Prelab conference. Topics for discussion:
1. Lysis tests (bacteriolysis, immune hemolysis). Mechanism, components, application.
2. The complement system. The pathways of complement.
3. The titration of complement.
4. Hemolytic system. Composition and application.
5. Complement-fixation test (CF-test). Mechanism, components and application.
¨ The Complement
At the beginning of 20th century it was recognized that a heat-labile component of normal serum was necessary for immune lysis of red blood cells. The term complement was given to this component since it functioned to «complement» the action of specific antibody in the destruction of bacteria and erythrocytes. Complement is a sequential, multimolecular system of plasma proteins which can be activated by a variety of immunological as well as nonimmunological stimuli. Complement activation, which can proceed via two different pathways, is mediated through a series of cascading reaction steps. The classical complement pathway is activated by Ig G- and IgM-containing immune complexes and is composed of eleven distinct plasma proteins which identified numerically as C1-C9. The alternative complement pathway is activated by plant, fungal, and bacterial polysaccharides and lipopolysaccharides in particulate form and is composed of six plasma proteins identified as factor D, factor B, properdin, factor H, factor 1, and C3. In addition, C5-C9 can be recruited to attack membrane surfaces as in the classical pathway. The C3 -C9 components are thus common to both pathways.
Activation of either pathway results in a variety of biological effects. These include: (1)the irreversible damage to biological membranes associated with complement dependent cytolysis, (2)the deposition of molecules on the surface of the particle under complement attack resulting in particle opsonization and clearance, and (3)the production of potent mediators of the inflammatory response.
¨ Lysis Tests
The term immune lysis reaction refers to lysis of the antigen conjugated with antibodies in the presence of a complement. Depending on the nature of antigens participating in the lysis reaction, it may be called spirochaetolysis, bacteriolysis, immune hemolysis, etc. Antibodies involved in the corresponding reactions are called spirochaetolysins, bacteriolysins, hemolysins, etc. Lysins exert their action only in the presence of a complement.
NOTE: Do not confuse immune hemolysis and nonspecific hemolysis which results from osmotic lysis.
The Bacteriolysis (Spirochetolysis) Test can be employed to detect specific antibodies against Vibrio cholerae and some spirochetes (Borrelia sp., Treponema sp.).
The Immune Hemolysis Test is used for complement titration and as an indicator system in the complement-fixation test (For Bacteriolysis Test and Immune Hemolysis Test procedures see «Practical Work» of LESSON 13).
¨ The Complement Fixation Test
During most antigen-antibody reactions, the complement binds to the antigen-antibody complex and is used up, or fixed. This process of complement fixation can be used to detect very small amounts of antibody or to detect the unknown antigen. Antibodies that do not produce a visible reaction, such as precipitation or agglutination, can be demonstrated by the fixing of complement during the antigen-antibody reaction. Complement fixation was ones used in the diagnosis of syphilis (Wassermann test) and is still used in the diagnosis of certain viral, fungal, and rickettsial diseases.
The complement-fixation test (CFT) belongs to the complex serological reactions. To be performed, it requires five ingredients; namely, an antigen, an antibody and complement (“the first system"), sheep erythrocytes and hemolytic antiserum (“the second, indicator system"). The specific interaction of the antigen and antibody is attended by fixation (binding) of the complement.
The execution of the complement fixation test requires great care and good controls.
The CFT is performed in two distinct steps. The first, the subject’s serum must be heated at 56oC for 30 minutes to inactivate its inherent complement. The inactivated serum is then diluted and mixed with a specific amount of known antigen and fresh complement. The mixture is then incubated for about 1 h. The antigen-antibody reaction cannot be observed at this point. To determine whether the complement present in the mixture is fixed by an antigen-antibody complex, another step of the CF-test must be performed.
The second step uses an indicator system to determine whether complement is free or combined (fixed). The indicator system consists of sheep red blood cells (the antigen) and specific antibodies in hemolytic antiserum (the hemolysins) that will interact with sheep erythrocytes. The exposure of these «hemolytic system» to the complement causes lysis of the red blood cells (hemolysis). Therefore, if the complement has been fixed by an antigen-antibody complex during the first step, it is not available to cause sheep erythrocytes lysis in the second step of CFT. In this case CFT is positive (Fig. 9). Thus, if the CFT is positive, no hemolysis occurs. But if the complement has not been fixed during the first step of reaction, then it is available to cause hemolysis in the second step. This negative test result means that there are no specific antigens or antibodies tested in the CFT. The negative result shows complete hemolysis, and the fluid in the tube (or well) becomes intensely pink and transparent ("varnish blood").
The CFT as well as other serologic tests can be used either for detecting specific antibodies in the patient’s serum by the known antigen or for identifying an antigen in a patient’s specimen by known antibodies.
The performance of CFT calls for special preparation. The glassware (test tubes, pipettes, vials) is thoroughly washed and care is taken not to use them for other purposes. All ingredients of the reaction are prepared and titrated prior to the main test by the following ways:
1) Before the test, the serum (either obtained from the patient or the diagnostic antiserum) is heated on a water bath at 56oC for 30 min in order to inactivate its inherent complement. Some sera, particularly those from immunized animals, possess anticomplement properties, i.e. they can bind complement even in the absence of a specific antigen. To prevent the anti-complement activity of sera tested and diagnostic antisera, they are kept in the lyophilized or frozen form at low temperature.
