Vol. 21, No. 1

INFECTION AND IMMUNITY, July 1978, p. 34-40

0019-9567/78/0021-0034$02.00/0

Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

Role of Antibody and Complement in Opsonization of Group B Streptococci ANN 0. SHIGEOKA,*l ROBERT T. HALL,2 VAL G. HEMMING,' CRAIG D. ALLRED,' AND HARRY R. HILL' 3 Division of Clinical Immunology, Department of Pediatrics,' and the Department of Pathology,3 University of Utah College ofMedicine, Salt Lake City, Utah 84132, and the Children's Mercy Hospital, University of Missouri, Kansas City, Missouri 641082

Received for publication 12 January 1978

A requirement for the classic complement pathway in opsonization of group B streptococci was observed by using both a chemiluminescence and a radiolabeled bacterial uptake technique. The classic pathway increased levels of opsonization for types Ia and II stock and wild strains and for some type III wild strains. In contrast, other type III wild strains and the type III stock strain had accelerated kinetics of uptake in the presence of an intact classic pathway, but the level of opsonization was unchanged from that with antibody alone. We could not demonstrate a significant role for the alternative pathway in opsonizing stock or wild strains of group B streptococci. Furthermore, electrophoretic and complement consumption analysis by hemolytic titration failed to reveal alternative pathway activation by the majority of strains of this group. Therapy aimed at supplying opsonins for these organisms will require the presence of type-specific antibody.

Such information would be essential in the proper selection of blood or blood products used in attempting to supply passive immunity to infected neonates. (This work was presented, in part, at the Plenary Sessions of the meeting of the Western Society for Pediatric Research, Carmel, Calif., 3 February 1977, and the meeting of the Society for Pediatric Research, San Francisco, Calif., 29 April 1977.)

Group B streptococci, in spite of their relative sensitivity to a number of antibiotics, are a major cause of morbidity and mortality in neonates and in other patients with compromised host defense mechanisms (1, 4, 6, 9, 11, 13, 25). Studies by Lancefield and co-workers (16, 17) have shown the protective effect in mice of passively administered type-specific antibody to these organisms. Such protection probably results from opsonic activity in these preparations, since a direct bactericidal effect has not been demonstrated. In a previous study we have shown that patients who develop group B streptococcal sepsis usually lack opsonic antibody to their infecting strain (10). Complement also contributes to the opsonization of most bacteria via the classic pathway when specific antibody is present and by the alternative pathway in the absence of antibody. Complement components of both pathways may be quantitatively and functionally diminished in neonatal serum compared with adult serum (8, 12, 19, 24; J. A. Winkelstein, L. Kurlandsky, and A. Swift, Pediatr. Res. 11:496). If complement is important in the opsonization of group B streptococci, such deficiencies may contribute to the susceptibility of the neonate to infection with these organisms. For these reasons, we examined the role of the classic and alternative complement pathways in opsonization of both stock and wild (isolated from infected infants) strains of group B streptococci.

MATERLALS AND METHODS Preparation of organisms. Reference (stock) strains of group B streptococci types Ia (090), II (18RS21), and III (D136) or group B streptococcal strains isolated from infected patients (wild strains) were cultured at 370C in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) for 18 h. Cultured organisms were centrifuged at 1,000 x g for 10 min at room temperature and washed three times in phosphatebuffered saline (PBS; 4,500 ml of distilled water, 5.2 g of Na2HPO4, 0.9 g of KCl, 0.9 g of KH2PO4, 36 g of NaCl). Standard suspensions were prepared by diluting the concentrated, washed organisms in sterile PBS to an optical density of 0.9 at 620 nm (Spectronic 20; Bausch & Lomb, Inc., Rochester, N.Y.). These suspensions contained 5.0 x 108 to 1.0 x 10" colony-forming units per ml.

