A radioimmunoassay for immunologic phenomena in pneumococcal disease and for the antibody response to pneumococcal vaccines. I. Method for the radioimmunoassay of anticapsular antibodies and comparison with other techniques

Journal of Immunological Methods, 33 (1980) 133--144 © Elsevier/North-Holland Biomedical Press

133

A RADIOIMMUNOASSAY FOR IMMUNOLOGIC PHENOMENA IN PNEUMOCOCCAL DISEASE A N D F O R T H E A N T I B O D Y R E S P O N S E T O PNEUMOCOCCAL VACCINES. I. M E T H O D F O R T H E RADIOIMMUNOASSAY OF ANTICAPSULAR ANTIBODIES AND COMPARISON WITH OTHER TECHNIQUES 1

GERALD SCHIFFMAN, ROBERT M. DOUGLAS, MARY JO BONNER, MIRIAM ROBBINS and ROBERT AUSTRIAN John Herr Musser Department of Research Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 and (G.S.) Department of Microbiology and Immunology, Downstate Medical Center, SUNY, Brooklyn, N Y 11203, U.S.A.

(Received 26 September 1979, accepted 30 October 1979)

A radioimmunoassay is described which uses a 14C biosynthetically internally labeled antigen. This modified Farr technique has been standardized by quantitative precipitation and compared with hemagglutination and mouse protection. Specificity was established by use of heterologous hyperimmune sera and by use of unlabeled pneumococcal polysaccharides for inhibition. Reproducibility has been evaluated for different preparations of antigens and varying storage conditions of sera. The system has been applied to a wide variety of studies requiring analysis of human and animal sera.

INTRODUCTION A n t i b o d i e s t o p n e u m o c o c c a l capsular p o l y s a c c h a r i d e s are d e t e r m i n e d f o r t h e f o l l o w i n g reasons: (1) to a s c e r t a i n t h e i m m u n e r e s p o n s e t o a v a c c i n e in o r d e r t o assess p r o t e c t i o n against p n e u m o c o c c a l t y p e s in t h e vaccine, (2) t o d e t e r m i n e t h e r e s p o n s e to t h e c o m p o n e n t s o f p n e u m o c o c c a l vaccine u n d e r a v a r i e t y o f c o n d i t i o n s , including genetic i m m u n o d e f i c i e n c i e s or s u p p r e s s i o n o f t h e i m m u n e s y s t e m b y s u r g e r y , r a d i a t i o n o r c h e m o t h e r a p y , (3) to aid in t h e diagnosis o f p n e u m o c o c c a l i n f e c t i o n s b y d e t e c t i o n o f a n t i g e n e m i a , circulating a n t i g e n ~ a n t i b o d y c o m p l e x e s o r a rise in t h e t i t e r o f a n t i c a p s u l a r antibodies. I m m u n o l o g i c m e t h o d s w h i c h have b e e n e m p l o y e d f o r t h e s e p u r p o s e s are: q u a n t i t a t i v e p r e c i p i t a t i o n ( H e i d e l b e r g e r a n d DiLapi, 1 9 4 9 ; S c h i f f m a n et al., 1 9 6 2 ; S c h i f f m a n , 1 9 6 6 ) , i n d i r e c t h e m a g g l u t i n a t i o n ( B a k e r et al., 1 9 6 9 ; A m m a n n a n d Pelger, 1 9 7 2 ) , c o u n t e r i m m u n o e l e c t r o p h o r e s i s ( K e n n y et al., 1 9 7 2 ; C o o n r o d a n d R y t e l , 1 9 7 3 ) , m o u s e p r o t e c t i o n ( M a c L e o d et al., 1 9 4 5 ; Douglas, 1 9 7 3 ; Castagna, 1978}, r a d i o i m m u n o a s s a y a n d , m o r e r e c e n t l y , 1 This study was partially supported by Contract Nos. PH 4367681, PH NO1 AI 32517 and NO1 AI 42541 from the National Institute of Allergy and Infectious Diseases.

