The following points highlight the nine important antigen-antibody reactions. They are: 1. Precipitation Reactions 2. Immunodiffusion Test 3. Counter Current Immunoelectroptioresis Test 4. Agglutination Reactions 5. Complement Fixation Reactions 6. Neutralization Reactions 7. Radioimmunoassay 8. Enzyme-Linked Immunosorbent Assay 9. Fluorescent Antibody Technique.
1. Precipitation Reactions:
The reaction of soluble antigens with IgG or IgM antibodies to form a large interlocking aggregates (lattices) is called precipitation reaction. The precipitates formed by antibodies are known as precipitins.
The precipitation reactions occur in two stages:
(i) Rapid interactions within a second between antigen and antibodies and formation of complex.
(ii) Slow rate of reaction completing even within a few minutes or hours and forming lattices from antigen-antibody complexes.
When the antibodies and antigens are in proper ratio, precipitation reactions normally occur. When there is excess amount of either of two, no visible precipitate is formed.
One can produce the optimal ratio of these two by putting antigens and antibody adjacent to each other and waiting for their diffusion together. In precipitation test, a precipitation ring appears which display the creation of optimal ratio. This zone is known as the zone of equivalence (Fig. 22.21).
2. Immunodiffusion Test (IDT):
Immunodiffusion tests are performed in a gelled agar medium. One of the IDTs is Ouchterlony test (Fig. 22.22). In Ouchterlony test wells are cut, into which a purified antiserum (a serum containing antibodies) is added, and to each surrounding well, soluble form of test antigens are added.
Thereafter, a line of visible precipitate is formed between the wells where after diffusion optimal ratio of antigen-antibody is formed. Through the Ouchterlony test, the presence of antibodies in the serum against more than one antigen at a time can be demonstrated. Through this test, identical, partially identical and different types of antigens can also be found out.
3. Counter Current Immunoelectroptioresis Test (Counter Innmunoelectroptioresis (cie)):
CIE not only depends entirely on diffusion of antigen and antibody in a gel, but also uses electrophoresis for their rapid movement (Fig. 22.23). By using this method protein can be separated within an hour. CIE is useful for the diagnosis of bacterial meningitis and the other diseases.
The principle of CIE is based on the movement of antigens and antibodies to opposite poles after applying electric current in buffers of correct electric strength and pH, because some of the antigens and antibodies have the opposite charges. If a reaction occurs, a precipitation line appears within an hour.
4. Agglutination Reactions:
Agglutination is the process of linking together of antigens by antibodies and formation of visible aggregates. Agglutination reactions involve particulate antigens i.e. soluble antigens adhering to particles. Agglutination reactions are very sensitive, readable and available in several varieties.
It is of two types, direct and indirect agglutination tests:
1. Direct Agglutination Test:
Direct agglutination test diagnoses antibodies against a large number of cellular antigens such as RBCs, bacteria and fungi. This test is carried out in plastic microtiter plates that have several small shallow wells. Each well acts as small test tube (Fig. 22.24).
Previously this test was done in test tubes. However, each well contains an equal amount of particulate antigen (e.g. RBCs) but the amount of antibodies in the serum is serially diluted in successive wells so that their concentration may be half of the previous well.
If one starts with more antibodies, more dilutions will be required to lower the amount to a point at which agglutination does not occur. This is the measure of titer or concentration of serum antibody.
In a positive reaction, agglutination occurs, and sufficient antibodies are present in the serum to link the antigen together. This results in formation of antibody-antigen mat which sinks to bottom of well (A). However, in the negative reaction, agglutination does not occur and insufficient antibodies are present to cause the linking of antigens.
The particulate antigens roll down the sloping sides of the well, and form a pellet at the bottom. In this example the antigen titer is only 80 since the well with a 1: 80 concentration is the most dilute concentration that gives a positive reaction. It may also be demonstrated that before illness, blood of persons does not have any antibody, whereas titer develops significantly with the progress of disease. This change in titer is called servotiter.
2. Indirect (Passive) Agglutination Tests:
This type of diagnostic tests are very rapid particularly for the detection of streptococci. If the antigens are adsorbed onto particles (e.g. RBCs, latex beads, bentomile clay), soluble antigens can respond to agglutination test. Antibody reacts with the soluble antigen adhering to the particles. Therefore, the particles agglutinate with each other as these do in the direct agglutination tests.
3. Haemagglutination:
Haemagglutination is the phenomenon of clumping of RBCs. When the RBCs are agglutinated by certain viruses such as those causing mumps, measles, influenza, etc. it is called viral haemagglutination. In the serum of a person, certain antibodies act against the antigens (of these viruses), the antibodies neutralize them after reaction. The haemagglutination test is widely used for the diagnosis of a number of viruses including those as above.
