Antigen thus prepared possesses, on the one hand, true antigenic power, that is to say, the power of binding complement in the presence of a syphilitic serum and thus inhibiting haemolysis, and, on the other hand, generally in a lesser degree, an anti-complementary power, that is to say, a power of destroying complement and thus inhibiting haemolysis without the intervention of a syphilitic serum. It must therefore be standardized with a view to determining the proper dosage to be used in the work to insure ample antigenic action and to exclude simple anti-complementary action. For this purpose a titration is carried out in the following manner: twenty small test-tubes are arranged in two rows in a suitable rack; one puts into each test-tube 1 c.c. of sheep corpuscle suspension and 0.1 c.c. of complement, prepared as described above; to each of the tubes In the front row one adds also 0.2 c.c. of serum from a syphilitic subject, known to give a positive reaction; one adds finally to the test tubes in both rows the antigen emulsion in amounts varying from 0.03 c.c. in the first test-tube to 1.0 c.c. in the tenth, as shown in the following sample titration.

The rack is then placed in the incubator for one hour, at the end of which time two units of amboceptor serum are added to each tube in both rows and the rack is again placed in the incubator; at the end of two hours of the second incubation the reading is taken.

Table 16

Amount of

Antigen

Emulsion.

Front row of tubes: inhibition of haemolysis due to true antigenic action.

Back row of tubes: inhibition of haemolysis due to simple anticomplementary action.

0.03 c.c.

Complete haemolysis

Complete haemolysis

0.05 c.c.

" "

" "

0.07 c.c.

Partial inhibition

" "

0.10 c.c.

Complete inhibition

" "

0.12 c.c.

" "

" "

0.20 c.c.

" "

" "

0.25 c.c.

"

" "

0.50 c.c.

" "

Partial inhibition

0.75 c.c.

" "

Complete inhibition

1.00 c.c.

" "

" "

The proper dosage of a specimen of antigen giving on titration results like those represented above would be 0.12 c.c.

It happens sometimes that a specimen of antigen is found on titration to possess either too feeble an antigenic power or too strong an anti-complementary power; in either case it cannot be used and another lot must be prepared.

Collection Of Specimens For Examination

The only-equipment required for obtaining a blood specimen is a test-tube, hollow needle about 1 1/2 inches long, gauge 19, with a short piece of rubber tubing attached to it, and a tourniquet consisting simply of a piece of rubber tubing about 16 inehes long. The tourniquet is applied well up on the arm tightly enough to impede the venous but not the arterial flow; it is more convenient to take the blood from the left arm. Having selected the largest sized superficial vein just above the bend of the elbow, the thumb of the left hand is placed on the vein partly to fix it and prevent its slipping and partly to guide the point of the needle; the needle then, held in the right hand with the rubber tube projecting into the test-tube which is also held in the right hand being grasped with the little and ring fingers, is thrust into the vein at a point as close as possible to where it is held by the thumb of the left hand; in doing so the needle is held with the bevel of its point upwards; the direction of the thrust is inwards and upwards in the direction of the vein. If the vein has been properly penetrated blood will begin to trickle into the test-tube either immediately or in a second or two.

If it seems that the needle has pierced through the vein instead of into it, blood may be started through it by withdrawing it slightly. About 6 or 7 c.c. of blood is allowed to flow into the test-tube, the needle withdrawn, and the puncture protected with a piece of sterile gauze fastened on with a strip of adhesive plaster. It goes without saying that the needle, test-tube, etc., are sterilized before being used and that the physician's hands and the patient's arm around the site of the puncture are scrubbed properly.

The test-tube containing the blood is stopped with a cotton plug and allowed to stand at room temperature for several hours, at the end of which time the serum may be examined for the reaction or it may be placed in the refrigerator to be examined on the following day.

Specimens of cerebro-spinal fluid are obtained by lumbar puncture, the technique of which has already been described.

Both the blood serum and the cerebro-spinal fluid should be examined if possible either on the same or on the following day after they have been obtained, as even if kept on ice they soon begin to undergo changes consisting most commonly of a development of non-specific anticomplementary power.

Technique Of The Reaction

A whole rackful of specimens may be examined together. It is most convenient to use a test-tube rack with spaces for two rows of test-tubes. Tubes 10 cm. long and 1 cm. in diameter are best for the purpose. For testing each specimen two tubes are used, a front tube for the reaction and a rear tube for control.

All the blood specimens to be examined are first inactivated by being placed for three-quarters of an hour in a thermostat at a temperature not exceeding 56° C. Spinal fluids do not require to be inactivated.

0.2 c.c. of the serum or spinal fluid to be examined is put in a front tube and the same amount in a corresponding rear tube. At the end of the rack two pairs of tubes are reserved respectively for the positive and negative controls: in the positive control tubes serum or cerebro-spinal fluid known to give a positive reaction is used; in the negative control tubes neither serum nor spinal fluid is used. To each tube is now added 0.1 c.c. guinea-pig complement. Finally to each front tube is added the proper dose of antigen emulsion as determined by the titration. It is well to dilute the antigen emulsion with 0.9% salt solution so that 1 c.c. of the dilution will contain the proper dose of antigen. Salt solution is now added to all the tubes, front and rear, so as to bring up the amount in each to 2 c.c, and the rack is placed in the incubator. At the end of one hour the rack is taken out and to each tube are added 1 c.c. of 5% sheep corpuscle suspension and the proper amount of anti-sheep haemolytic amboceptor as determined by the titration. As in the case of the antigen, it is well to dilute the amboceptor with 0.9% salt solution so that 1 c.c. will contain the proper amount of amboceptor.

Each test-tube is thoroughly shaken and the rack is returned to the incubator for two hours longer, during which time the tubes are frequently taken out, inspected, and shaken, and at the end of which time the readings are to be taken. The positive and negative control sets are inspected first, and if these are found to be all right the readings in the other tubes are taken and recorded. The rear tubes, containing no antigen, should in every case show complete haemolysis; if any rear tube shows inhibition of haemolysis it is probably due to non-specific anti-complementary power in the specimen of serum or cerebro-spinal fluid, as the case may be, and any inhibition of haemolysis in the front tube in such a case, being attributable to the same cause, is, therefore, inconclusive. If the rear tubes show complete haemolysis, inhibition of haemolysis in any front tube indicates a positive reaction, partial haemolysis indicates a slight or doubtful reaction, and complete haemolysis indicates a negative reaction.