134 enzyme-linked immunosorbent assay (ELISA) (Engvall and Perlmann, 1972; Beauvery et al., 1977; Berntsson et al., 1978; Dolana et al., 1979; Gray, 1979; Russell et al., 1979). This paper describes a radioimmunoassay which is based on the use of biosynthetic, internally labeled (14C) capsular polysaccharide antigens. The Farr technique (Farr, 1958) for precipitation of antigen-antibody complexes permits the determination of total antigenbinding capacity to be made rapidly, specifically and with a sensitivity in the nanogram range. MATERIALS AND METHODS

Strains of pneumococcus and sources of antisera Pneumococcal strains were either isolated from patients, obtained from the American Type Culture Collection, or received from Dr. E. Lund, Statens Seruminstitut, Copenhagen, Denmark. Cultures were monitored for purity by use of the Gram stain, the capsular precipitin (Quellung) reaction and the appearance of growth on the surface of blood agar plates. Typing sera for the Quellung reaction were obtained from the Statens Seruminstitut or prepared by standard procedures (Schiffman, 1966). Pneumococcal polysaccharides Unlabeled pneumococcal capsular polysaccharides prepared by E.R. Squibb and Sons were received from the late Dr. C.M. MacLeod, others prepared by Dr. R. Brown, New York State Department of Public Health, were provided by Dr. K. Amiraian. Other generous supplies were from Merck, Sharp and Dohme and from Lederle Laboratories. Preparation of antigens Uniformly labeled [14C]glucose (240 mCi/mmole) (ICN Pharmaceuticals, Inc., Irvine, CA) was incorporated into the capsular polysaccharide of each pneumococcal type during growth of the organism. Stock cultures of Streptococcus pneumoniae of the types required for assay are grown in fresh beef heart infusion medium (Austrian, 1974) and subcultured on blood agar. One ml of the culture is used to inoculate 50 ml of medium containing 2mCi of 14C uniformly labeled [14C]glucose (240 mCi/mmole) and is grown at 37°C for 18 h. Indicator, 0.5% phenol red in 95% ethanol, is added and the culture is neutralized with 3 N NaOH added dropwise. Sodium deoxycholate is added up to 1.0 mg/ml. The lysed culture is neutralized as needed and dialyzed against at least 10 vol of deionized water, changed 3 times. The non-dialyzable material is recovered by lyophilization, dissolved in 5 ml of 5% sodium acetate, and centrifuged to remove insoluble material; 2.5 ml of absolute ethanol is added with mixing. The polysaccharide is purified by fractionation with alcohol. After 18 h at 4°C the precipitate is centrifuged. An additional 2.5 ml of ethanol is added to the supernatant fluid and this is repeated twice. The precipitates

135 are assayed for incorporated counts and precipitability with specific antiserum by the method to be described. Additional purification consists of gel filtration on Sepharose 4B, fractionation with ammonium sulfate, and ion exchange chromatography on DEAE cellulose (Whatman CM52). Technique. Antibodies to each of the 14 capsular antigens in the currently licensed pneumococcal vaccine are assayed as follows: the radiolabeled capsular polysaccharides are diluted to 20,000 cpm/ml. Aliquots of serum, 5--50 gl, are mixed with 0.5 ml (10,000 cpm) of labeled antigen and incubated at 37°C for 15 min. The antigen-antibody complexes are precipitated with 0.5 ml of ammonium sulfate solution saturated at 37°C and centrifuged at 27,000 × g at 4°C for 15 min. The resulting pellet is resuspended in 50 pl of 10% aqueous Triton X-100 (Rohm and Haas, Philadelphia, PA), 10 ml of scintillant are added, and the activity is measured in a liquid scintillation counter. Dilutions of a serum standardized by quantitative precipitation (Schiffman et al., 1962; Schiffman, 1966) are treated similarly. From a curve constructed b y plotting counts per minute in excess of a blank sample containing no antibody against nanograms of antibody nitrogen added, antibody nitrogen per ml in the test serum is determined b y reference to the standard curve. Each assay includes controls with aliquots of the reference antibody and others with labeled antigen only. The within-assay coefficient of variation of standard samples measured repeatedly was 2--4%. Diluent. The diluent used is 0.01 M phosphate-buffered saline, pH 7.2 (Kabat, 1961) to which is added fetal calf serum (GIBCO, Grand Island, NY) to a final concentration of 2.5%, inactivated by heating 30 min at 56°C. Five ml of 5% phenol in 0.15 M saline is added to each 100 ml of diluent to maintain sterility. Scintillant. The scintillant consists of a solution of toluene and Triton X-100 in a 2 : 1 ratio, each liter of which contains 4 g of 2,5-diphenyloxazole (PPO). RESULTS

Specificity Specificity was evaluated b y several methods, the first of which was to test precipitability of the 14C-labeled pneumococcal capsular polyaccharides by homologous and heterologous rabbit antisera. The results show that essentially all the added counts (90--100%) are precipitated b y homologous antibody and less than 10% b y antibody to other pneumococcal capsular polysaccharides. The second m e t h o d was inhibition of the binding of 14C-labeled antigen to specific antibody by unlabeled homologous and heterologous pneumococcal capsular polysaccharides. Each system is completely inhibitable b y 1 pg of unlabeled homologous polysaccharide and much less b y polysaccharides of other types. Pneumococcal C polysaccharide did n o t inhibit significantly any system tested, indicating that C polysaccharide is n o t a major impurity in the 14C-labeled polysaccharides. Other

136

determinations of specificity will be described later when the values obtained b y RIA have been compared with quantitative precipitation, hemagglutination inhibition and mouse protection.

