The Main Properties of Human Immunoglobulins

Reactions of antigens and antibodies are highly specific. An antigen will react only with antibody elicited by its own kind or by a closely related antigen. Because of this high specificity, reactions between an antigen and an antibody can be used to identify one by means of the other. This specificity is the basis of serologic reactions.

Antigen-antibody reactions are used to identify specific components in mixtures of either one. Microorganisms and other cells possess a variety of antigens and may thus react with many different antibodies. Countless in vitro diagnostic techniques detect specific reactions between antibodies and their corresponding antigens. Any diagnostic technique that depends on specific reactions between antigen and antibody is called immune or serological (= serologic) test. Immune tests are usually used in the following cases:

1) to identify an «unknown antigen» (bacteria, virus, toxin, etc. in a specimen) with the help of the «known antibody» (diagnostic antibody-containing antisera);

2) to determine an «unknown antibody» (in a patient’s blood serum) with the help of the «known antigen» (diagnosticum).

Thus, one component (ingredient) in serological tests should always be known.

Although most serological tests depend on the same theoretical principle, methods for detecting specific antibody-antigen reactions differ. Agglutination, precipitation, complement-fixation test, neutralization, IF-test, ELISA and RIA are the most important of them.

¨ The Agglutination Tests

A particulate antigens are employed in the agglutination tests. A particulate antigens are relatively large cellular antigens, such as bacteria or their large antigens, fungi etc., or some soluble antigens adhering to particles (latex, erythrocytes, charcoal, etc.). They can be linked together by specific antibodies to form visible aggregates. This process is called agglutination. Agglutination reactions are very sensitive, relatively easy to read, and available in great variety.

There are three main components of agglutination test:

1) the antigen (agglutinogen);

2) the antibody (agglutinin);

3) the electrolyte (isotonic solution).

There are two stages in the development of the agglutination results. The first, the specific stage, is a specific interaction between the antigen and antibody tested. It is invisible. The next one is the nonspecific stage that shows the visible aggregates formation in the presence of the electrolyte solution (see Fig. 3).

The Agglutination tests are classified as either direct or indirect (or passive).

Ø The Direct Agglutination Tests allow either to identify unknown particulate antigens (the slide agglutination test) or to detect antibodies against particulate antigens (the serial dilution test). The appearance of the agglutination test depends on the type of antigen and the size of cells. In bacteria, the interaction between somatic antigens (O-antigens) and specific antibodies is slow and a fine granular sediment can be seen after 20-24 h. incubation, and these small grains do not break upon shaking. This phenomenon is called O-agglutination. The flagellar H-antigens induce a rapid development of agglutination (5 min. to 2 h.), and a large readily breakable flakes are observed. This is termed H-agglutination.

v The Slide Agglutination Test is usually used to identify the unknown antigen (pathogen, isolated from a specimen). This test is performed on the slide glass. Using a Pasteur pipette, a drop of diagnostic antiserum and a drop of isotonic solution as a control, are applied on a grease-free slide glass. Then a loopful of bacterial pure culture is inoculated into each drop and thoroughly mixed. The reaction takes place at room temperature within 5-10 min. A positive test is indicated by the appearance of a large flakes (H-agglutination) or small grains (O-agglutination) in the drop with antiserum. In a negative test, the fluid remains uniformly turbid (see Fig. 3B).

The slide agglutination test is usually a qualitative test, but sometimes it can be a quantitative method as a presumptive agglutination test (e.g., Huddleson’s test used to identify an antibodies in a patient’s serum in brucellosis diagnosing).

v The Serial Dilution Agglutination Test (the Standard AT) is carried out in a series of test tubes, or in plastic microtiter plates, which have many shallow wells to take the place of individual test tubes. This test is usually employed to detect the unknown antibodies (i.e. to measure of serum antibody titer) in a patient’s serum. Serum is diluted in a simple numerical ratios such as 1:50, 1:100, 1:200, 1:400, etc. Then 1-2 drops of the suspension of dead microorganisms or their particulate antigens (diagnosticum) are transferred into each tube with diluted serum and into the antigen control with isotonic solution instead of serum. One tube is served as the serum control (it contains a mixture of the serum tested and isotonic solution). The inoculated tubes are placed into the 37 ^o C incubator and are kept there for 2 h. to 24 h.

The results estimation begins with the control tubes. In the antigen control a mixture remains uniformly turbid. The liquid in the serum control is completely transparent. Positive results in the test tubes show the presence of large or small flakes.

