Complement Fixation Test and Agglutination Test

Complement fixation test

Here complement is fixed to an antigen-antibody complex. For this reason, an indicator system is involved in the test to detect fixation of complement. In the absence of either antigen or antibody complement will not be fixed. This test is mainly used for detection of antibodies in patient’s serum.

Complement fixation test involves a test system and an indicator system, both of which are capable of activating complement. The test system consists of patient’s serum and known antigen. The indicator system consists of sheep red cells coated with antibody. These cells will lysis in the presence of complement.

Principle

• The Complement fixation test is a technique that has been used over many years to detect and quantify antibody that doesn’t agglutinate or precipitate when reacted with its antigen but can be demonstrated by its use or fixation of complement.
• When complement takes part in antigen – antibody reaction, it is bound or fixed to the antigen- antibody complex cells.
• When these complexes are on bacteria, red cells or other cells, the complement brings about the lysis of cell involved.

CFT consists of two following steps:

1. Complement fixation step
2. Indicator step

Complement fixation step

A known antigen and patient’s serum (which may contain antibody to the antigen) are reacted with a limited amount of reagent complement. If specific complement activating antibodies present in the serum, complement will be activated or fixed by the antigen-antibody complex. On the other hand, if there is no antibody in patient’s serum, there will be no fixed, but will remain free.

Indicator step

In the second step, the indicators system is added to the test. If complement is fixed in the first step, due to the presence of antibody in patient’s serum, there will be no Ag-Ab complex and therefore, complement will not be fixed. This on fixed complement will bring about the lysis of red cells. Therefore, no lysis of sheep red cells indicates a presence of antibody in the test serum and lysis of sheep red cells shows a negative test for ab in the serum.

Complement fixation test is complex procedure and requires five reagents:

• Antigen

The antigen may be soluble or particulate.

• Antiserum

The patient's serum which has to be tested for the presence of ab should be inactivated at 56⁰C for 30 minutes to destroy its complement activity so that it will not interfere with the test. The inactivation also removes known specific inhibitors of a complex which may be present.

• Complement

The commonest source of fresh complement is the guinea pig serum.

• Sheep red cells
• Haemolysin

This is an antibody to sheep red cells raised in rabbit.

Complement fixation tests are more complicated to perform but much more sensitive than precipitation and agglutination test. The tests are very valuable in the diagnosis of many viral, rickettsial and mycotic infections.

Uses of Complement fixation tests

1. It is used for serial diagnosis of diseases of disease such as:

• Gonorrhea, Brucellosis- Bacterial diseases
• Typhus fever- Rickettsial diseases
• Syphilis- Spirochetal disease
• Kalazar, Hydatid cyst, Amoebiasis- Parasitic disease

Agglutination test

Agglutination is the observable clumping which occurs when a particulate antigen is mixed with its antibody. This reaction takes place at a suitable temperature, PH, and in the presence of electrolytes. Thus reaction produces visible clumping of cells or particles. That is called agglutinates. It is the clumping of particulate antigen (bacterial cells, RBCs etc) mediated by an antibody (agglutinin).Agglutination test is more sensitive than precipitation test since agglutination reaction can even detect less amount of antigen i.e. it can detect less than 1ug antigen.

Agglutination reaction is the same basic reaction as with precipitation the differences is nature of antigen that is antigen is insoluble in agglutination reaction whereas soluble antigen in the precipitation reaction.

Agglutination reaction is a factor affecting the reaction. Factors responsible for agglutination reactions are:

1. Temperature: Reaction is conducted at room temperature (42-27° C).Antigen-antibody being proteins they are heat sensitive.
2. Electrolyte: Commonly used electrolyte is NaCl with the concentration of 145mmol/lit and a buffer can also be used. It is important for strong covalent bond formation so that clumping will be properly formed as that of precipitation.
3. PH: optimum pH is 7.2.It is important for strong covalent bond formation.
4. Mixing: Proper mixing or shaking for the movement of antigen or antibody in only one direction gives proper agglutination by giving visible clumping.

Agglutination can be performed by following ways:

• Qualitative agglutination
• Quantitative agglutination

1. Qualitative agglutination test

• This test is used to assay for the presence of antigen or antibody.
• This antibody is mixed with antigen and a positive test is indicated by the agglutination of the particulate antigen.
• Example: patients RBC mix with antibody to blood group antigen group antigen to determine a person’s blood type. That is: a patient serum is mixed with RBC of known blood type to assay for the presence of antibody to that blood type in patients serum.