Reproducibility of the radioimmunoassay In epidemiologic studies of pneumococcal disease and in assessment of the immunologic responses of man to pneumococcal vaccines, it is important to have data regarding the reliability and reproducibility of the method. Four facets of this problem have been investigated: (1) reproducibility of results obtained from the repeated assay of a single serum specimen with a single antigen; (2) reproducibility of the assay of a single serum with different lots of labeled antigen of the same capsular type; (3) reproducibility of results with a single serum stored under varying conditions and reacting with a single antigen; and (4) stability of radiolabeled antigens with the passage of time. Table 1 records the results of repeated assays with each of 11 labeled capsular antigens of a single serum from a volunteer vaccinated with a dodecavalent vaccine of pneumococcal capsular polysaccharides. Except with t y p e 4, the standard deviations of more than 25 replicate assays for each t y p e fall within 20% of the means. The standard deviation for type 4 is 25% of the mean, reproducibility of the assay of antibody to this antigen being technically more difficult because of its precipitation b y less ammonium sulfate than that required for the other capsular antigens. Aliquots of the same sera when assayed with different lots of radiolabeled

TABLE 1 REPRODUCIBILITY OF ANTIBODY LEVELS IN REPLICATE RADIOIMMUNOASSAYS OF A SINGLE SERUM FROM A NORMAL HUMAN IMMUNIZED WITH DODECAVALENT PNEUMOCOCCAL VACCINE. Assays with a single lot of antigen of each of 11 types. Pneumococcal type

Number of assays

Mean /lg AbN/ml

S.D.

Lowest value

Highest value

1 2 3 4 6A 7F 8 12F 14 18C 19F

29 32 30 28 30 30 30 30 30 30 30

1.20 3.61 2.79 1.44 1.54 0.85 2.20 0.25 0.32 0.58 0.62

0.16 0.30 0.26 0.36 0.12 0.10 0.34 0.05 0.05 0.07 0.10

0.9 2.9 2.5 0.9 1.4 0.6 1.7 0.2 0.2 0.4 0.4

1.60 4.90 3.30 1.80 1.7 1.0 2.9 0.3 0.4 0.7 0.8

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antigen show good agreement when these radioactive antigens are prepared in a reproducible manner. The effect of the storage of serum for 6 months on its content of antib o d y is given in Table 2. There appears to have been little alteration regardless of whether the serum was stored at 4°C, at room temperature, or was frozen and repeatedly thawed and refrozen. Radiolabeled capsular antigens stored at 4°C are stable for 6 months to a year. Stability can be monitored and loss in specific activity detected b y comparison with results obtained with the standard reference antiserum used as a control. Even when specific activity has begun to decline, the antigens can be recalibrated in this way and used.

Correlation of the results o f the radioimmunoa~ay, quantitative precipitin test, indirect hemagglutination test and mouse protection in the assay To obtain sera for this correlation 8 young adult volunteers were bled 250 ml prior to immunization with a dodecavalent vaccine containing the capsular polysaccharides of pneumococcal types 1, 2, 3, 4, 5, 6A, 7F, 8, 12F, 14, 18C and 19F and again 3 weeks later. The sera from the paired bleedings were coded and assayed for type-specific antibody to types 1, 2,

TABLE 2 LACK OF E F F E C T OF STORAGE AND MODE OF HANDLING ON THE ASSAY OF HUMAN ANTIBODIES TO EACH OF 3 PNEUMOCOCCAL CAPSULAR POLYSACCHARIDES IN PAIRED SERA OF TWO V O L U N T E E R S BEFORE AND A F T E R IMMUNIZATION WITH POLYVALENT PNEUMOCOCCAL VACCINE Antibody to type :