The amount of particulate antigen in each tube (or well) is the same, but the amount of serum containing antibodies is diluted so that each successive well has half the antibodies of the previous well. Clearly, the more antibody we start with, the more dilutions it will take to lower the amount to the point at which agglutination does not occur. This is the measure of titer, or concentration of serum antibody. In general, the higher the serum antibody titer, the greater the immunity to the disease. The titer alone is of limited use in diagnosing an existing illness. There is no way of knowing whether the measured antibodies were generated in response to the immediate infection or to an earlier illness. For diagnostic purposes, a rise in titer is significant; that is, the titer is higher later in disease than at it onset. If it is possible to demonstrate that the person’s blood had no antibody titer before the illness but has a significant titer while the disease is progressing; this change in titer, called seroconversion, is also diagnostic.

NOTE: A paired patient's sera taken at 7 -10 days interval during the course of infection are usually taken from a patient. A fourfold rise in antibody titer between the acute and the convalescent stages of the disease verifies the etiologic role of the corresponding microorganism.

The standard agglutination test is a quantitative test, and it is usually employed to determine the antibody titer, but sometimes this test is used to identify the unknown antigen (e.g., to identify the E. coli serogroup).

v The Direct Hemagglutination Test. The surfaces of blood cells contain many molecules that can act as antigens. When individual needs a blood transfusion it is important that the donor blood cells not elicit an immune response in the recipient. To minimize such an occurrence, the donor and the recipient blood types are determined and only cells with similar antigens are used for transfusion. The ABO blood group and the Rh blood group are the most important antigens to match. The direct hemagglutination test is employed to identify ABO blood group and the Rh blood group. The person's erythrocytes serve as an antigen in this test. (For details see the relevant LECTURE).

Table 13

Blood Type	Antigens on Cell	Serum Antibodies	May Donate to	May Accept from
A	A	anti-B	A or AB	A or O
B	B	anti-A	B or AB	B or O
AB	A and B	None	AB	AB, A, B, or O
O	None	anti-A and anti-B	O, A, B, or AB	O

Hemolytic disease of the newborn usually results from incompatibility between an Rh-negative mother and an Rh-positive fetus. IgG antibodies to the Rh antigen cross the placenta from the mother's circulatory system and destroy fetal blood cells. This antibody-mediated death of red blood cells may cause severe anemia or even death if toxic compounds released from cell destruction are deposited in the brain.

Hemolytic disease rarely occurs during a first pregnancy since an Rh-negative mother may develop Rh-specific antibodies only after being exposed to Rh-positive cells. After her first Rh-positive child, the mother is sensitized to the Rh antigen, so subsequent pregnancies with an Rh-positive fetus elicit an antibody response that is sufficient to damage the fetus. This can be prevented by treating the mother by anti-Rh-antibody immediately after her first delivery. The injected antibody neutralizes any of the baby's Rh-positive blood cells (antigens) that entered the mother's circulation during the removal of the placenta, thereby preventing the fetal blood cells from sensitizing the immune system. The antibody must be administered after each pregnancy (even after miscarriage and abortion).

The antibody production occurs in two distinct stages. The primary response follows exposure to an antigen that the host never before encountered. During this time, antigen-specific B-lymphocytes proliferate, and the host becomes sensitized to that antigen. Once sensitized, each subsequent exposure to the same antigen stimulates a secondary response. Initial exposure to the antigen is followed by lag period of 3-30 days. During this time no antibody is detectable in the blood. The length of the lag period depends on several variables, including the nature and concentration of the antigen, its route of administration, and the immunologic competence of the host. After the lag period, the antibody titer (concentration) increases for a short period of time.

Since, antibodies are usually undetectable for several days following onset of initial infection, routine serological tests early in the disease can produce false negative results. During lag period of disease so-called incomplete antibodies can be detected in the blood serum. These antibodies differ from the ordinary antibodies because they are monovalent, that is they possess only one reactive site and therefore they can couple only with one identical antigenic determinant without visible agglutination. The Coombs’ test is employed to detect incomplete antibodies and so to diagnose early infection.

v Coombs’ Test. The procedure of this test consists of two stages. The antigen (certain diagnosticum) is added into each tube with diluted patient’s serum. After 30 min. incubation the antiglobulin diagnostic antiserum is applied. The antiglobulin antiserum contains antihuman antibodies and it is obtained by rabbit's immunization with human immunoglobulins. Thus, patient's serum incomplete antibodies serve as an antigen and rabbit’s antibodies couple with them forming diagnosticum-human antibody-rabbit's antiglobulin complex and the results of agglutination become readily visible (see Fig. 4).

