In the precipitin test a reaction

In the precipitin test a reaction takes place between a soluble antigen and a solution of its homologous antibody. The reaction is manifested by the formation of a visible precipitate at the interface of the reactants but may be inhibited by an excess of either antigen or antibody. The most useful precipitin test therefore provides for diffusion of the reactants until optimum concentration is reached. The equivalence zone represents the concentrations of antigen and antibody where complete precipitation occurs. Culture filtrates or animal serums contain a number of different antigens. If they are used for immunization, they give rise to a correspondingly large number of different antibodies.
Unless special precaution is taken to remove all but one precipitin from the solution, several layers of precipitate should be formed, one for each precipitin system present. This is not necessarily a disadvantage since the fact is used to identify and distinguish between different antigens and antibodies in a solution. Physical factors which influence precipitin tests are electrolytes, pH, temperature and time. Precipitin tests are not limited to the laboratory diagnosis of bacterial infections of man and other animals by means of specific antigens but are also useful in many other ways, such (1) the laboratory diagnosis of syphilis where a nonspecific antigen not related to the spirochete is usually used; (2) typing of hemolytic streptococci and other microorganisms; (3) identification of blood or seminal fluid in stains on clothing, weapons, or other exhibits for medicolegal purposes; (4) postmortem diagnosis of anthrax from tissue of a dead or decomposed animal by the Ascoli test; (5) determination of the kind of animal a mosquito has recently fed on (this information helps entomologists and epidemiologists to prevent the spread of arthropod-borne diseases); (6) detecting the adulteration of foods. Antigen for precipitin tests is prepared by making an extract from bacterial cells, tissues or other suitable material. For example, in the Ascoli test for anthrax, a small amount (1 or 2 grams) of spleen is boiled for 5 to 10 minutes in a few millimeters of physiological salt solution. The test consists in layering a millimeter or so of the extract over a similar amount of antiserum prepared by injecting a rabbit with killed anthrax bacilli. Precipitating antigens for streptococcus typing and diagnosis of certain other bacterial infections are made from 24- to 48- hour cultures that are extracted to yield a clear solution of precipitable material. The RING TEST The ring test is the simplest of the precipitin tests. In a small-bore tube an antigen solution is layered on a solution of serum containing antibody. The two solutions diffuse until they are in optimum concentration for precipitation at which point a white disk or ring of precipitate appears in the two otherwise clear solutions. BACTERIOLYTIC (CYTOLYTIC) ANTIBODIES AND COMPLEMENT-FIXTAION TESTS Complement-fixation tests are based on the presence of cytolytic or complement-fixation antibodies of serum. The cytolytic antibodies that lyse bacteria are called bacteriolytic amboceptors and are produced in an animal body stimulated by bacterial antigens. In the presence of complement, bacteriolytic amboceptor causes lysis of the specific bacterial cells. This purpose of the complement-fixation test is to determine whether specific bacterial amboceptors are present in serum. In the actual test, two systems are involved. One is the bacteriolytic or complement-fixing system in which serum, bacterial suspension 9or other antigen) and complement are mixed. If the antigen and antibody in the serum are capable of union, the complement is said to be fixed. The hemolytic system is simply an indicator system in the test. Hemolytic amboceptor is prepared by immunizing rabbits with the red blood cells of sheep. Serum from sheep-cell-immunized rabbits is mixed with red blood cells are lysed. If, however, the complement was fixed by being used in the reaction between the bacteriolytic amboceptor and antigen, no hemolysis will occur. Therefore, a hemolytic reaction indicates a negative test. Obviously, all reactants in the complement-fixation test must be accurately adjusted. The complement-fixation test is widely used in the laboratory diagnosis of many infectious diseases, including those of bacterial, virus, rickettsial, protozoan and fungal etiology. It is also used for the identification of many microorganisms. On of the best-known applications of the complement-fixation test is the Wassermann test for syphilis, which differs from many other serological reactions in that the antigen is not prepared from the causative organism of the disease but is made by extracting beef-heart powder with ether and alcohol. This extract contains a complex phospholipid called cardiolipin. Cholesterol and lecithin are added to the extract to increase the sensitivity and specificity of the antigen. Since this material is not capable of including antibody formation in an animal, it is not a true antigen, but because it combines with syphilitic ‘antibodies” in the complement-fixation and flocculation tests (Kahn), it is spoken of as a nonspecific antigen. The antibody produced to Treponema pallidum is called regain. However, we must qualify the term antibody as applied to regain, since there is no evidence that it protects the individual from infection. It does, however, react with the antigen described above to bind complement in the diagnostic test. Te Wassermann and other tests for the serodiagnosis of syphilis which employ nonspecific cardiolipin antigens were in use for many years before it was possible to prepare an antigen from the specific organism. Since techniques for cultivating virulent Treponema pallidum have been developed, several new tests have been introduced. One is the Treponema pallidum complement-fixation (TPCF) test, in which the antigen is an extract of the virulent treponemas. The principle is the same as that of the other complement-fixation tests, but the actual techniques differ somewhat. Another test is the Reiter protein complement-fixation (RPCF) test, which differs from the regular procedure principally in the antigen, which is a protein extract from the avirulent Reiter strain of T.pallidum. The RPCF antigen is relatively simple and inexpensive to produce and is highly specifi