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The following points highlight the three main types of serological reactions that occur in human body. The types are: 1. Neutralization 2. Precipitation 3. Agglutination.
Serological Reaction: Type # 1. Neutralization:
Neutralization is a type of serological reaction which occurs between a toxin and its antibody (antitoxin) or between a virus and its antiserum. The reaction is so named, because it leads to neutralization or removal of harmful effects of the antigens.
Among the practical applications of neutralization reaction is detection of botulin toxin in contaminated food, and the Schick test for diagnosis of diphtheria. The spore-forming obligate anaerobe, Clostridium botulinum, produces a deadly poisonous proteinaceous toxin, known as botulin toxin, in canned food when the food has not been properly sterilized. For detection of the presence of botulin toxin in food suspected to be contaminated with C. botulinum, the test material is mixed with botulin-antitoxin (immune serum containing anti-botulin antibodies) and injected into some experimental animals.
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In a control set of animals, the test material alone is injected. If the food contains botulin toxin, the animals injected with test material alone succumb, while the animals receiving test material + anti-botulin antiserum survive. If, the animals of the latter group also die, it would indicate that the food is contaminated with some other poison, but not botulin-toxin.
A. Test material prepared from contaminated food
B. Test material + botulin-antitoxin
I. Injection of A alone into experimental animal —> Death A contains some poisonous material
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II. Injection of A + B into experimental animals —> No death —> Food contains botulin toxin
III. Injection of A + B Death —> Food contains some poison, but not botulin toxin.
The Schick test is for detection of diphtherial antitoxin in the serum of a patient suspected to have contracted diphtheria caused by Corynebacterium diphtheriae. The test is conducted by intradermal injection of a minute quantity of diphtherial exotoxin to the suspected patient. A positive Schick test is indicated by a local inflammatory response characterized by erythema, swelling and tenderness at the injection spot.
Such a response happens because the person has no antitoxin and is free of diphtheria, but, at the same time is, susceptible to an attack of diphtheria because of absence of antitoxin in serum. On the other hand, a negative Schick test indicates the person has diphtherial antitoxin in serum which neutralizes the injected toxin. This may be due to previous immunization or an active infection.
Serological Reaction: Type # 2. Precipitation:
This type of serological reaction occurs between a soluble antigen and antiserum. The resultant antigen-antibody complex forms a lattice which forms a precipitate when the lattice becomes so large that it can no longer remain in a soluble state.
Precipitation reaction can be used for detection of specific antibodies in the serum of a person suspected to be infected by a pathogen. Obviously, the antigenic substance produced by the pathogen must be present in soluble form, e.g. a toxin or a capsular polysaccharide etc.
Precipitation reaction can be carried out either in a liquid system or an agar gel. In a liquid system, precipitation reaction leads to formation of a ring of flocculation at the interface of antigen and antibody solutions. In agar-gel, precipitation zones are produced where the antigen and antibody meet with each other as they diffuse through the gel.
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Liquid precipitation reaction is carried out in narrow tubes (4 to 5 mm in diameter). Equal quantities of the antiserum to be tested are distributed in a series of tubes to form the bottom layer. Then equal quantities of serial dilutions of a known antigen solution in normal saline are added on the top without disturbing the bottom layer.
A zone of flocculation detected by its turbidity appears at the interface within a few hours. With a potent antiserum, a dilution of 10-6 to 10-7 can produce a visible ring. The ring results from combination of a large number of antigen molecules with antibodies belonging to IgG and IgM classes present in the antiserum of the patient.
In the Ouchterlony double-diffusion technique, the precipitation reaction is carried out in petridishes containing agar or agarose gel. Wells of 1 cm diameter and 5mm depth are scooped out from a solidified gel. Antisera and solutions of antigens are placed in different wells. On incubation, both antiserum and antigen diffuse into the agar. They meet each other and form visible zones of precipitation (Fig. 10.45).
Serological Reaction: Type # 3. Agglutination:
Agglutination is another serological reaction occurring when the antigen present on particulate bodies reacts with antibodies to form clumps or agglutinates. This happens because particulate bodies, like bacterial cells, erythrocytes etc. having surfaced antigens are linked to each other by binding with antibodies to form a mat of antigen-antibody complex.
Previously agglutination tests were carried out in small tubes. The antiserum to be tested was diluted in steps of 2 and distributed in a series of tubes. The particulate antigen was added and incubated for 2 to 24 hr. Agglutinates of the antigen, if formed, settled at the bottom. A practical application of agglutination test was the Widal test for diagnosing typhoid. It was performed in hanging-drops. The test is no longer used.
The procedure of Widal test was to mix a drop of the diluted serum of the patient with a standard suspension of Salmonella typhi on a coverslip which was then inverted over a grooved slide. After varying periods of incubation, presence of clumps of bacteria was examined. Formation of clumps indicated that the serum contained specific antibody for typhoid bacilli. The procedure has now been replaced by a more sophisticated method.
At present, microtitre plates made of plastic are used for agglutination or other tests. These plates have many shallow wells of equal capacity. Agglutination tests are conducted by two methods — one is direct and the other is indirect passive.
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In the direct agglutination test, equal quantities of two-fold dilutions of the test serum are taken in the wells of a microtitre plate, so that each well contains half the amount of antibody (presumably present in the serum) of the preceding well and double the amount of the succeeding well. Next, the particulate antigen is added to each well in equal volumes having the same concentration. The highest dilution of the serum which gives visually detectable agglutination is considered as the antibody titre of the test serum (Fig. 10.46).
The passive indirect agglutination test is a further refinement of the technique. In this method, extremely fine polystyrene particles having an average diameter of one-tenth of that of bacterial cells are used for adsorbing either soluble antigens or antibodies present in serum. The particle-bound soluble antigen reacts with antibody forming agglutinates. Alternatively, polysterene-bound antibody reacts with particulate antigen and agglutination occurs (Figs. 10.47A and 47B).
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Agglutination of erythrocytes is known as haemagglutination. Erythrocytes have surface-bound antigens characteristic for different blood groups. When incompatible blood samples are mixed, these surface antigens bind to the antibodies present in serum and results in haemagglutination. Such antigen- antibody reaction is made use of in blood typing.
Haemagglutination due to mixing of blood samples of groups A and B of ABO system is schematically shown in Fig. 10.48:
Another practical application of haemagglutination is the Coombs test for detection of Rh- antibodies. These antibodies are formed in the body of an Rh-negative person when Rh-positive erythrocytes enter. Rh-antibodies can interact with Rh-antigens present on the erythrocytes of Rh- positive blood.
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To test the presence of Rh-antibodies in a person’s serum, it is mixed with Rh-positive erythrocytes resulting in coating of the erythrocytes by Rh-antibody (if present). But this does not lead to haemagglutination. Next, anti-human globulin is added to the mixture resulting in haemagglutination indicating the presence of Rh-antibody in the test serum.
The principle involved in coombs test is shown in Fig. 10.49:
Some viruses, like those causing mumps, measles and influenza are capable of haemagglutination without involving antibodies. The phenomenon is known as viral haemagglutination. These enveloped viruses can directly bind to the erythrocytes and cause their clumping. Specific antiviral antibodies can inhibit the haem-agglutinating activity of the viruses by combining with their attachment sites. This can be used for diagnosing the disease caused by a virus of mumps, measles and influenza group.