2. Quantitative agglutination test

• It can be used to quantify the level of antibody to a particulate antigen.
• It is used to find out titer value.
• In this test, one makes serial dilutions of a sample be tested for antibody and then adds a fixed number of RBC or bacteria or other such particulate antigen and determines the maximum dilution which gives agglutination.
• The maximum dilution that gives visible agglutination is called the titer.
• The results are reports as the reciprocal of the maximal dilution that gives visible agglutination

Mechanism of agglutination

The mechanism is based on the same principle as that of precipitation that is the formation of the lattice by antigen and antibody combining in optimal concentration (proportion). However, due to the particulate nature of the antigen in agglutination, the reaction takes place in two steps (phases):

• Primary phase
• Secondary phase

1. Primary phase: Here, an antibody molecule binds with an antigenic determination on the particulate antigen.
2. Secondary phase: Here, the same antibody molecule must bind to an antigenic determinant on another particle of antigen to facilitate the formation of a lattice.

Types of agglutination

• Active agglutination (Direct)
• Passive agglutination (Indirect)

1. Active agglutination

It is also known as direct agglutination which uses the antigen where the antigen determinant is inherent to the particles itself.

2. Passive agglutination

It is also known as indirect agglutination. This test uses inert particles coated with antigen (soluble). By passive agglutination, even soluble antigens can be used for agglutination test. The inert particles commonly employed are polystyrene latex, charcoal, red cells etc. They are:

• Tests using latex particles
• Test using RBC
• Tests using carbon particles

Test using latex particles

In this test, polystyrene latex particles are commonly used. For example polystyrene, latex particles are used for detection of antibody to streptolysin 0 of streptococci by coating the latex particles with the streptococcal antigen.

Test using RBC

Tests using RBC agglutination are called hemagglutination test. Tryponema palladium hemagglutination (TPHA) test is one of the examples of hemagglutination test which uses chicken erythrocytes/RBC coated with an extract of Tryponema palladium. These red cells are agglutinated by antibody in the serum of a patient with syphilis.

Tests using carbon particles

Carbon particles mixed with cardiolipin antigen are used in rapid plasma reagin (RPR) test. Patient’s serum is mixed with this antigen and agglutination reaction is made visible by the presence of carbon particles. This is screening test for syphilis.

Hemagglutination inhibition (HAI) test

Some viruses have the ability to induce hemagglutination when to combine in vitro with erythrocytes from specific animal sources. For example, Rubella virus can agglutinate red cells from born chicken. This activity can be neutralized by neutralizing antibodies to the virus.

Antibody to a hemagglutinating virus can be detected first by reacting patient’s serum with the virus. After appropriate incubation, the red cell suspension of susceptible species is added to the suspension of susceptible species is added to the mixture. If an inhibiting antibody is present in the patient's serum hemagglutination will not occur.

Character of HAI test

• This reaction involves agglutination using RBC.
• Here, an antigen is attached to a surface of RBC and then patient’s serum is added.
• Antibody binds with antigen coated with RBC then appear visible clumping.
• It may take several hours to perform a test.
• The specificity and sensitivity depend on antigen used and how cells are prepared.
• This test can be used for detection of antibody.
• Example: Tryponema palladium Hemagglutination for the diagnosis of syphilis (TPHA)

Prozone effect

If increasing quantity of antigen is added to the same amount of antibody in a different tube, precipitation is found to occur most rapidly and abundantly in the middle tubes in which antigens and antibodies are present in optimal or equivalent proportion.

Prozone is important in some serological reaction when an antibody is present in very high titer in the serum because, in such cases, the test result may be wrongly interpreted as a negative test.

• Occasionally, one observation that when the concentration of antibody is high (i.e. low dilution), there is no agglutination and then as the sample is diluted agglutination occurs.
• The lack of agglutination at high concentration of antibody of is called the prozone effect.
• Lack of agglutination in the prozone is due to antibody excess resulting in very small complexes which don’t clump to form visible agglutination.

Uses of agglutination test

1. To perform blood typing
2. For cross matching performance
3. For diagnosis of diseases
4. Serological diagnosis of infection
5. To perform Widal test
6. For identification of types of organisms.

For example: For serotyping of Streptococcus, Salmonella, E. coli.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.

D, Grenwood, Slack RCB and Peutherer J. Medical Microbiology. Dunclude Livingstone: ELBS, 2001.