Serum specimen

pg AbN/ml Initial assay

Storage 4°C 6 months

Storage r o o m temp, 6 months

Freeze-thaw 48 cycles 6 mouths

1

la a lp 2a 2p

0.3 2.4 0.1 0.7

0,3 2.6 0.1 0.8

0.3 2.0 0,1 0.8

0.3 2.4 0.1 0.7

2

la lp 2a 2p

0.4 7.3 0,3 3.1

0.4 8,3 0,2 3,2

0.3 6.3 0.2 2.9

0.1 7.2 0 1.2

8

la lp 2a 2p

0.1 6.0 0.2 3.0

0.1 4,5 0,2 2.1

0 5.0 0.2 2.8

0 3.5 0 2.4

a Sera l a and 2a obtained before immunization; sera l p and 2p, 3 weeks after immunization,

138 TABLE 3 DOUBLE BLIND ASSAY OF ANTIBODIES TO TYPE 1 CAPSULAR POLYSACCHARIDE IN SERA OF 8 HUMAN SUBJECTS BEFORE AND 3 WEEKS AFTER IMMUNIZATION For Tables 3, 4, 5 and 6, Quant. precip. = quantitative precipitin test (Schiffman et al., 1962; Schiffman, 1966), results i n ng AbN/ml; RIA = radioimmunoassay, results in ng AbN/ml; HA = hemagglutination assay. The technique for the assay of type specific capsular antibodies by agglutination of erythrocytes coated with pneumococcal capsular polysaccharides was that of A m m a n n and Pelger (1972); Mouse protection test. Serial 3-fold dilutions of the serum to be assayed were made and 0.3 ml of undiluted serum or of each of 5 serial dilutions was mixed with 50--100 MLD of the pneumococcal challenge to give a final volume of 1.5 ml. Immediately following the admixture of serum and organisms, 3 mice were injected intraperitoneally with 1.5 ml of each preparation. Mice were observed for 6 days, and deaths were recorded. To quantify the protective value of a serum, the following procedure was adopted. If all mice died, the protective value assigned to the serum was zero. If 2 or 3 mice injected with undiluted serum survived and all others died, the value assigned was 1. The protective value assigned when higher dilutions prevented death was the denominator of the highest dilution protecting 2 of 3 mice; i.e., 3, 9, 27, 81, or 243. Animals succumbing were autopsied, and bacteriologic studies were performed to assure the cause of death was pneumococcal infection. Subject

Serum

Test

no.

Quant. precip,

RIA

Mouse protection

HA

1

Pre Post

400 6300

800 2300

0 27

256 256

2

Pre Post

500 3600

800 190

0 3

32 512

3

Pre Post

800 3800

900 1400

0 3

512 512

4

Pre Post

800 3600

700 1300

0 9

32 64

5

Pre Po~

500 4700

400 1050

0 9

64 1024

6

Pre Post

600 3400

500 1840

0 9

32 512

7

Pre Po~

300 2200

200 840

0 9

32 256

8

Pre Post

600 3000

2060 2750

1 1

32 1024

a n d 8 b y r a d i o i m m u n o a s s a y , q u a n t i t a t i v e p r e c i p i t i n t e s t ( S c h i f f m a n e t al., 1 9 6 2 ; S c h i f f m a n , 1 9 6 6 ) , i n d i r e c t h e m a g g l u t i n a t i o n ( B a k e r e t al., 1 9 6 9 ; A m m a n n a n d P e l g e r , 1 9 7 2 ) a n d m o u s e p r o t e c t i o n ( M a c L e o d e t al., 1 9 4 5 ; Douglas, 1 9 7 3 ; Castagna, 1978). All assays were p e r f o r m e d w i t h o u t t h e

139 TABLE 4 A S S A Y O F A N T I B O D I E S TO T Y P E 2 C A P S U L A R P O L Y S A C C H A R I D E IN T H E SERA OF 8 HUMAN SUBJECTS BEFORE AND 3 WEEKS AFTER IMMUNIZATION F o r legend see T a b l e 3. Subject

Serum

Test

riO.