Ø The Indirect (Passive) Agglutination Tests. The antibodies against small bacterial or viral antigens, and soluble antigens can be detected by agglutination tests if the antigens are adsorbed onto particles such as red blood cells, bentonite clay, or minute latex spheres. Such tests are commonly used for rapid detection of antibodies against many human pathogens. In such indirect or passive agglutination tests, the antibody reacts with the soluble antigen adhering to the particles. The particles then agglutinate with one another much as particles do in the direct agglutination tests (see Fig. 5). The same principle can be applied in reverse by using particles coated with antibodies to detect the antigens against which they are specific. A test to detect the botulinum toxin in food products makes useful the antibody-coated erythrocytes (see Reversed IHA).

v The Indirect Hemagglutination Test (IHA-test). When agglutination reactions involve the clumping of red blood cells, the reaction is called hemagglutination. Erythrocytes have the highest adsorptive capacity. Sheep, horse, rabbit, chicken, mouse, human, and other red blood cells can be used to adsorb even the highly dispersed antigens (e.g., viruses or Rickettsia) to make the results of agglutination test readily visible. Erythrocytes with adsorbed antigens are usually called erythrocytic diagnosticums. The Indirect Hemagglutination Test (IHA) is the agglutination test with the application of erythrocytic diagnosticums used to detect the unknown antibody. In this test the antibody of a patient's serum couples with the antigen adhering to the erythrocytes, i.e. indirect clumping of red blood cells takes place (see Fig. 5A). The IHA-test is routinely employed in the diagnosis of viral infections and epidemic typhus.

 

v The Reversed Indirect Hemagglutination Test (RIHA). To perform RIHA-test, the antibody-containing erythrocytic diagnosticum is employed. In this case the unknown antigen binds to the specific antibody absorbed to the erythrocytes and the indirect hemagglutination also takes place (see Fig. 5B). The botulinum toxin in food products or in a patient’s specimens can be demonstrated by RIHA-test.

 

¨ The Precipitation Tests

Precipitation tests involve the reaction of soluble antigens with specific antibodies (in general, Ig G or Ig M) to convert the soluble antigen to a solid precipitate called lattice. Using the precipitation test (PT), one can demonstrate the antigen in such tiny amounts which can not be detected by chemical tests. The use of the PT for sanitary and hygienic control of foodstuffs makes it possible to uncover adulteration of meat, fish, and flour products, as well as admixtures in milk, etc.

Precipitation tests occur in two distinct stages. First, there is the rapid interaction between antigen and antibody to form small antigen-antibody complexes. This interaction occurs within seconds and is followed by a slower reaction, which may take minutes to hours, in which the antigen-antibody complexes form lattices that precipitate from solution.

Precipitation reactions normally occur only when there is an optimal ratio of antigen to antibody. The optimal ratio is produced when separate solutions of antigen and antibody are placed adjacent to each other and allowed to diffuse together. In a precipitin ring test (see PRACTICAL WORK), a cloudy line of precipitation (ring) appears in the area in which the optimal ratio has been reached (the zone of equivalence) - Fig. 6 and 7.

 

 

Immunodiffusion tests are precipitation reactions carried out in an agar or gel medium. In one such test, the Ouchterlony test, wells are cut into a purified agar gel in a Petri plate. A serum containing antibodies (antiserum) is added to one well, usually centrally located and soluble test antigens are added to each surrounding well. A line of visible precipitate develops between the wells at the point where the optimal antigen-antibody ratio is reached. The Ouchterlony test is most useful in determining whether the antigens are identical, partially identical, or totally different.

The Ouchterlony test (the gel-diffusion plate technique) is used to demonstrate toxigenicity of diphtheria bacilli. A filter paper strip saturated with antitoxin- containing antiserum(antitoxinsas an antibodies) is placed on the culture medium, which favors the production of diphtheria toxin (antigen). Several strains of Corynebacterium diphtheriae are then seeded by streak technique. The toxigenic strains (the diphtheria toxin-producing strains) develop V-shaped lines of precipitate in the zone of optimal antigen-antibody proportion.

The Mantchini test is commonly used to measure the concentration of immunoglobulins in patient’s serum. The monoclonal or animal antibodies (“ known antibodies ”) against corresponding class of human immunoglobulins are mixed with melted purifies agar and poured into Petri plate. Wells are cut in the agar and the patient’s serum (certain class of immunoglobulins as “ unknown antigens ”) is added to one well. Two-fold dilutions of standard immunoglobulins are prepared and added to other wells. A zone of precipitation surrounds each well due to the antigen-antibodies complexes production. The diameter of zone precipitation depends on the concentration of immunoglobulins in the patient’s serum: the lager zone demonstrates the lager concentration. To determine the immunoglobulins concentration the diameter of precipitation zone around the test well is compared with precipitation zone around the standard wells.

