mixing in a test tube suspension of bacteria, complement, and bacteriolytic amboceptor we wish to determine whether bacteriolysis has taken place, we may do so simply by testing for the presence of complement; its absence would prove that it has been used up and that the immunity reaction has taken place, while its presence would prove that such reaction has not taken place.

The test for complement is done simply by adding blood corpuscles and hæmolytic amboceptor; in the presence of complement hæmolysis will occur, in its absence it will, of course, not occur.

The application of the phenomenon of fixation of complement with resulting inhibition of hæmolysis, known as the Bordet-Gengou phenomenon, in a test for syphilis is due to Wassermann.

In the case of syphilis the ingredients of the immunity reaction are:

1

syphilitic antigen 1 + complement + syphilitic amboceptor.

The actual test is performed in two stages. In

1 Antigen is a general term applied to all bodies, such as bacteria, blood corpuscles, etc., which are capable of exciting the generation of specific antibodes. The Treponema pallidum, not having as yet been successfully cultivated on artificial media, Wassermann employed as syphilitic antigen watery extract of livers from congenitally syphilitic infants. It has since been found that certain lipoid substances which may be extracted from normal body tissues, curiously enough, possess, like true syphilitic antigen, the property of binding complement. Such lipoids are now frequently employed as "artificial syphilitic antigen." It is to be judged from this that the Wassermann reaction is not really an instance of the Bordet-Gengou phenomenon, but a purely empirical and unexplained test for syphilis which, moreover, is not strictly specific

the first stage syphilitic antigen, complement, and the serum to be tested are brought together; if the serum contains syphilitic amboceptor the reaction will take place and complement will, consequently, be used up; if the serum does not contain syphilitic amboceptor the reaction will not take place and complement will therefore remain free. The second stage of the reaction consists simply in the addition of blood corpuscles and hæmolytic amboceptor to test for complement; in the case of a syphilitic serum, complement, having been used up in the first stage of the reaction, will not be available for the hæmolytic system and there will be no hemolysis; in the case of a non-syphilitic serum, complement will remain free after the first stage of the test; it will therefore be available for the hæmolytic system, and hæmolysis will take place.

Preparation of Reagents. Complement is derived from fresh guinea pig serum, the following being the most convenient way. A full-grown guinea pig is held by an assistant over a large Petri dish in a hyperextended position by grasping the head with one hand and all the four legs with the other. A long slender sharp knife is introduced into the neck at the side just in front of the vertebral column until it is thrust through on the other side, when the edge of the blade is turned ventrally and all the tissues in the front part of the neck are cut through. The blood is caught in the Petri dish, which is then covered and set aside in a corner out of direct sunlight and allowed to stand at room temperature for about two hours, at the end of which time the serum

may be poured off and used; or the Petri dish may at the end of two hours be placed in the refrigerator where it may be kept over night and used on the following morning; but standing over night at room temperature renders the serum inactive. If kept on ice the activity of the serum is reduced much more slowly, so that it usually remains good for about forty-eight hours.

In performing the test 0.1 c.c. of this serum is used. Guinea-pig serum is very rich in complement, so that the amount used in the test is really in excess of that actually required for complete hæmolysis.

It is customary to use sheep corpuscles in the hæmolytic system. The blood of a freshly slaughtered sheep is collected in a sterile vessel, defibrinated, centrifuged, and the corpuscles washed at least five times with 0.9% sodium chloride solution in distilled water, by pouring off the supernatant serum or salt solution, adding fresh salt solution, shaking the centrifuge tube, and centrifuging again. The washed sheep corpuscles are used in immunizing rabbits for the preparation of anti-sheep amboceptor; for this purpose one adds to the corpuscles in the sedimentation tube only about as much salt solution as would suffice to bring the corpuscle suspension to the original concentration of the blood, i.e., two parts by volume of the corpuscles in the sedimentation tube to one part of salt solution. The sheep corpuscles are also used as a reagent in the reaction; for this purpose a weaker suspension is prepared containing but five parts by volume to ninety-five of salt solution.

Anti-sheep haemolytic amboceptor is derived from the blood serum of a rabbit which has been immunized by two injections of 5 and 8 c.c. of sheep corpuscles, respectively, in the above-mentioned concentration, at an interval of five days. A full-grown male rabbit weighing about five pounds is preferred, and the injections are made into the ear vein with a 10 c.c. syringe. To facilitate the injections the assistant holding the rabbit places his thumb at the root of the ear thus impeding the blood return and rendering the vein prominent. A needle about two inches long, gage 20, is used. Subcutaneous injection is useless and may simply result in a slough; therefore, if, as the injection is begun, a swelling forms, the needle must be either readjusted or reinserted until proper penetration into the vein is assured. On the ninth day after the second injection a small amount of blood is withdrawn, centrifuged, and the serum tested for hæmolytic power. If a dilution of 1: 1500 is capable of hæmolyzing with the aid of guinea pig complement a 5% suspension of sheep corpuscles in about half an hour, then the rabbit is ready for bleeding. If not, it may be necessary to give a third injection of sheep corpuscles and again wait eight or nine days. When this preliminary test gives a satisfactory result, the rabbit is exsanguinated, the blood being collected in a sterile bowl, covered, and allowed to stand at room temperature for twelve or sixteen hours. The serum is then distributed in small sterile test tubes, putting about 2 c.c. in each and adding salt solution containing tricresol in small amount so that the

concentration of the latter does not exceed 1: 2000. The tops of the tubes are sealed with a blow-pipe and they are placed on ice. In this way the amboceptor serum may be preserved for three or four months.

Kaplan has pointed out that the preliminary standardization of the amboceptor serum does not suffice to gauge its hæmolytic power under the conditions of the Wassermann reaction, owing to the slight, but appreciable, non-specific inhibiting power of normal blood serum and of whatever antigen may be used. It will, therefore, tend to eliminate error if, on each day when the examination of a series of specimens is undertaken, the amboceptor serum is standardized anew in the presence of a non-syphilitic serum and the usual amount of antigen. This has the further advantage of making possible the allowance for any change that may have taken place in the strength either of the amboceptor or of the antigen.

The standardization is carried out as follows. Six test tubes, about 10 cm. long and 1 cm. in diameter, are placed in a rack, and into each are put 0.2 c.c. non-syphilitic serum, the usual quantity of antigen, 0.1 c.c. complement, and 1.0 c.c. 5% sheep corpuscle suspension; the rack is then placed in the incubator for one hour, at the end of which time the amboceptor serum is added in amounts varying from a concentration of 1 : 200 to one of 1 : 6400, as shown in the following sample titration; the rack is returned to the incubator and the reading taken at the end of two hours.