Quant. precip,

RIA

Mouse protection

HA

1

Pre Post

200 8000

130 5500

0 81

16 2048

2

Pre Post

1200 8300

200 9900

0 27

4 128

3

Pre Post

400 5100

0 7500

1 27

64 512

4

Pre Post

400 5900

100 3300

0 9

4 128

5

Pre Post

700 4700

100 3400

0 27

4 128

6

Pre Post

900 5100

100 5400

1 27

4 1024

7

Pre Post

200 3200

100 1200

0 3

8 256

8

Pre Post

400 2600

200 1800

0 27

8 128

knowledge of the experimenter as to donor and time in relation to immunization. The results are recorded in Tables 3, 4 and 5 and show general agreement. Rank order coefficients of correlation were derived for all 4 methods for each individual t y p e and for the combined data from the assays of the 3 pneumococcal types (Table 6). Rank orders for the quantitative precipitin test and mouse protection test correlated well for all 3 types. Coefficients of correlation of the radioimmunoassay and quantitative precipitation and of the radioimmunoassay and mouse protection showed good correlation for types 2 and 8 b u t were less good for type 1. Excellent correlation was observed between hemagglutination and mouse protection for type 1. In general, the radioimmunoassay correlated slightly better with b o t h quantitative precipitation and mouse protection than did indirect hemagglutination. The results indicate that the radioimmunoassay is suitable for the assay 1of antibodies to pneumococcal capsular polysaccharides although values obtained in the assay of antibodies to t y p e 1 capsular polysaccharide m a y be less than optimal. Correlation of the R I A and quantitative precipitin test was also carried

140 TABLE 5 ASSAY OF ANTIBODIES TO TYPE 8 CAPSULAR POLYSACCHARIDE IN THE SERA OF 8 HUMAN SUBJECTS BEFORE AND 3 WEEKS AFTER IMMUNIZATION For legend see Table 3. Subject no.

1 2 3 4 5 6 7 8

Serum

Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post

Test Quant. precip,

RIA

Mouse protection

HA

300 7900 1300 3700 2600 8900 900 1300 0 4900 700 6600 900 3500 1000 3000

100 6000 100 1800 800 6000 100 1800 100 3800 200 6000 100 2400 500 1000

0 81 1 9 1 27 0 3 0 27 0 27 0 9 0 1

16 33,000 16 512 128 4096 32 512 128 8192 16 8192 32 1024 32 128

o u t w i t h a n t i b o d i e s f r o m s e q u e n t i a l bleedings o f r a b b i t s i m m u n i z e d w i t h vaccines o f w h o l e p n e u m o c o c c i . Bleedings t a k e n e a r l y in i m m u n i z a t i o n a n d c o n t i n u e d f o r m a n y m o n t h s s h o w e d n o d i v e r g e n c e b y e i t h e r m e t h o d . Sera o f vaccinees a n d o f p a t i e n t s c o n v a l e s c i n g f r o m p n e u m o c o c c a l p n e u m o n i a w e r e f r a c t i o n a t e d o n S e p h a d e x G - 2 0 0 (Osler et al., 1 9 6 6 ) . H e m a g g l u t i n a t i n g activi t y is l o c a t e d p r i m a r i l y in f r a c t i o n s c o n t a i n i n g IgM w h e r e a s b i n d i n g o f radiolabeled antigens o c c u r s in b o t h IgM a n d I g G . I f t h e a m o u n t o f IgG is large, s o m e h e m a g g l u t i n a t i n g a c t i v i t y is seen in t h e I g G region as well. T h e smaller c o e f f i c i e n t s o f c o r r e l a t i o n b e t w e e n t h e results o f t h e r a d i o i m m u n o a s s a y a n d indirect hemagglutination probably reflect the disparate behavior of the several i m m u n o g l o b u l i n s in t h e tests, as well as t h e n o n - u n i f o r m distrib u t i o n o f a n t i c a p s u l a r a n t i b o d i e s a m o n g i m m u n o g l o b u l i n s o f t h e sera o f m a n f o l l o w i n g v a c c i n a t i o n or p n e u m o c o c c a l i n f e c t i o n . DISCUSSION T h e r a d i o i m m u n o a s s a y d e s c r i b e d , like t h o s e e m p l o y e d f o r t h e m e a s u r e m e n t o f a n t i c a p s u l a r a n t i b o d i e s t o o t h e r bacterial species ( B r a n d t et al., 1 9 7 2 ; J o h n s t o n et al., 1 9 7 3 ) , p r o v i d e s a r a p i d a n d sensitive m e t h o d f o r

141 TABLE 6 COEFFICIENTS OF CORRELATION AMONG RESULTS OF 4 TECHNIQUES FOR ASSAY OF ANTIBODIES TO PNEUMOCOCCAL CAPSULAR POLYSACCHARIDES For legend see Table 3.

Type 1 Quant. precip.

