the performance of CFT.

Procedure for CFT

I. Complement stage

  1. Add known Abs in a test tube.
  2. Add unknown Ags in a test tube.
  3. Add complement in a test tube.
  4. If the reaction is positive, complement protein will degrade but if the reaction is negative C-protein will not degrade.

II. Indicator stage

  1. Add 1% sheep RBCs and amboceptors in a test tube.
  2. RBCs will bind with Ag and there is the formation of hemolysis. It indicated the test is positive.
  3. If the test is negative, there is no hemolysis, and button formation will occur.

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