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
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Bacterial or other diagnosticums, tissue lipids, viruses and
virus-containing materials, etc. may be used as antigens for CFT.
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.
Sheep erythrocytes are employed as a 3 per cent suspension in isotonic sodium chloride solution.
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).
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.