Quant. precip,

RIA

Mouse protection

HA

1.00

0.51 (P = 0.02) 1.00

0.81 (P < 0.001) 0.60 (P < 0.007) 1.00

0.70 (P < 0.001) 0.42 (P < 0.55) 0.51 (P < 0.022) 1.00

RIA Mouse protection HA Type 2 Quant. precip. RIA Mouse protection HA

1.00

0.89 1.00

0.85 0.81 1.00

0.71 0.77 0.86 1.00

0.89 1.00

0.85 0.81 1.00

0.71 0.77 0.86 1.00

0.84 1.00

0.88 0.83 1.00

0.75 0.78 0.75 1.00

P values for all correlations <0.001. Type 8 Quant. precip. RIA Mouse protection HA

1.00

P values for all correlations <0.001. Combined data for types 1, 2 and 8 Quant. precip. 1.00 RIA Mouse protection HA P values for all correlations <0.001.

the measurement of antibodies to many pneumococcal capsular polysaccharides. Incorporation of the 14C-radiolabel into the capsular polysaccharide during growth of pneumococcus avoids possible modification of the antigen which might result from the chemical treatment required to label it extrinsically, and the antigens manifest considerable stability when stored at 4°C. The procedure calls for no more than 0.1 ml of serum and is 1000fold more sensitive than the quantitative precipitin test (Schiffman, 1966). The radioimmunoassay was not suitable for antibodies to the capsular polysaccharides o f pneumococcal types 5 and 11 because both are precipitated by the concentration of ammonium sulfate used to precipitate antigen-antibody complexes. Because of the ease with which the labeled capsular antigens of most pneumococcal types can be standardized, the

142 radioimmunoassay is devoid of difficulties of quantification inherent in agglutination tests performed with differing serotypes, the cells of which produce unequal quantities of capsular polysaccharide, and in the direct hemagglutination test, which measures mainly antibodies of the IgM class. It is much cheaper and more humane than the mouse protection test, which, besides, can be used to assay antibody only to those pneumococcal types which are virulent for the mouse. Methods for the preparation of labeled capsular antigens of pneumococcus have been designed to minimize inclusion of C or cell wall polysaccharide of pneumococcus, antibody to which is found in the sera of most normal humans (Heidelberger and Anderson, 1944). Neither the C polysaccharide of the strains used as sources of capsular antigens nor the Cs polysaccharide of a pneumococcai mutant producing a capsule of cell wall-like polysaccharide (Bornstein et al., 1968) incorporates significant amounts of 14C from labeled glucose into its structure. However, removal of C-polysaccharide seems necessary since Higginbotham et al. (1970) reported that removal of pyrurate from the capsular polysaccharide of pneumococcus type 4 results in a product which reacts with antibody to C polysaccharide and with C-reactive protein. Reproducibility of the results of the radioimmunoassay has been examined with a variety of types and under several conditions and has been found comparable to that of other methods. Correlation of results by the radioimmunoassay with those of the quantitative precipitin test, mouse protection and indirect hemagglutination indicates that the radioimmunoassay is suitable for the quantification of antibodies to the type-specific capsular antigens of many pneumococcal types. Correlation with mouse protection test is rather poor but this is to be expected because the mouse protection test is notoriously inaccurate. Because of its sensitivity and the small quantities of reagents required, the radioimmunoassay lends itself well to studies of the antibody responses of man and of animals to pneumococcal vaccines and infection, including measurement of antibodies to pneumococcal polysaccharides of patients with pneumococcal infections (Dee et al., 1977), in patients splenectomized for trauma (Sullivan et al., 1978) or Hodgkin's disease (Siber et al., 1978; Minor et al., 1979), and in patients with sickle cell disease (Sullivan et al., 1978). The RIA has also been used to study the immune response in children with lipid nephrosis (Fikrig et al., 1978), systemic lupus erythematosus (Klippel et al., 1979), renal dialysis (Simberkoff et al., 1979), and renal allografts (Dailey et al., 1979). The immune response to polysaccharides in the vaccine has been evaluated in normal adults (Borgono et al., 1978), infants (Borgono et al., 1978; Sell et al., 1978) a n d in children with otitis media (Sloyer et al., 1979). By adding unlabeled antigen to the 14C4abeled antigen before adding antibody one can construct a curve of per cent inhibition by added unlabeled antigen, making it possible to determine quantitatively: (1) purity of vaccine, (2) antigenemia in patients with pneumococcal infections, (3) quantita-

143

tive determination of antigen-antibody complexes (Preheim et al., 1979) after dissociation of the complexes and proteolysis of the antibody, (4) subtyping of pneumococci in the absence of monospecific serum (Schiffman et al., 1975). ACKNOWLEDGEMENTS

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