usually contain somatic protein as the principal antigen, determination of the Heidelberger and Kendall (1932) developed a method for the quantitative

Transcription

1 A MOUSE PROTECTION METHOD FOR THE ESTIMATION OF ANTIGENIC PNEUMOCOCCAL POLYSACCHARIDE IN SOLUTION CURTIS SANDAGEl AND ORTON K. STARK Miami University, Oxford, Ohio Received for publication May 5, 1947 In a study involving the preparation and testing of pneumococcus capsular polysaccharide, the need for a rapid method of assay of preparations of unknown antigenic potency became evident. Most bacterial vaccines are standardized on the basis of cell counts or total nitrogen determinations. Since such vaccines usually contain somatic protein as the principal antigen, determination of the number of cells or of the quantity of nitrogen is often a reliable criterion of antigenicity. Obviously, neither procedure can be used for the standardization of capsular oarbohydrate preparations. Heidelberger and Kendall (1932) developed a method for the quantitative estimation of specific carbohydrate in solution which is based on the precipitation of this material by homologous type-specific antiserum. By this method, they were able to determine as little as 0.01 mg of type III pneumococcus polysaccharide. The procedure is quite satisfactory for many purposes, but it does not necessarily measure the antigenicity of the substance under test. For example, Felton (1934) found that heating type I pneumococcus polysaccharide in acid solution destroyed from 50 to 87 per cent of its immunizing activity, although the precipitin titer was not altered. Avery and Goebel (1933), working with type I pneumococcal capsular polysaccharide (subsequently referred to as SI), found that the deacetylated product precipitated specific antibody from homologous antiserum but that it was not antigenic when tested in mice. It is evident in this case that the method based on the precipitin reaction is not a reliable index of antigenicity. The method of Heidelberger and Kendall is applicable only when the specific carbohydrate is obtainable in a state of purity. Even then, a given weight of the pure material may vary widely in antigenicity owing to the effect of different methods of purification. Sevag (1934) reported that mice treated with mg of SI survived 1,000 fatal doses of type I pneumococcus. Schiemann et al. (1931), using highly purified SI, found that the minimum amount necessary to produce demonstrable active immunity when injected into white mice was 0.01,ug. Since mice are so responsive imunologically to antigenic SI, it seemed that a mouse protection method might offer an extremely sensitive means of evaluating the antigenicity of experimental preparations. EXPERIMENTAL A sample of highly purified SI was obtained2 and tested in white mice. Animals weighing 18 to 22 grams were injected intraperitoneally on 4 successive 1 The Wm. S. Merrell Co., Fellow. M 2 Dr. Michael Heidelberger, College of Physicians and Surgeons, Columbia University, kindly supplied this material. 8383

2 334 CURTIS BANDAGE AND ORTON K. STARK [VOL. 54 days using a daily dose of 0.25 ml of tenfold dilutions of the SI in saline. Five days after the last immunizing dose, the mice were challenged by intraperitoneal injections using a type I pneumococcus culture. The virulence of the test culture was such that 1.0 ml of a 10- dilution was regularly fatal to control animals within 60 hours. Dilutions of 107, 10(8, and 10-s were used in order to be certain that some animals would receive 100 or more MLD. In this work, there has been no need to use the LD5o method. The data shown in table 1 indicate that the minimum quantity of SI affording complete protection is 0.01,g. Using this material as a standard of potency, various crude and partially purified preparations were assayed by determining the highest dilution of the unknown which gives corresponding protection against type I pneumococcus. The results of a representative test are shown in table 2. The material under test was a partially purified SI solution obtained from a broth culture. It is evident that 1: 5,000,000 is the highest dilution of the unknown affording mouse TABLE 1 Determination of the minimal quantity of standard SI which provides immunity against D. pneumoniae, type I DIUNWIZING DOSE OF STANDARD SI DILUTION OF CHALLENGING CULTURE None (control) 0/6 0/6 6/6 1.0gg 6/6 6/6 6/6 0. 1,ug 6/6 6/6 6/ p&g 6/6 6/6 6/ pg 0/6 0/6 2/6 protection in this case. Since this dilution contains a quantity of SI equal in antigenic activity to 0.01 ug of the standard, the quantity of active SI in the original solution can be calculated as 50 mg per ml. Both tables 1 and 2 illustrate the definite end point which has been obtained routinely by this method. In table 1 it can be seen that whereas quantities of the standard SI from 1.0 ug to 0.01 Mug are completely effective in protecting mice, Mg is ineffective. The lowest concentration of SI affording complete protection is considered to be the end point. No significance is attached to the average survival time for individual groups, even though mice receiving less than 0.01 Lg occasionally appear to survive somewhat longer than control animals. The necessity for statistical treatment of data is thus eliminated. In order to obtain these definite end points, it is necessary to standardize the conditions of culture and the virulence of the organism used for challenging. Mice of the proper weight from four sources have been used for these tests with completely consistent results. The results obtained in an attempt to apply this method for the estimation of