5. Complement Fixation Reactions:
A group of 20 or more serum protein is collectively known as complement. During reaction, the complement binds to antigen-antibody complex and is used up or fixed. This process of complement fixation may be used to measure even very small amount of antibody that does not produce a visible reaction such as precipitation or agglutination. Therefore, it is necessary to use indicator system.
This method is used in diagnosis of diseases such as leptospirosis, mycoplasmal pneumonia, Q fever, polio, rubella, histoplasmosis, coccidiodomycosis and streptococcal infections. The test requires patient’s serum, test antigen, complement from guinea pig and antibodies of sheep RBCs to determine whether sheep RBCs may be lysed by guinea pig complement.
The test is accomplished in the following two stages:
Stage 1:
The patient’s serum is heated at 56°C for 30 minutes so that the complement should be inactivated. The heated serum is diluted and then added to known amount of specific antigen and complement (Fig. 22.24). The test antigen may correspond to the diseases.
For example, if a patient is suffering from a disease caused by streptococci the test antigen would be the streptococcal antigen. If the patient’s serum contains antibodies against streptococci, the test antigen will form complement sequence. This mixture is again incubated for about 30 minutes. At this point, no antigen-antibody reaction occurs.
Stage 2:
In stage 2, the complement fixed by antigen-antibody reaction is detected by an indication system. This system consists of sheep RBCs containing specific antibodies attached to their surfaces.
When these are added to complement, haemolysis of RBCs occurs that impart changes in colour of the mixture. This shows that the complements have not been fixed during the first stage; therefore, these become available to cause haemolysis (Fig. 22.25). This indicates that the patient has no streptococcal pneumonia.
However, if the guinea pig complements are destroyed, they will not be able to cause the lysis of RBCs. On the other hand, if the complements are fixed (by antigen- antibody reaction) during the first stage, these will not be available to cause haemolysis during the second stage. This indicates that the patient has the infection of streptococci.
6. Neutralization Reactions:
The neutralization reactions are the reactions of antigen- antibody that involve the elimination of harmful effects of bacterial exotoxins or a virus by specific antibodies. These neutralizing substances i.e. antibodies are known as antitoxins. This specific antibody is produced by a host cell in response to a bacterial exotoxin or corresponding toxoid (inactivated toxin).
The antitoxin reacts with exotoxin and neutralizes it. These antitoxins can be artificially induced in animals such as horses. Thus, the antitoxin of animal sources in turn can be injected into human which provides a passive immunity against a toxin present in human body produced by the pathogens causing diphtheria, tetanus, etc.
1. Diagnosis of Viral Infections:
Neutralization test is very useful in diagnosis of viral infections in humans. After introduction of a virus, antibodies are produced in response and bind to receptor sites present on the viral surfaces. After binding of antibodies, viral particles fail to reach to the cells. Thereafter, the virus is destroyed.
Artificially, the virus is capable of destroying their cell-damaging effect in cell culture or embryonic eggs can be used to determine the presence of antibodies against them. However, when serum contains antibodies against a particular virus, the antibodies will not allow the virus to infect the cell in cell culture; consequently the cells will not be damaged.
2. Schick Test:
Schick test measures the level of immune system of a person to the infection of diphtheria. When testing the status of immunity, a small amount of diphtheria exotoxin is inoculated in the skin of a person. Depending on ability and quantity of antitoxin, positive or negative responses develop.
If serum antitoxin in body would be in sufficient amount to neutralize the exotoxin, no visible reaction will occur. In control, when serum antitoxin is in insufficient amount the exotoxin will damage the tissues at the site where incision was made, and will produce a swollen and reddish area which is converted into brown within 4 or 5 days. This shows that the immune response is not present to a satisfactory level.
7. Radioimmunoassay (RIA):
It is such a technique which is highly sensitive and can measure even the less concentration (i.e. 0.001 µg/ml) of antigen or antibody. In 1960, for the first time this technique was developed by S.A. Berson and R. Yalow when they were engaged in determining the concentration of insulin and anti-insulin complexes in diabetics. Thereafter, Berson died, and significance of this technique was realised. In 1977, Yalow was awarded a Nobel Prize.
There are two methods of measuring RIA: the liquid phase and the solid phase RIAs.
1. Liquid Phase RIA:
The liquid phase RIA is based on competitive binding of radiolabeled antigen and un-labelled antigen, to a high affinity antibody. The antigen labelled with 125I is mixed with such a concentration of antibody that can just saturate the antibody. Therefore, the increasing amount of antigen (un-labelled) of unknown concentration is added. The two types of antigens now compete for available sites of the antibody.