Other precipitation tests do not depend entirely on the passive diffusion of antigen and antibody in a gel but instead use electrophoresis to speed up their movement. Protein mixtures can be separated rapidly, sometimes in less than an hour, with this method. A modification of the precipitation reaction combines the techniques of immunodiffusion and electrophoresis in a procedure called immunoelectrophoresis. This procedure is used in research to separate proteins in human serum and is the basis of certain diagnostic tests (for example, countercurrent immunoelectrophoresis - CIE). CIE is based on the fact that some antigens and antibodies have opposite charges when they are placed in buffers of correction strength and pH. When an electrical current is applied the antigens and antibodies move toward the pole with the electrical charge opposite to the one they carry. They pass through each other to do so, and precipitation line appears within an hour if a reaction occurs (Fig. 8).

¨ Diagnosticums. Antibody-containing antisera

The suspension of dead (and occasionally living) microorganisms or their antigens is called diagnosticums. Diagnosticums of killed microbes are fairly stable, retaining their properties for several years and present no risk of contamination. Sometimes bacterial antigens of different microbes may produce false positive results with non-absorbed antisera because they possess relative antigens (e.g., bacteria of genera Escherichia and Shigella are the members of the same family Enterobacteriaceae. Therefore non-pathogenic E.coli may produce false positive results with Salmonella -antiserum). To prevent “cross-reactivity” of identical antigens of various bacteria O-, H- and K-bacterial diagnosticums are prepared.

Erythrocytes sensitized with antigens are called erythrocytic diagnosticums. Sheep red blood cells which possess high adsorptive capacity are the most commonly used erythrocytes for preparing erythrocytic diagnosticums. Diagnosticums contain known antigens (bacterial, viral, etc.) and are usually used to detect unknown antibodies in the patient’s serum. Sometimes antibodies can be adhered to the surface of erythrocytes. Preparation, that contains erythrocytes coated with known antibodies is called erythrocytic antibody-containing diagnosticum.

NOTE: The antigens labeled with fluorochromes, enzymes and radioactive agents will be discussed later (see LESSON 14 ).

Several antibody-containing antisera are employed to determine the unknown antigen. There are different types of diagnostic antibody-containing antisera. For example, agglutinin-containing antisera (agglutinating antisera) for agglutination tests, precipitin-containing antisera (precipitating antisera) for precipitation tests, etc. These diagnostic antibody-containing antisera are usually prepared by hyperimmunization of animals (rabbits, goats or donkeys) with certain antigen (agglutinogen or precipitinogen, etc.) and by concentration of specific antibodies collected from the animal serum several weeks later.

The titer of the agglutinating antiserum (agglutinin-containing antiserum) is the minimum concentration of serum antibodies (=maximum antiserum dilution) which can interact with the corresponding antigen to produce readily visible clumps.

The titer of the precipitating antiserum, in contrast to the titer of agglutinating antiserum, is determined by the maximum dilution of the antigen which is precipitated by the specific antiserum.

This is explained by the fact that the antigen participating in the precipitation reaction has an infinitesimal magnitude and that in a volumetric unit of the serum there are much more antigens than antibodies.

NOTE: One should not mix up the terms “SERUM” and “ANTISERUM”!!! Antiserum contains known antibodies and is used for diagnosis, treatment or immunoprophylaxis. It is obtained by immunizing animals with corresponding known antigen.

The term “ serum ” is commonly used for patient’s tested serum with the “ unknown antibodies ”. These antibodies are identified by diagnosticums with known antigens in the serologic tests.

The agglutinating antiserum, obtained by immunizing a rabbit (or other animal) with the whole bacterial cells, is called native non-absorbed antiserum. This antiserum contains antibodies which can false react with identical antigens of other microorganisms (these antibodies are usually termed «cross-reactive antibodies»). Possible cross-reactions between related antigens can limit the test’s specificity. That is why the immune pre-absorbed antisera are more preferable. The Castellani technique is used to prepare these antisera. Antibodies, which can cause false-positive result with the related antigens are adsorbed on the identical antigens (agglutinogens) of another microbes and then eliminated from the serum. Therefore, the pre-absorbed antiserum is more specific and it can couple only with certain antigen (or antigens). There are monovalent pre-absorbed antisera, which can react only with one antigen or receptor, and polyvalent pre-absorbed antisera, that contain more than two types of antibodies and are able to bind with several antigenic determinants of the microorganism.