Opsonizing procedures. Strains of group B streptococci (types Ia, II, and III) were opsonized by mixing 0.5 ml of the bacterial suspension with 0.1 ml of the test serum in a sterile capped tube (12 by 75 mm; Falcon Plastics, Division of Bioquest, Oxnard, Calif.). 34

VOL. 21, 1978

OPSONIZATION OF GROUP B STREPTOCOCCI

This mixture was rotated at 370C for 30 min. After incubation, tubes were centrifuged again at 1,000 x g for 10 min at room temperature, and the bacteria were washed twice in PBS and resuspended to the original volume (0.5 ml) in PBS. Opsonization for assays of factor B activation and for bacterial immunofluorescence was performed by suspension of 0.5 x 109 to 1 x 109 bacteria in 0.1 ml of serum with incubation at 370C for 60 min. Serum treatment. Serum was used immediately after collection or was frozen at -70'C. Inactivation of both complement pathways was accomplished by heating serum at 560C for 30 min. The classic complement pathway was inactivated by treating serum with 10 mM Mg2'Cl and 10 mM ethylene glycol tetraacetic acid (EGTA; Sigma Chemical Co., St. Louis, Mo.) (5, 12). Immunoelectrophoretic analysis revealed that Mg2+-EGTA treatment under the conditions in our assay (room temperature for 10 min before opsonization of bacteria) did not lead to detectable activation of factor B. Addition of Zymosan (Schwarz/Mann, Orangeburg, N.Y.) to serum treated in this manner resulted in factor B activation comparable to that observed with fresh, untreated serum. Reconstitution of heated serum was attempted with 0.025 td of whole human complement (Cordis Laboratories, Miami, Fla.). This amount of complement has previously been found to maximally enhance the opsonic activity of heated rabbit or human serum (10). The whole human complement preparation had a 50% hemolytic complement of 40 U, with 96 mg of C3 per 100 ml, and 20 mg of C3PA per 100 ml. Chemiluminescence (CL) technique. (i) Preparation of leukocytes. Leukocyte-rich plasma was obtained by gravity sedimentation of erythrocytes from heparinized (10 U/ml) fresh whole blood obtained from healthy adult volunteers. It was centrifuged at 150 x g for 5 min at room temperature, and the leukocytes were washed twice with PBS and suspended in PBS to a final concentration of 107 polymorphonuclear leukocytes (PMN) per ml. The leukocyte suspension generally contained 60 to 70% PMN, 25 to 35% lymphocytes, and a few monocytes. (ii) Scintillation counting. Beckman Poly Q (Beckman Instruments, Fullerton, Calif.) vials were wrapped in aluminum foil and stored in the dark for at least 18 h before use. Scintillation counting was performed in ambient light and temperature in a Beckman LS-100c liquid scintillation system out of phase, with one photomultiplier tube disconnected. The bacterial suspension (0.5 ml) and a portion (0.5 ml) of the PMN were mixed, and the volume was adjusted to 3.5 ml with sterile PBS. The vial was capped, mixed well, placed in the scintillation counter immediately, and counted for 1 min at approximately 10-min intervals for 100 min. The CL detected is expressed in counts per minute. Since it was impossible to perform these studies in the complete absence of light, the first counts obtained immediately after the introduction of the vial are not presented. After 10 min, the counts per minute due to the introduction of light had returned to base-line levels. Giemsa-stained smears of the postphagocytic mixtures were performed and examined for visual evidence of phagocytosis. These results confirmed our previous findings correlating

35

phagocytic uptake with CL peaks of 6,500 cpm or greater (10). As illustrated in Fig. 1, visual examination of 104 separate bacteria-serum-leukocyte preparations revealed evidence of phagocytic uptake in all preparations in which the peak in CL equaled or exceeded 6,500 cpm. Conversely, phagocytosis was not observed when the peak in CL was less than 5,500 cpm. Several mixtures had peaks between 5,500 and 6,500, but these showed only minimal phagocytic uptake. This led us to select 6,500 cpm as the level which indicated the presence of functional opsonins. The standard deviation of triplicate sampling averaged 5% of the mean, whereas the same opsonizing serum tested on three occasions had mean peaks in CL with a standard deviation that averaged 7 ± 6% of the mean (mean variance, 6 + 6% of the mean). Radiolabeled bacterial uptake technique. In the radiolabeled bacterial uptake technique (22; C. 0. Solberg, C. D. Allred, and H. R. Hill. Acta Pathol. Microbiol. Scand. Sect. C, in press), bacteria were prepared by incubation in Todd-Hewitt medium containing [3H]leucine (5 ,ICi/ml) at 370C for 24 h, heat killed, washed three times in PBS, and adjusted to 3.3 x 108/ml. Portions were frozen at -20'C before use and then were opsonized at 37°C for 30 min as described above. Opsonized bacteria were washed once and resuspended to the original volume in medium

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FIG. 1. Peak neutrophil CL elicited by group B streptococci correlated with the presence or absence ofphagocytosis as determined by microscopic examination of each of 104 neutrophil-bacteria reaction mixtures (10). Reprinted with permission of the publisher.