2) Bacterial or other diagnosticums, tissue lipids, viruses and virus-containing materials, etc. may be used as antigens for CFT.
3) Guinea pig serum collected immediately before a reaction is used as a complement. Dry commercially prepared complement may also be employed. To obtain the basic solution for subsequent titration, the complement is diluted 1:10 with isotonic solution. The complement titer is its minimum concentration (= maximum dilution) that causes complete hemolysis in hemolytic system.
4) Sheep erythrocytes are employed as a 3 per cent suspension in isotonic sodium chloride solution.
5) The hemolytic antiserum for CFT is obtained in the following manner. Rabbit is immunized by suspension of washed sheep erythrocytes. Seven days after injection, the rabbit’s blood serum is obtained. Then the obtained antiserum is heated for 30 min at 56oC. This antiserum contains anti-erythrocytic antibodies (hemolysins).
6) The hemolytic system consists of hemolytic antiserum and 3 per cent suspension of sheep erythrocytes, mixed in equal volumes. To sensitize the erythrocytes by hemolysins the mixture must be incubated at 37oC for 30 min.
¨ The Complement Titration
Before the test, the basic solution of complement (1:10) is dispensed into a series of the test tubes in quantities varying from 0.05 to 0.5 ml, and then isotonic solution is added to each tube, bringing the volume of the fluid to 1.5 ml (see PRACTICAL WORK, item 3). Then the hemolytic system is added to them and the tubes are incubated for 30 min at 37oC. After incubation the complement titer is measured. The titer of the complement is defined as its minimum concentration causing complete hemolysis in hemolytic system. To perform the test, one should take the working dose of the complement exceeding the titer by 20-30 per cent (the next tube that demonstrates hemolysis). The higher complement concentration is need (the abundance of the complement) to inhibit the anti-complement activity of the CF-test components.
¨ The Neutralization Reactions
The Neutralization Tests are antigen-antibody reactions in which the harmful effects of a bacterial exotoxin or a virus are eliminated by specific antibodies. Neutralization reactions are used both in diagnostic tests and in disease treatment, especially for diphtheria, botulism and tetanus. In such reactions a neutralizing antibodies called antitoxins combine with the exotoxin to neutralize it. Theses specific antibodies are produced by a host in response to exposure to the antigens of bacterial exotoxin or its corresponding toxoid (inactivated toxin) - see LESSON 15.
The neutralization tests are used in the diagnosis of toxemic bacterial and viral infections. Neutralization tests also allow to identify bacterial exotoxins. The antigenic type of toxin is identified by neutralization with specific antitoxin in mice (in vivo test, biological examination). For in vivo test exotoxin-containing specimen and several types specific antitoxic antisera are mixed in tubes respectively. Control tube contains the mixture of tested sample and isotonic solution. After 30 minutes incubation at room temperature 1 ml of mixture from each tube is injected intraperitoneally to mice. Mice injected with non-neutralized toxin die rapidly (a negative result of neutralization test). If toxin-antitoxin neutralization takes place (with the corresponding type of antitoxin), mouse will survive (positive result).
A more frequent form of neutralization test is a skin test such as the Schick test, which determines the status of a person's immunity to diphtheria. In this test, a small amount of diphtheria exotoxin is inoculated into the person's skin. If there is sufficient serum antitoxin to neutralize the exotoxin, there is no visible reaction, indicating that the person would be immune to diphtheria (positive test of neutralization). If antitoxin is insufficient, the exotoxin damages the tissues at the site of injection and produces a swollen, tender, reddish area that turns brown in four to five days (negative result).
PRACTICAL WORK
1. The Bacteriolysis Test. In carrying out the bacteriolysis reaction, the immune serum (patient’s serum) is heated for 30 min at 56oC to inactivate the complement present in it. Suspension of microorganisms is prepared from their 24-hour culture (1 milliard (billion) CFU/ml). This suspension is added to the serum diluted 1:10 (or 1:20, 1:40, etc.). To the serum control tube no complement is added. In the complement control the immune serum is replaced with isotonic solution.
The bacteriolysis test may be performed both in vitro and in vivo. To perform the in vitro test after incubation at 37 OC for 2 h., the material of each tube is streaked with inoculating loop on MPA. After incubation at 37oC for 24 h the bacterial growth/no growth on MPA is checked. If the serum to be tested contained bacteriolysins, no growth of bacterial colonies is seen. And the growth in the control tubes should be observed.
For in vivo test, the contents of all tubes after first step of examination (mixing the components and their incubation at 37oC for 2 h.) are injected intraperitoneally into the mice (or guinea pigs). The peritoneal fluid is then examined microscopically 10 min, 20 min, and 1 h. after injection.
The Procedure of the Bacteriolysis Test. This test is performed in four tubes. The test tube contains the immune antiserum, suspension of the microorganisms, complement and isotonic solution. The control tubes do not contain either one component: the immune antiserum (2), the complement (3), or both immune antiserum and the complement (4) (see Table 13-1).
Table 13-1