Complement Fixation Test- Principle, Procedure, Results, Types

Complement fixation is a technique that can be used to detect the presence of specific antibodies or antigens in a patient`s serum. It is based on the ability of complement proteins to bind to antigen-antibody complexes and mediate their destruction. Complement proteins are a group of serum proteins that are involved in the immune response and can cause lysis of cells, opsonization of pathogens, inflammation and immune clearance.

Complement fixation was widely used in the past to diagnose infections and autoimmune diseases, but it has been largely replaced by newer methods such as ELISA and PCR. However, it is still useful for some applications, such as detecting antibodies against certain pathogens that are difficult to culture or identify by other means.

The principle of complement fixation is that if a patient`s serum contains antibodies against a specific antigen, they will form complexes with the antigen and consume the complement proteins. This will leave no complement available for the indicator system, which consists of sheep red blood cells coated with antibodies. These cells will not be lysed by complement and will remain intact. On the other hand, if the patient`s serum does not contain antibodies against the antigen, the complement proteins will not be used up and will be able to lyse the indicator cells, causing hemolysis.

The procedure of complement fixation involves several steps:

• The patient`s serum is heated to inactivate any endogenous complement proteins and then diluted.
• The antigen of interest is added to the serum and incubated at 37°C for 30 minutes.
• A source of exogenous complement proteins (usually from guinea pig serum) is added to the mixture and incubated again.
• The indicator system (sheep red blood cells and anti-sheep antibodies) is added and observed for hemolysis.

The result of complement fixation is interpreted as follows:

• If hemolysis occurs, it means that the patient`s serum does not contain antibodies against the antigen and the test is negative.
• If no hemolysis occurs, it means that the patient`s serum contains antibodies against the antigen and the test is positive.

Complement fixation can also be used to detect antigens in a patient`s serum by adding a known antibody instead of an antigen in the first step. The rest of the procedure is similar.

Complement fixation can be made semi-quantitative by performing serial dilutions of the patient`s serum and determining the highest dilution that still gives a positive result. This dilution factor corresponds to the titer of antibodies or antigens in the serum.

Complement fixation has some advantages and limitations as a diagnostic method:

• Advantages: It can detect antibodies or antigens that are not easily detected by other methods; it can measure low levels of antibodies or antigens; it can be used for various types of infections and diseases; it has good sensitivity.
• Limitations: It is more complex and time-consuming than other methods; it requires specific reagents and equipment; it can be affected by factors such as temperature, pH, concentration and quality of reagents; it has lower specificity than other methods.

Complement fixation is a technique that measures the presence and amount of antigen-antibody complexes in a sample by using a complement protein and an indicator system. The complement protein is a part of the immune system that can bind to the antigen-antibody complex and activate a cascade of reactions that lead to the destruction of the target. The indicator system is usually composed of red blood cells (RBCs) coated with antibodies that can also bind to the complement protein. The indicator system serves as a way to detect whether the complement protein has been fixed by the antigen-antibody complex or not.

The principle of complement fixation is based on the fact that the complement protein is limited in quantity and can only bind to one antigen-antibody complex at a time. Therefore, if the sample contains a specific antigen or antibody of interest, it will form a complex with its complementary reactant (antigen or antibody) and fix the complement protein. This will prevent the indicator system from binding to the complement protein and cause no change in the appearance of the sample. This is considered a positive test result.

On the other hand, if the sample does not contain the specific antigen or antibody of interest, it will not form a complex with its complementary reactant and leave the complement protein free. This will allow the indicator system to bind to the complement protein and trigger a reaction that causes the lysis (breakdown) of the RBCs. This will change the appearance of the sample and make it clear or pink. This is considered a negative test result.

Complement fixation can be used to detect various types of infections caused by bacteria, viruses, fungi, or parasites. It can also be used to measure the level of immunity or exposure to certain diseases. Complement fixation is one of the oldest and most classical methods of immunological testing, but it has some limitations such as complexity, low sensitivity, and cross-reactivity. Therefore, it has been largely replaced by newer and more advanced techniques such as enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR).