36

SHIGEOKA ET AL.

199. Phagocytic uptake by human PMN was determined by a modification of the method previously described by Mandell (19). Monolayers of PMN were prepared by incubating 0.3 ml of leukocyte suspension on glass cover slips (18 by 18 mm) at room temperature for 30 min. Nonadhering cells were washed off with PBS, and 0.3 ml (10' organisms) of the opsonized, labeled bacterial suspension was added to each cover slip. Control suspensions contained bacteria alone and unopsonized bacteria plus PMN. At intervals of 1, 10, 20, 30, 40, and 50 min, cover slips were washed with PBS, placed in scintillation vials with 5.0 ml of Aquasol, and counted for 5 min in a Beckman LS-lOOc scintillation counter. The monolayers appeared evenly distributed under microscopic examination. The number of adherent cells (5 x i05 to 1.5 x 106) was estimated by counting cells in five random grids at X400 and multiplying the average by the number of grids (20,227) per cover slip. The accumulated counts per minute per 106 cells were calculated at each interval and plotted against time. Controls with bacterial suspensions added to cover slips without cell monolayers or with cells but unopsonized bacteria showed no accumulation of radioactivity. Immunofluorescence procedure. A 0.1-ml amount of fluorescein isothiocyanate-conjugated antihuman immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), or antihuman C3 antiserum (concentration, 1:3; Behring Diagnostics, American Hoechst Corp., Somerville, N.J.) was added to cell buttons of opsonized or unopsonized bacteria. Each tube was mixed, incubated on ice for 30 min, and then washed three times with PBS. After being washed, 0.1 ml of a 1:2 mixture of normal saline and glycerine was added to the bacterial button, and the tube was mixed. A drop of the mixture was placed on a clean microscope slide, a cover glass was placed over the drop, and the mixture was examined immediately with a Zeiss UV microscope by using an epi-illuminator (Carl Zeiss, Inc., New York). The amount of immunofluorescence on the bacterial surface was graded from trace (small) to 4+ (heavy). Electrophoretic analysis for factor B activation. Immunoelectrophoresis was performed by using rabbit antihuman C3 activator (Behring Diagnostics) and agarose gel (Pol-E-Film; 0.15 film of 1% agarose, and 5% sucrose in 0.075 M barbital buffer [pH 8.6] with ethylenediaminetetraacetic acid and polyvinylalcohol preservative and stabilizer; Pfizer Diagnostics Division, Pfizer Inc., New York, N.Y.). Serum was run in this system at 90 mV for 16 h in saline. Plates were washed with saline for 4 h, followed by distilled water for 1 h; they were then stained with amido black lOB for 10 min and decolorized with 5% glacial acetic acid. Controls included fresh serum (negative control) and serum activated with zymosan (10 mg of zymosan per ml, incubated with rotation at 370C for 60 min-positive control). Factor B migrated toward the cathode and factor B (unactivated) migrated toward the anode. Complement consumption assay of alternative pathway activation. Hemolytic complement was measured in 50% hemolytic complement units (15) by a microtiter plate technique utilizing sheep erythrocytes sensitized with hemolysin (Cordis Laboratories,

INFECT. IMMUN.

Miami, Fla.) and various dilutions of serum in Veronal buffer (pH 7.4). Untreated serum and serum preincubated with various strains of group B streptococci were studied in this assay for residual complement activity after removal of bacteria by high-speed centrifugation. Sera for these studies included samples without type-specific opsonins as well as Mg2e-EGTAtreated samples. The latter were recalcified with 0.5 ml of 100 mM CaCl2 per ml of serum. Quantitation of C3 and factor B. Factor B and C3 were quantitated by radial immunodiffusion in antibody-containing agar plates (Behring Diagnostics). Clinical specimens. Serum specimens were obtained from 45 adults and tested immediately or frozen at -70'C. Included in the adult sera were five with antibody to type Ia, 10 with antibody to type II, and 23 with antibody to type III. These specimens were utilized in studies of the role of the classic complement pathway in opsonization of these organisms. In addition, a number of sera lacking heat-stable antibody were examined to determine the role of the alternative complement pathway in opsonization. Seven sera lacking antibody to type Ia, 15 lacking antibody to type II, and 22 without antibody to type III were used in these studies.