The antibody does not differentiate the labelled antigen from the un-labelled one. Upon gradually increasing concentration of un-labelled antigen, the labelled antigen could be displaced from the binding sites available on antibody. The labelled antigens are made free in the solution. The amount of labelled antigen in solution is measured, and the concentration of un-labelled antigen can be determined.
2. Solid Phase RIA:
In solid phase RIA, either antigen or antibody is immobilized on a solid phase matrix. It is simple and easy in handling as compared to liquid phase RIA.
8. Enzyme-Linked Immunosorbent Assay (ELISA):
The principle of ELISA is similar to RIA, but differs slightly. In RIA radiolabelled antigen is used, whereas in ELISA enzyme is used that reacts with a colourless substrate and develops a coloured reaction product. There is a large number of enzymes such as alkaline phosphatase, horse radish peroxidase, and p-nitro-phenyl phosphatase which are employed in ELISA. As compared to RIA, this assay is both cheaper and safer.
On the basis of known concentration of antigen or antibody a standard curve is prepared from which the unknown concentration of sample is measured. A microliter plate with numerous shallow wells is used in this method. It is very useful in testing for AIDS antibodies. However, now-a days a number of ELISA kits have been developed and are in current use.
1. Indirect ELISA:
It is used to measure antibody. Known antigen is coated on the plastic lining of the wells of microtiter plate which is made up of polystyrene latex. To test for the presence of antibodies against this antigen in the patient, his blood serum is added to the wells (Fig. 22.26A). If the patient’s serum contains antibody specific to antigen, the antibody will bind to the absorbed antigen otherwise not.
After incubation the wells are washed and the enzyme, labelled with antihuman gamma globulin (anti-Hgg), is added to the wells. Anti-Hgg can react with antigen antibody complex. The mixture of wells is washed to remove the excess of unbound labelled anti- Hgg.
Finally the correct substrate for the enzyme is added which is hydrolysed by the enzyme and develops a colour. Varying concentrations of antibody in serum shows changes in the intensity of colour. This method is very useful in detection of antibodies to HIV, Salmonella, Yersinia, Brucella, Treponema and streptococci.
2. Double Antibody Sandwich ELISA:
This method detects antigen. In this case antibody (antiserum) is immobilised on the surface of wells of microtiter plate (Fig. 22.26B). A test antigen is added to each well and allowed to react with the bound antibody. It is incubated during this period. If antigen combines specifically with antibody absorbed to wells, the antigen will be retained even after washing and unbound antigen would be made free.
Thereafter, a second enzyme-linked antibody (e.g. alkaline phosphatase tagged to antibody) is added to react with bound antigen. It is again incubated for a few seconds, the enzyme labelled antibody reacts with the antigen-antibody complex already formed in the wells and results in the development of a “sandwich’.
The mixture in wells is washed again to remove the excess of labelled enzyme. A chromogenic substrate e.g. nitro-phenyl phosphate is added which reacts with enzyme and develop yellow colour.
The reaction can be stopped simply by changing the pH or denaturing the enzyme. The change in colour is measured visually or spectrophotometrically. Change in colour shows the presence of desired antigen in the sample. This technique is useful in detection of toxins of Vibrio cholerae, E. coli, Staphylococcus enterotoxin-A and antigens of rotavirus.
9. Fluorescent Antibody (FA) Technique:
The FA technique is used to detect the microorganisms present in clinical specimens, and specific antibodies present in serum. If the antibodies bind to cell or tissues, it can be observed by tagging the antibody with a fluorescent dye such as fluorescein isothiocyanate and rhodamine.
Both the dyes can conjugate the FC region of antibody without affecting the specificity and make the antibody fluorescent when exposed to UV light. Fluorescein absorbs blue light (490 nm) and emits yellow green fluorescence (517 nm). Similarly, rhodamine absorbs the yellow green light (515 nm) and emits deep red fluorescence (546 nm). The FA technique is very useful in testing for rabies within a few hours with 100% accuracy.
There are two methods of FA test, direct FA test and indirect FA test. Direct FA test is used to identify the microorganisms present in clinical specimen. The specimen containing antigen is fixed onto a slide and, thereafter, fluorescein-labelled antibodies are added on the specimen. It is incubated for a few minutes.
The slide is washed to remove unbound antibody and observed under the UV microscope for yellow-green fluorescence. The indirect FA test is useful for the detection of specific antibodies in serum formed by a microorganism.
This method follows the following steps:
(a) Fix a known antigen onto a slide,
(b) Add a test serum (microorganism-specific antibody reacts with antigen and forms a bound complex,
(c) Add fluorescein-labelled anti-Hgg to the slide,
(d) Incubate and wash the slide,
(e) Examine the slide under fluorescence microscope.
The development of fluorescence confirms the presence of antibody specific to antigen fixed on slide.