A complement fixation test requires the following components to perform the analysis:

• Samples such as serum or cerebrospinal fluid (CSF) that may or may not contain the specific antigens or antibodies of interest. The samples are usually heated at 56°C for 30 minutes to inactivate any endogenous complement proteins that may interfere with the test.
• Known complementary antigens or antibodies based on the component desired to be detected. For example, if the test is for detecting antibodies against a certain infectious agent, then the known antigen of that agent is added to the sample. Conversely, if the test is for detecting antigens of a certain infectious agent, then the known antibody against that agent is added to the sample.
• Complement proteins that are obtained from an exogenous source, usually guinea pig serum. The complement proteins are essential for mediating the antigen-antibody reaction and triggering the indicator system. The amount of complement added to the sample is standardized and limited, so that any excess or deficiency of complement can affect the outcome of the test.
• Indicator system that consists of sheep erythrocytes (red blood cells) coated with antibodies (mainly derived from rabbit serum) on their surface. These erythrocytes are also called sensitized erythrocytes. The indicator system is used to detect whether the complement proteins have been fixed by the antigen-antibody complex or not. If the complement proteins are fixed, then there will be no hemolysis (rupture) of the erythrocytes. If the complement proteins are free, then they will bind to the antibodies on the erythrocytes and cause hemolysis .

The principle of complement fixation test is based on the ability of antigen-antibody complexes to activate or fix a limited amount of complement proteins in the serum. Complement proteins are a group of molecules that participate in the immune response and can cause cell lysis, inflammation, and opsonization. When an antigen and its specific antibody bind together, they form a complex that can attach to the complement proteins and trigger a cascade of reactions that result in the consumption or fixation of the complement. This means that there will be no free complement left in the serum to react with other molecules.

The complement fixation test uses this principle to detect the presence of either specific antigen or specific antibody in a patient`s serum. The test consists of two stages: the complement fixation stage and the indicator stage.

In the complement fixation stage, the patient`s serum is first heated to inactivate any endogenous complement proteins that may interfere with the test. Then, a known amount of standardized complement (usually obtained from guinea pig serum) is added to the serum, along with a known antigen or antibody (depending on what is being tested for). The mixture is incubated for a certain period of time to allow any antigen-antibody complexes to form and fix the complement. If the patient`s serum contains the specific antibody or antigen of interest, there will be no free complement left in the mixture. If not, there will be some free complement remaining.

In the indicator stage, a system that can detect the presence or absence of free complement is added to the mixture. This system consists of sheep red blood cells (sRBCs) that have been coated with antibodies (usually obtained from rabbit serum) on their surface. These sRBCs are also called sensitized RBCs because they are sensitive to lysis by complement. If there is free complement left in the mixture, it will bind to the antibodies on the sRBCs and cause them to lyse, releasing hemoglobin into the solution. This will result in a color change that can be observed visually or measured spectrophotometrically. If there is no free complement left in the mixture, it means that it has been fixed by antigen-antibody complexes, and there will be no lysis of sRBCs. The solution will remain clear and colorless.

Therefore, the outcome of the complement fixation test depends on whether hemolysis occurs or not. Hemolysis indicates a negative test result, meaning that there is no specific antibody or antigen in the patient`s serum. No hemolysis indicates a positive test result, meaning that there is specific antibody or antigen in the patient`s serum.

The test can be made semi-quantitative by performing serial dilutions of the patient`s serum and determining the highest dilution factor that still yields a positive result. This dilution factor corresponds to the titer of the antibody or antigen in the serum.

The complement fixation test (CFT) is based on the principle that the antigen-antibody complex can only fix the complement and its effect on the hemolysis of RBC used in the indicator system. Hemolysis refers to the lysis or rupture of RBC, which releases hemoglobin into the solution and changes its color. The presence or absence of hemolysis indicates whether the test is positive or negative for the specific antibody or antigen of interest.

• Positive CFT: If no hemolysis is observed, it indicates a positive complement fixation test. This means that the sample contains the specific antibody or antigen of interest, and that the antigen-antibody reaction and complement fixation occurred in the first stage of the test. As a result, no free complement is available to lyse the RBC in the indicator system. The solution remains clear and red, indicating that the sensitized RBC are intact .
• Negative CFT: If hemolysis of RBC is observed, it indicates a negative complement fixation test. This means that the sample does not contain the specific antibody or antigen of interest, and that no antigen-antibody reaction and complement fixation occurred in the first stage of the test. As a result, free complement is available to lyse the RBC in the indicator system. The solution becomes pink or colorless, indicating that the sensitized RBC are lysed .

The degree of hemolysis can be measured by comparing the color of the solution with standard color charts or by using a spectrophotometer. The intensity of hemolysis is inversely proportional to the amount of specific antibody or antigen in the sample. To determine the titer or concentration of specific antibody or antigen in the sample, serial dilutions can be performed and subjected to the same test procedure. The highest dilution that still shows a positive result (no hemolysis) is considered as the titer.