RESULTS Antibody and the classical pathway. Fresh serum containing antibody had maximum opsonic activity for type Ia stock and wild strains. As shown in Fig. 2, the peak in CL induced by a type Ia wild strain of group B 12-

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OPSONIZATION OF GROUP B STREPTOCOCCI

VOL. 21, 1978

streptococci opsonized in fresh serum from an adult donor was significantly higher than that produced by bacteria opsonized in the same serum after heat treatment at 560C for 30 min. Addition of whole human complement to heated serum containing antibody restored opsonic activity to the level of the original fresh serum. Bacteria incubated with the complement preparation alone resulted in CL similar to that induced by unopsonized bacteria. Inactivation of the classic complement pathway with 10 mM Mg2+-EGTA resulted in CL approximately equal to that produced by bacteria opsonized in heated serum. Indirect immunofluorescent studies revealed significant (3+ to 4+) deposition of IgG and C3 on the bacterial surface in fresh serum, whereas only IgG was present on bacteria opsonized in heated or Mg2e-EGTA serum. Similar results were obtained with all sera containing antibody to type Ia and II stock or wild strains and with four of five type III wild strains, indicating a major role for the classic complement pathway in opsonizing these organisms. The results of the CL assay and the radiolabeled bacterial uptake technique (Fig. 3) were comparable in each instance. In contrast to types Ia and II, levels of opsonization for type III stock and one of five wild strains were not enhanced significantly by complement. Two wild, untypable strains yielded

37

similar results. The peak in CL observed with fresh serum was not different from that observed with heated or Mg2+-EGTA-treated serum (Fig. 4). The kinetics of the CL curve were somewhat different, however. In the experiment shown in Fig. 4, organisms opsonized in fresh serum induced a peak in CL of 8,200 cpm within 20 min after addition to PMN. In contrast, organisms opsonized in serum lacking an intact classic complement pathway induced a CL peak of 8,200 cpm only after 60 min of incubation with control PMN. Similar results were observed with all of the sera examined containing antibody to these organisms. In addition, serum dilutions demonstrated persistence of these kinetics at lower antibody titers and a tendency toward further delay in peak CL to 70 to 90 min. Thus, the classic complement pathway significantly influenced the kinetics of phagocytosis with these strains. Indirect immunofluorescent studies revealed that the antibody to these strains in human sera was also IgG. Alternative complement pathway. As indicated above, a number of fresh adult sera lacking antibody to a streptococcal type were examined for their effect on the opsonization of these organisms. In 44 instances, sera lacked opsonic activity as measured by the CL procedure in heated samples and failed to deposit IgG on the bacterial surface as determined by indi-

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38

SHIGEOKA ET AL.

INFECT. IMMUN.

rect immunofluorescence. Type Ia, II, and III wild (or stock) strains, opsonized in serum lacking antibody, failed to induce a significant peak in CL (phagocytosis correlated with CL - 6,500 cpm) (Fig. 5). Moreover, visual evidence of phag-

ocytosis was not observed with any of these sera, and only trace amounts of C3 were observed on the bacterial surface. Immunoelectrophoretic analysis of six sera lacking group B opsonins failed to show activation of factor B after exposure to type Ia, II, and III wild strains. In addition, preincubation of serum (either lacking opsonins, or Mg2e-EGTA-treated and recalcified) with various wild strains of group B streptococci (Ia, two strains; II, three strains; and III, four strains) showed no significant decrease in 50% hemolytic complement titer compared with similarly treated serum not incubated with bacteria. Two of 11 wild-type Ill strains tested were found to activate factor B in fresh serum. Little C3 was deposited on these organisms, however, and they did not elicit a significant peak in CL or show evidence of phagocytic uptake.