3 1947] ESTIMATION OF PNEUMOCOCCAL POLYSACCHARDE 335 SI in body fluids are shown in table 3. A rabbit weighing 3 kilograms was injected intravenously using 25 mg of SI. One hour later the rabbit was bled TABLE 2 Determination of the minimal dose of unknown SI solution which provides immunity against D. pneumoniae, type I DIIlUTION O CHALLENGING CULTURE Controls Saline 0/6 0/6 6/6 Standard SI, 0.01,Pg 6/6 6/6 6/6 Dilutions of unknown SI 1:500,000 6/6 6/6 6/6 1:5,000,000 5/6 6/6 6/6 1:50,000,000 0/6 0/6 1/6 TABLE 3 Estimation of SI in rabbit serum by determining active immunity developed in mice in response to injection of the serum DILUTION OF CEALLENGING CULTURE 10-i I 10 I 10 I 10-1 Controls Saline 0/6 0/6 6/6 Normal serum 1:10 0/6 0/6 1/6 SI*standard, 0.05* pg 6/6 6/6 6/6 Dilution of serumt from rabbit injected with 25 mg SI 1:10 6/6 6/6 6/6 1:1,000 6/6 6/6 6/6 1:10,000 4/4 4/4 4/4 1:100,000 0/4 0/4 0/4 * Diluent was normal serum 1:10. t Serum from blood obtained by cardiac puncture 1 hour following intravenous injection of SI. from the heart. Basing the calculations on body weight, the serum obtained should have contained approximately 200,ug per ml. Calculations based on the results given in table 3 indicate that it contained more than 100,ug but less than 1,000,ug per ml. No attempt was made to determine the quantity more accu-

4 336 CURTIS BANDAGE AND ORTON K. STARK [vol.54 rately, though it could doubtless be done by using additional dilutions of the unknown. It appears that the presence of body fluids does not interfere with the test, although naturally occurring immune substances in serum must be considered. DISCUSSION The usual mouse protection tests, both active and passive, emphasize the determination of the number of lethal doses which treated animals resist rather than the determination of the quantity of antigen required to produce significant immunity. The passive mouse protection test of immune serum is, nevertheless, an indirect means of determining the quantity of antibody present (Heidelberger, Sia, and Kendall, 1930). The principle involved in active mouse protection tests is quite different in that the response of the mouse to the antigenic stimulus determines the amount of antibody formed. It would appear that the determination of the minimal quantity of an antigen which elicits active immunity may be more significant as a measure of antigenicity than the number of lethal doses of the test organism which the mouse will resist. The method presented accomplishes this objective and gives easily interpreted end points. In preliminary work it appears that the time necessary to complete such a test can be shortened considerably. In one experiment it was found that a single injection of 1.0 ml of the material under test produced the same results as did 4 consecutive daily injections of 0.25 ml. This would shorten the time required for a determination by 3 days. It may not be necessary to allow 5 days between immunization and challenging of mice. In another experiment comparable results were obtained after a 4-day waiting period. Since significant deaths occur within 72 hours, this schedule permits a test to be completed within 8 days. The method described has been limited in application to pneumococcal materials which are antigenic in mice and for which an acceptable comparison standard can be obtained. There is no reason to believe that it could not be applied to the evaluation of other antigenic materials of at similar nature. The choice of dosage schedule, waiting period, and the standard to be used is arbitrary and can be planned to suit individual needs. SUMMARY A mouse protection method for the estimation of antigenic pneumococcal polysaccharide in solution has been described. The principle of the test is based on the immune response of white mice to minute quantities of antigenically active polysaccharide. The procedure should be a useful supplement to methods based on the precipitin reaction because of its sensitivity and technical simplicity. Furthermore, the method described does not require standardization of antisera or purification of the antigen under test. This procedure provides a measure of antigenic potency rather than a measure of precipitable polysaccharide.

5 1947] ESTIMATION OF PNEUMOCOCCAL POLYSACCHARIDE 337 REFERENCES AVERY, 0. T., AND GOEBEL, W. F Chemo-immunological studies on the soluble specific substance of pneumococcus. I. The isolation and properties of the acetyl polysaccharide of pneumococcus type I. J. Exptl. Med., 58, FELTON, L. D The effect of heat on alkaline and acid solutions of the inununizing substances of pneumococci type I and type II. J. Immunol., 27, 336. HEIDELBERGER, M., AND KENDALL, F. E Quantitative studies on the precipitin reaction. The determination of small amounts of a specific polysaccharide. J. Exptl. Med., 55, HEIDELBERGER, M., SIA, R. H. P., AND KENDALL, F. E Specific precipitation and mouse protection in type I antipneumococcus serum. J. Exptl. Med., 52, SCHIEMANN, O., LOEWENTHAL, O., AND HACKENTHAL, H tjber die immunisierende und shockerzeugende Wirkung von C-haltigen Fraktionen, gereinigter C-Substanz und Lipoiden aus Pneumokokken. Z. Hyg. Infektionskrankh., 112, SEVAG, M. G Eine neue physikalische Enteiweissungsmethode zur Darstellung biologisch wirksamer Substanzen. Isolierung von Kohlenhydraten aus Hithnereiweiss und Pneumococcen. Biochem. Z., 273, Downloaded from on August 23, 2018 by guest