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 overnight and used on the following morning; but standing overnight at room temperature renders the serum inactive. If kept on ice the activity of the serum is reduced much more 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.
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 haemolysis.
It is customary to use sheep corpuscles in the hemolytic 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 hoemolytic 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 5 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 2 inches long, gauge 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 hsemolytic power. If a dilution of 1: 1500 is capable of hsemolyzing 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 hsemolytic 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.
Amboceptor serum 1: 200.......
" " 1: 400.......
" " 1: 800.......
" " 1:1600.......
" " 1:3200.......
The rule for actual work is to use double the amount of amboceptor which is sufficient to give complete haemolysis under conditions such as those of the above titration. Accordingly one would select in this case an amount of amboceptor serum to give a concentration of 1: 800 or 1: 1000.
Antigen may be prepared in many different ways, and it is immaterial which of these is chosen, the serviceableness of the product depending not so much on the method of preparation as on the care and results of its standardization. The method that seems capable of yielding a most uniform product is that of Noguchi: thoroughly mashed beef liver or kidney is steeped in ten times its volume of absolute alcohol in the incubator for seven days, at the end of which time it is filtered and the filtrate evaporated with the aid of an electric fan to the consistency of a thick, sticky mass; this mass is dissolved in a small quantity of ether, the solution is filtered, and to the filtrate is added five times its volume of acetone; the precipitate which is thrown down immediately is allowed to settle and is taken up after the supernatant fluid has been decanted. 0.2 gram of this precipitate is dissolved in 5 c.c. of ether and to this 100 c.c. of 0.9% salt solution is gradually added; the resulting emulsion is filtered through paper to remove flocculi or solid particles.
This emulsion can be kept on ice for weeks without deteriorating, and the stock mass of antigen can be kept even for months under acetone also on ice.