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DISCUSSION The present study indicates that antibody is essential in the opsonization of type Ia, II, and III group B streptococci. The classical complement pathway was required for maximal opsonization of types Ia, II, and many strains of type Ill. Strains which did not require complement for maximal levels of opsonization demonstrated significantly accelerated kinetics in the presence of the classical pathway. We could not demonstrate a significant role for the alternative complement pathway in opsonizing stock or wild strains of types Ia, II, and III group B strepto-

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TIME (min) FIG. 4. Effects of serum containing antibody on opsonization of group B streptococci type III. Symbols: *-*, Fresh or heated serum; A-A, Mg2"EGTA-treated serum;. 0, heated serum plus 0.25 Id of whole human complement. CL - 6,500 cpm correlates with phagocytic uptake. -

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VOL. 21, 1978

OPSONIZATION OF GROUP B STREPTOCOCCI

cocci. These results indicate the necessity for specific antibody in opsonizing these organisms. Clinical studies relating neonatal and maternal antibody deficiency with overwhelming neonatal infection (2, 10) have previously suggested the importance of antibody in host defense against group B streptococci. The explanation for the differing roles of complement in opsonizing the three streptococcal types may relate to the characteristics of the antibody or to the specific antigens involved. Studies employing erythrocytes and Staphylococcus aureus have indicated that IgG antibodies to different antigens differ in their ability to attach to phagocytes or to activate complement (20, 21). Thus, antibody directed against the type HI group B capsular polysaccharide may be sufficient for attachment and ingestion of these organisms by phagocytes. Complement may accelerate the process, as has been reported (21, 22) with both particles and antibody-coated bacteria, without significantly affecting the overall level of uptake. Type Ill strains opsonized in the absence of complement produced a CL peak which was delayed to 60 to 90 min. At that time, phagocytic uptake as determined visually was comparable with that in fresh serum. Baltimore et al. (3), by using an opsonophagocytic assay whose end point was measured in log reduction of colony counts, also found that the classic pathway was required for optimal killing of group B streptococcal strains. Thus, like some pneumococci, these group B streptococcal strains may be maximally phagocytized only after sequential coating with antibody: C1, C4, C2, and C3 (14). In contrast to most other bacterial species, neither wild nor stock strains of type Ia, II, and III group B streptococci were significantly opsonized by the alternative complement pathway. Furthermore, we could not demonstrate significant (3+ to 4+) deposition of C3 on the bacterial surface of any of these strains; none of the stock strains and only two of 11 wild group B strains tested resulted in activation of factor B of the alternative pathway in fresh sera. The two strains which showed some activation were not opsonized as determined by the CL assay or direct measures of phagocytic uptake. These results were similar to the strain specificity of alternative pathway activation for S. pneumoniae (24). Such strain specificity appears to implicate factors on the bacterial surface in determining the potential role of the alternative pathway in opsonizing group B streptococci. The difficulties of assaying rate and degree of phagocytic ingestion are well known, although it is generally recognized that both factors are important determinants of adequate host de-

39

fense (23). Our study and that of Baltimore et al. (3) indicate a requirement for antibody in opsonizing group B streptococci and show a significant role for the classical pathway of complement. In contrast, the alternative pathway does not have a major role in opsonizing the strains of these streptococci. This information may be of use in determining immunological adjuncts to antibiotic therapy for severe group B streptococcal infections. ACKNOWLEDGMENTS This study was supported by Public Health Service grants A113150 from the National Institute of Allergy and Infectious Diseases and AM21140 from the National Institute of Arthritis, Metaboliam and Digestive Diseases. H. R. Hill is an Investigator of the Howard Hughes Medical Institute.

LITERATURE CITED 1. Baker, C. J., F. F. Barrett, R. C. Gordon, and M. D. Yow. 1973. Suppurative meningitis due to streptococci of Lancefield group B: a study of 33 infants. J. Pediatr. 82:724-729. 2. Baker, C. J., and D. L Kasper. 1976. Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infection. N. Engi. J. Med. 294:753-756. 3. Baltimore, R. S., D. L Kasper, C. J. Baker, and D. K. Goroff. 1977. Antigenic specificity of opsonophagocytic antibodies in rabbit antisera to group B streptococci. J. Immunol. 118:673-678. 4. Bayer, A. S., A. W. Chow, B. F. Anthony, and L B. Guze. 1976. Serious infections in adults due to group B

5.

6.

7. 8.

9. 10.

