A number of tests are employed for the serodiagnosis of syphilis. Some of these are based on the complement-fixation technique; others are flocculation tests.
The most important ones are known as:
(1) The Wassermann test and its many modifications,
(2) The Kahn test, and
(3) The Kline test.
1. Wassermann Test:
A complement-fixation test was proposed by Wassermann in which the antigen consisted of an aqueous extract of syphilitic fetus. It was found later that the aqueous extract of syphilitic fetus could be replaced by an antigen prepared by extracting with alcohol beef-heart powder, previously extracted with ether, and adding cholesterol or a mixture of cholesterol and lecithin to the alcoholic extract.
The lipoidal suspension is not an antigen in the true sense. It is not capable of inciting the production of immune bodies when injected into an animal. However, since it is capable of binding syphilitic antibodies in the serum of the patient, it is generally spoken of as an antigen.
A type of antibody activity is developed as a result of infection with Treponema pallidum, the causative agent of syphilis. Since it is not known if this is a true antibody, it is generally referred to as a reagin. It has the power to react with the lipoidal antigen prepared from beef heart and reinforced by the addition of cholesterol and lecithin.
It has been suggested that T. pallidum damages tissue cells of the host and causes a lipoidal fraction to split off which acts as a hapten, and this combines with the protein of the bacteria to stimulate the production of antibodies (reagin).
Cardiolipin is the name given to a complex phospholipid first isolated by Pangborn (1941) from beef heart in essentially chemically pure form. It is a single substance and on alkaline hydrolysis yields oleic and linoleic acids, a polyester of glycerophosphoric acid, and glycerol.
Cardiolipin, purified lecithin, and purified cholesterol used singly are not antigenic, but certain mixtures of the three act well as antigens in the complement-fixation test for syphilis. Mixtures reproducible to a high degree of accuracy may be prepared from different lots of the components.
Cardiolipin reinforced with lecithin and cholesterol appears to be generally superior in both sensitivity and specificity to the lipoidal suspension prepared by extracting, with alcohol, powdered beef heart previously extracted with ether, and adding lecithin or a mixture of lecithin and cholesterol to the alcoholic extract.
2. Kahn Test:
Precipitation tests for the diagnosis of syphilis have gained in popularity during the past several years. The first practical procedure of this type is the Kahn test. It is now the most commonly employed precipitation test for the diagnosis of syphilis.
The Kahn antigen is prepared by extracting dried beef-heart powder with ether and then alcohol. Cholesterol is added to the alcoholic extract. The cholesterolized extract must be mixed with salt solution to produce an unstable antigen suspension. The antigen suspension contains lipid aggregates. These aggregates disperse when in contact with serum. Then, in syphilitic serum, new aggregates appear in the form of a precipitate, whereas in non-syphilitic serum no new aggregates appear.
Agitation markedly influences the formation of precipitates in syphilitic serum with antigen suspension. While strongly positive serum will show flocculation immediately on mixing with an appropriate amount of antigen suspension, weakly positive serum requires agitation for several minutes for the formation of a precipitate.
3. Kline Test:
The Kline test is demonstrated as a microscopic slide reaction.
It is performed as follows:
Pipette 0.05 ml. of heated serum in a ringed area on a slide.
Add 1 drop (0.008 ml.) of antigen emulsion to the serum.
Rotate the slide for 4 min.
Examine under the microscope at a magnification of 100. for the presence of clumps.
Results are recorded as negative, weakly positive (± and +), positive (++ and +++), and strongly
positive (++++), depending upon the degree of clumping and the size of the clumps.
The antigen originally employed by Kline was made from the alcohol soluble acetone-insoluble fraction of beef heart, concentrated by evaporation at 50°C.
Kline now recommends a cardiolipin antigen reinforced with lecithin in place of alcohol-soluble acetone-insoluble fraction of beef heart. This antigen shows greater specificity and much greater sensitivity than the original preparation and is now recommended as the standard antigen for the Kline test.
Other flocculation tests and modifications of the Kahn and Kline tests include:
(1) The Hinton test,
(2) The Mazzini test,
(3) The Rein-Bossak test, and
(4) The V.D.R.L. test.
The Treponema pallidum immobilization (TPI) test, discovered by Nelson and Mayer (1949), is based on the principle that the serum of patients with syphilis contains a specific humoral antibody capable of sensitizing the Nichols strain of T. pallidum so that the spirochetes become non-motile in the presence of active guinea-pig complement. The test itself, while simple in theory, has presented many difficulties in its practical application.