The complement fixation test consists of two stages: the complement fixation stage and the indicator stage. The following steps describe the general procedure for the test :

1. A serum sample is taken from the patient and heated at 56 °C for 30 minutes to inactivate any endogenous complement proteins that may interfere with the test.
2. The serum is then adsorbed with washed sheep red blood cells (RBCs) to remove any cross-reactive anti-RBC antibodies that may be present in the serum.
3. The test antigen (the antigen of interest) and a known amount of complement proteins (usually obtained from guinea pig serum) are added to the serum and incubated at 37 °C for 30 minutes. This allows the formation of antigen-antibody complexes and the fixation of complement proteins by these complexes, if the serum contains specific antibodies against the test antigen.
4. The indicator system, which consists of sheep RBCs coated with anti-sheep RBC antibodies (also called sensitized RBCs), is added to the serum and incubated at 37 °C for another 30 minutes. This allows the detection of any free or unbound complement proteins in the serum by their ability to lyse the sensitized RBCs.
5. The serum is then observed for any change in color or appearance due to hemolysis (the rupture of RBCs and release of hemoglobin) or lack thereof.

The complement fixation test results are based on the presence or absence of hemolysis in the indicator system. Hemolysis is the rupture of red blood cells and the release of hemoglobin into the solution, which gives it a red color. The degree of hemolysis can be visually assessed or measured by a colorimeter.

• If the sample contains the specific antibody or antigen of interest, there will be no change in the solution after the test and it will be considered a positive test. The non-hemolyzed sensitized RBCs will remain intact and settle down in the sample. This means that the complement was fixed by the antigen-antibody complex in the first stage and was not available for the indicator system in the second stage .
• If there is some change in the appearance of the solution during the test due to hemolysis, it will be considered a negative test. The hemolyzed sensitized RBCs will give a red color to the solution. This means that the complement was not fixed by the antigen-antibody complex in the first stage and was available for the indicator system in the second stage .

The amount of hemolysis can be graded as follows:

• 0: No hemolysis, clear supernatant
• 1+: Slight hemolysis, pink supernatant
• 2+: Moderate hemolysis, red supernatant
• 3+: Complete hemolysis, clear supernatant

Some samples may show partial or incomplete fixation of complement, which results in partial hemolysis. This can be due to low antibody or antigen concentration, low complement activity, or nonspecific fixation of complement by other factors.

The complement fixation test can also be used to measure the amount of specific IgG antibody in the serum by titrating different dilutions of the serum and finding the highest dilution that still gives a positive result. The reciprocal of this dilution is called the complement fixation titer and it reflects the level of antibody response to an infection . For example, a serum with a complement fixation titer of 1:32 has more IgG antibodies than a serum with a titer of 1:8.

The interpretation of complement fixation test results should also take into account the clinical history, symptoms, and other laboratory findings of the patient. Some factors that may affect the accuracy and reliability of the test are:

• The quality and specificity of the antigen and antibody reagents
• The presence of anticomplementary substances or inhibitors in the serum
• The cross-reactivity or heterogeneity of antigens or antibodies

• The timing and frequency of sample collection

  Applications of Complement Fixation Test

Complement fixation test (CFT) is a versatile technique that can be used to detect the presence of either specific antigen or specific antibody in a patient`s serum, based on whether complement fixation occurs. It can also be used to measure the amount of antibody in serum by determining the highest dilution factor that will still yield a positive CFT. CFT has been widely used to diagnose infections, particularly with microbes that are not easily detected by culture methods, and in rheumatic diseases. However, in clinical diagnostics labs it has been largely superseded by more sensitive and specific methods such as ELISA and PCR.

Some of the applications of CFT are:

• Wasserman`s test is one of the CFTs for the detection of syphilis. It is an antibody detection test that uses cardiolipin antigen derived from beef heart. A positive test indicates the presence of anti-cardiolipin antibodies in the patient`s serum, which are produced in response to Treponema pallidum infection. However, this test is not very specific, as other conditions such as leprosy, malaria, tuberculosis, and pregnancy can also cause false-positive results.
• Treponema pallidum immobilization test (TPI) is another CFT for the diagnosis of syphilis. It is an antigen detection test that uses live T. pallidum as antigen and patient`s serum as antibody source. A positive test shows serum to contain treponemal antibodies that immobilize the spirochetes in the presence of complement. This test is considered the gold standard for the serodiagnosis of syphilis, as it is highly specific and sensitive.
• Vibriocidal antibody test is a CFT for the detection of anti-cholera antibodies. It is a cytolytic test that uses live Vibrio cholerae as antigen and patient`s serum as antibody source. A positive test shows serum to contain vibriocidal antibodies that lyse the bacteria in the presence of complement. This test is used to measure the immune response to cholera vaccination or infection.
• Mycoplasma pneumoniae complement fixation test (MPCFT) is a CFT for the diagnosis of M. pneumoniae infection, which causes atypical pneumonia. It is an antibody detection test that uses M. pneumoniae antigen derived from culture filtrates. A positive test indicates the presence of anti-M. pneumoniae antibodies in the patient`s serum, which are usually detectable 10-14 days after onset of symptoms. However, this test has low sensitivity and specificity, and cross-reactions with other mycoplasmas can occur.
• Bordetella pertussis complement fixation test (BPCFT) is a CFT for the diagnosis of B. pertussis infection, which causes whooping cough. It is an antibody detection test that uses B. pertussis antigen derived from whole-cell lysates or purified components. A positive test indicates the presence of anti-B. pertussis antibodies in the patient`s serum, which are usually detectable 2-3 weeks after onset of symptoms. However, this test has low sensitivity and specificity, and cross-reactions with other Bordetella species can occur.

• Fungal complement fixation tests are CFTs for the diagnosis of various fungal infections, such as histoplasmosis, coccidioidomycosis, blastomycosis, cryptococcosis, and aspergillosis. They are antibody detection tests that use fungal antigens derived from culture filtrates or cell extracts. A positive test indicates the presence of anti-fungal antibodies in the patient`s serum, which are usually detectable 2-6 weeks after onset of symptoms. However, these tests have low sensitivity and specificity, and cross-reactions with other fungi can occur.

  Advantages and Limitations of Complement Fixation Test

Complement fixation test (CFT) is a classical technique for detecting antigen-antibody complexes in a sample. It has some advantages and limitations that should be considered before using it.

Advantages of CFT

• CFT can screen against a large number of viral and bacterial infections at the same time by using a pool of different antigens .
• CFT is economical as it uses relatively crude antigens (such as infected cell lysates) and inexpensive reagents (such as sheep erythrocytes and guinea pig complement) .
• CFT can detect both immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against specific microbial antigens, which can help in diagnosing acute and chronic infections .
• CFT can measure the antibody titer by using serial dilutions of the sample and determining the highest dilution that still yields a positive result .

Limitations of CFT

• CFT is not sensitive enough to detect low levels of antibodies or antigens, which limits its use for immunity screening or early diagnosis .
• CFT is time-consuming and labor-intensive as it involves multiple steps of heating, adsorption, incubation, and observation .
• CFT is often non-specific as it may show cross-reactivity between different antigens or antibodies, such as herpes simplex virus (HSV) and varicella-zoster virus (VZV) .
• CFT may be affected by anticomplementary factors in the sample, such as immune complexes, cryoglobulins, bacteria, or endotoxins, which can consume complement and interfere with the test result .
• CFT has been largely replaced by newer and more reliable methods of serological testing, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), which are more sensitive, specific, rapid, and easy to perform .

Types of Complement Fixation Tests

There are several different types of complement fixation tests (CFTs) that can be used to diagnose different conditions. Some examples include:

• Syphilis CFT: This test is used to diagnose syphilis, a sexually transmitted infection caused by the bacterium Treponema pallidum. The patient`s serum is tested for the presence of antibodies against the Treponema pallidum antigen. A positive test indicates that the patient has been exposed to syphilis .
• Indirect CFT: This test is used when the patient`s serum cannot fix guinea pig complement, which is usually the source of complement in CFTs. This may happen with some avian or mammalian sera. In this test, a standard antiserum against a known antigen that can fix complement is added to one set of tubes, while the patient`s serum is added to another set. The antigen and the complement are then added to both sets. If the patient`s serum contains antibodies against the antigen, they will bind to it and fix the complement, leaving no complement for the standard antiserum to react with. This will result in no hemolysis of the indicator system (sensitized sheep red blood cells). On the other hand, if the patient`s serum does not contain antibodies against the antigen, the standard antiserum will bind to it and fix the complement, leading to hemolysis of the indicator system .
• Conglutinating Complement Absorption Test (CCAT): This test uses horse complement, which is non-hemolytic, instead of guinea pig complement. The indicator system consists of sensitized sheep red blood cells mixed with bovine serum, which contains a beta-globulin called conglutinin. Conglutinin can bind to complement and cause agglutination of the sheep red blood cells. If the patient`s serum contains antibodies against the antigen, they will bind to it and fix the horse complement, preventing it from binding to conglutinin. This will result in no agglutination of the indicator system. However, if the patient`s serum does not contain antibodies against the antigen, the horse complement will remain free and bind to conglutinin, causing agglutination of the indicator system .
• Immune Adherence Test: This test uses some bacteria (such as Vibrio cholerae or Treponema pallidum) that can adhere to erythrocytes or platelets when they combine with their specific antibodies in the presence of complement. This phenomenon is called immune adherence and it facilitates phagocytosis of bacteria. The patient`s serum is mixed with bacteria and complement and then added to erythrocytes or platelets. If the patient`s serum contains antibodies against the bacteria, they will bind to them and cause immune adherence, resulting in visible clumping of erythrocytes or platelets. If the patient`s serum does not contain antibodies against the bacteria, no immune adherence will occur and no clumping will be observed .
• Immobilization Test: This test uses live bacteria (such as Treponema pallidum) that can be immobilized by their specific antibodies in the presence of complement. The patient`s serum is mixed with bacteria and complement and then observed under a microscope. If the patient`s serum contains antibodies against the bacteria, they will bind to them and fix the complement, resulting in immobilization of bacteria. This test is considered as the gold standard for serodiagnosis of syphilis .
• Cytolytic Test: This test uses live bacteria (such as Vibrio cholerae) that can be lysed by their specific antibodies in the presence of complement. The patient`s serum is mixed with bacteria and complement and then incubated at 37°C for some time. The bacterial lysis is measured by a decrease in turbidity or optical density of the solution. This test is used to measure anti-cholera antibodies .

There are several different types of complement fixation tests (CFTs) that can be used to diagnose different conditions. Some examples include:

• Syphilis CFT: This test is used to diagnose syphilis, a sexually transmitted infection caused by the bacterium Treponema pallidum. The patient`s serum is tested for the presence of antibodies against the Treponema pallidum antigen. A positive test indicates that the patient has been exposed to syphilis .
• Indirect CFT: This test is used when the patient`s serum cannot fix guinea pig complement, which is usually the source of complement in CFTs. This may happen with some avian or mammalian sera. In this test, a standard antiserum against a known antigen that can fix complement is added to one set of tubes, while the patient`s serum is added to another set. The antigen and the complement are then added to both sets. If the patient`s serum contains antibodies against the antigen, they will bind to it and fix the complement, leaving no complement for the standard antiserum to react with. This will result in no hemolysis of the indicator system (sensitized sheep red blood cells). On the other hand, if the patient`s serum does not contain antibodies against the antigen, the standard antiserum will bind to it and fix the complement, leading to hemolysis of the indicator system .
• Conglutinating Complement Absorption Test (CCAT): This test uses horse complement, which is non-hemolytic, instead of guinea pig complement. The indicator system consists of sensitized sheep red blood cells mixed with bovine serum, which contains a beta-globulin called conglutinin. Conglutinin can bind to complement and cause agglutination of the sheep red blood cells. If the patient`s serum contains antibodies against the antigen, they will bind to it and fix the horse complement, preventing it from binding to conglutinin. This will result in no agglutination of the indicator system. However, if the patient`s serum does not contain antibodies against the antigen, the horse complement will remain free and bind to conglutinin, causing agglutination of the indicator system .
• Immune Adherence Test: This test uses some bacteria (such as Vibrio cholerae or Treponema pallidum) that can adhere to erythrocytes or platelets when they combine with their specific antibodies in the presence of complement. This phenomenon is called immune adherence and it facilitates phagocytosis of bacteria. The patient`s serum is mixed with bacteria and complement and then added to erythrocytes or platelets. If the patient`s serum contains antibodies against the bacteria, they will bind to them and cause immune adherence, resulting in visible clumping of erythrocytes or platelets. If the patient`s serum does not contain antibodies against the bacteria, no immune adherence will occur and no clumping will be observed .
• Immobilization Test: This test uses live bacteria (such as Treponema pallidum) that can be immobilized by their specific antibodies in the presence of complement. The patient`s serum is mixed with bacteria and complement and then observed under a microscope. If the patient`s serum contains antibodies against the bacteria, they will bind to them and fix the complement, resulting in immobilization of bacteria. This test is considered as the gold standard for serodiagnosis of syphilis .
• Cytolytic Test: This test uses live bacteria (such as Vibrio cholerae) that can be lysed by their specific antibodies in the presence of complement. The patient`s serum is mixed with bacteria and complement and then incubated at 37°C for some time. The bacterial lysis is measured by a decrease in turbidity or optical density of the solution. This test is used to measure anti-cholera antibodies .

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