11.

streptococci, clinical and serotypic characterization. Am. J. Med. 61:498-503. Des Prez, RI M., C. S. Bryan, J. Hawiger, and D. G. Colley. 1975. Function of the classical and alternate pathways of human complement in serum treated with ethylene glycol tetraacetic acid and MgClrethylene glycol tetraacetic acid. Infect. Immun. 11:1235-1243. Eickhoff, T. C., J. 0. Klein, A. K. Daly, D. Ingall, and M. Finland. 1964. Neonatal sepsis and other infections due to group B beta-hemolytic streptococci. N. Engl. J. Med. 271:1221-1228. Fine, D. P. 1975. Pneumococcal type-associated variability in alternate complement pathway activation. Infect. Immune. 12:772-778. Fireman, P., D. A. Zuchowski, and P. M. Taylor. 1969. Development of the human complement system. J. Immunol. 103:25-31. Franciosi, RI A., J. D. Knostman, and R. A. Zimmerman. 1973. Group B streptococcal neonatal and infant infections. J. Pediatr. 82:797-718. Hemming, V. G., R. T. Hall, P. G. Rhodes, A. 0. Shigeoka, and H. R. Hill. 1976. Assessment of group B streptococcal opsonins in human and rabbit serum by neutrophil chemiluminescence. J. Clin. Invest. 58:1379-1387. Hemming, V. G., D. W. McCloskey, and H. R. Hill. 1976. Pneumonia in the neonate associated with group B streptococcal septicemia. Am. J. Dis. Child.

130:1231-1233. 12. Hill, H. R., N. A. Hogan, J. F. Bale, and V. G. Hemming. 1977. Evaluation of nonspecific (alternative pathway) opsonic activity by neutrophil chemiluminescence. Int. Arch. Allergy Appl. Immunol. 53:490-497. 13. Howard, J. B., and G. H. McCracken, Jr. 1974. The spectrum of group B streptococcal infections in infancy. Am. J. Dis. Child. 128:815-818.

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14. Johnston, R. B., Jr., M. R. Klemperer, C. A. Alper, and F. S. Rosen. 1969. The enhancement of bacterial phagocytosis by serum: the role of complement components and two cofactors. J. Exp. Med. 129:1275-1290. 15. Kabat, E. A., and M. M. Mayer (ed.). 1961. Experimental immunochemistry, 2nd edition, p. 149-153. Charles C Thomas, Springfield, Ill. 16. Lancefield, R. C. 1934. A serological differentiation of specific types of bovine hemolytic streptococci (group B). J. Exp. Med. 59:441-458. 17. Lancefield, R. C., M. McCarty, and W. N. Everly. 1975. Multiple mouse-protective antibodies directed against group B streptococci. J. Exp. Med. 142:165-179. 18. McCracken, G. H., Jr., and H. F. Eichenwald. 1971. Leukocyte function and the development of opsonic and complement activity in the neonate. Am. J. Dis. Child. 121:120-126. 19. Mandell, G. L. 1975. Effect of temperature on phagocytosis by human polymorphonuclear neutrophils. Infect. Immun. 12:221-223.

INFECT. IMMUN. 20. Mesner, R. P., and J. Jelinek. 1970. Receptors for human gamma globulin on human neutrophils. J. Clin. Invest. 49:2165-2171. 21. Scribner, D. J., and D. Fahrney. 1976. Neutrophil receptors for IgG and complement: their roles in the attachment and ingestion phases of phagocytosis. J. Immunol. 116:892-897. 22. Stossel, T. P. 1973. Quantitative studies of phagocytosis: kinetic effects of cations and heat labile opsonins. J. Cell. Biol. 58:346-356. 23. Stossel, T. P. 1975. Phagocytosis: recognition and ingestion. Semin. Hematol. 12:83-116. 24. Stossel, T. P., C. A. Alper, and F. S. Rosen. 1973. Opsonic activity in the newborn: role of properdin. Pediatrics 52:134-136. 25. Wilkinson, H. W., R. R. Facklam, and E. C. Wortham. 1973. Distribution by serological type of group B streptococci isolated from a variety of clinical material over a five-year period (with special reference to neonatal sepsis and meningitis). Infect. Immun. 8:228-235.

Role of antibody and complement in opsonization of group B streptococci.

Vol. 21, No. 1 INFECTION AND IMMUNITY, July 1978, p. 34-40 0019-9567/78/0021-0034$02.00/0 Copyright i 1978 American Society for Microbiology Print...
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