INFnCTION AND IMMUNITY, Feb. 1976, p. 407-412
Vol. 13, No. 2 Printed in U-SA.
Copyright © 1976 American Society for Microbiology
Antigens of Group A Streptococci Involved in Passive Hemagglutination Reactions ALAN L. BISNO,* ITZHAK OFEK,
AND
EDWIN H. BEACHEY
Departments of Medicine and Microbiology, University of Tennessee Center for the Health Sciences,* and Memphis Veterans Administration Hospital, Memphis, Tennessee 38163 Received for publication 15 September 1975
Antibodies to streptococcal extracellular products were detected in rabbit sera by passive hemagglutination tests as early as 1 week after intravenous injection of live group A streptococci (strain C203S). Using these antibodies in immunoadsorbent columns, we prepared an antigenic fraction from crude concentrates of streptococcal extracellular products. The specific activity of this fraction in sensitizing glutaraldehyde-treated erythrocytes for passive hemagglutination tests and in absorbing passive hemagglutination antibodies from immune sera was increased by 80-fold and at least 24-fold, respectively, as compared with crude extracellular products. The fraction has been found to contain at least three serologically active antigens, which are of considerable interest because: (i) they gave rise to early and consistent immune responses both in humans and in experimental animals; (ii) they appear so far to be produced only by betahemolytic streptococci; and (iii) antibodies to these antigens are present in high titers in patients with acute rheumatic fever. Our data suggest that passive hemagglutination antigens may be distinct from those extracellular enzymes and hemolysins ordinarily employed in streptococcal serological studies.
During the course of human infection, group A streptococci elaborate a variety of extracellular products (ECP), many of which are antigenic (3, 5). Only a few of these ECP antigens are characterized precisely enough to allow their use in antibody tests for clinical and basic immunological purposes. Conventionally, the serodiagnosis of streptococcal infection is based upon titration of antibodies that neutralize the enzymatic or hemolytic activities of streptococcal extracellular antigens. Tests currently in use include: antistreptolysin 0 (ASO), anti-deoxyribonuclease B (ADNase B), antihyaluronidase, antistreptokinase and anti-nicotinamide adenine dinucleotidase (ANADase). Recently, considerable attention has been given to measurement of immune responses to streptococcal infection by means of an alternative assay procedure: passive hemagglutination (PHA) of erythrocytes sensitized with ECP of group A streptococci. Although this approach is currently being subjected to clinical evaluation (1, 2, 6, 9, 10), there is little available information regarding the identity of the antigens and antibodies involved. This study focuses upon streptococcal antigens that participate in PHA reactions. Some of the immunochemical properties of these antigens, as well as the kinetics of the immune response induced by them in rabbits, will be described.
MATERIALS AND METHODS Bacterial strains. Group A streptococcus, strain C203S, has been used extensively in many laboratories and is known to be a good producer of streptolysin 0, DNase B, NADase, and streptokinase (3). Strains B.P. (group C) and E.W. (group G) were isolated from the throats of patients with pharyngitis. Diplococcus pneumoniae, type 24, is a laboratory stock strain. The Streptococcus viridans strain used in this study was isolated from the blood of a patient with subacute bacterial endocarditis. Rabbit antisera. Bacteria were harvested after 18 h of growth in Todd-Hewitt broth at 37 C, washed twice with 0.9% NaCl, and adjusted to a turbidity of 100 U on a Klett-Summerson colorimeter (green filter). A 0.5-ml portion of the suspension of live organisms was injected intravenously into rabbits at zero time, and 1 ml was injected 3 days later. In some experiments a 1-ml booster injection was given during week 5. Bleedings were obtained from all rabbits before immunization and thrice weekly thereafter. The sera were separated, heated at 56 C for 30 min, and stored at -20 C until assayed for antibody. Human sera. Sera were collected from patients hospitalized at City of Memphis Hospitals with acute rheumatic fever (ARF) and acute glomerulonephritis (AGN). ECP. ECP were obtained from broth filtrates (5 to 10 liters) of 18-h growth of the various bacteria. The filtrates were dialyzed against distilled water and then lyophilized. In some experiments, the filtrates of group A strain C203S were precipitated with 60%
407
408
BISNO, OFEK, AND BEACHEY
INFECT. IMMUN.
ammonium sulfate, redissolved in .0O2 M phos- serum was absorbed with packed RBC obtained by phate-O.i5 M NaCl, pH 7.4 (PBS), dialyzed against centrifuging 1 ml of STZ reagent. Controls consisted distilled water, concentrated, and lyophilized (3). of unsensitized, glutaraldehyde-fixed sheep RBC. Antibody assays. The conventional streptococcal After incubation at 37 C for 30 min, the mixture was antibody tests were ASO, antihyaluronidase, ADN- centrifuged and the supernatant was separated for ase B, antistreptokinase, and ANADase. Detailed antibody assay. protocols for the performance of these tests have Antigenic activity. Serial dilutions of ECP or been described elsewhere (3). Assays for PHA anti- ACF were used to absorb A-STZ antibodies from an bodies were performed using streptozyme (STZ) re- ARF serum. Conditions of absorption were identical agent (Wampole Laboratories, Stamford, Conn.). to those described above. The ARF serum had an AThe reagent consists of aldehyde-stabilized sheep STZ titer of 1:1,600 and was employed at a dilution of red blood cells (RBC) sensitized with streptococcal 1:100 in these experiments. Antigenic activity was ECP. ECP were obtained from modified Todd-Hew- defined as the minimum amount of antigen (in miitt broth media by precipitation with ammonium crograms) that completely inhibited agglutination. sulfate at 60% saturation (information supplied by 2-ME. A 20-,ul portion of 0.03 M 2-mercaptoethathe manufacturer). The procedure for performance nol (2-ME) was mixed with an equal volume of rabof this test in our laboratory has been published (2). bit antiserum for 30 min at 37 C to inactivate immuLots of STZ reagent employed were as follows: 2574, nogobulin M (IgM) antibody. The mixture was then 121373, and 12573 were gifts of Wampole Laborato- further diluted to determine antibody titer in the ries; ST92, ST71, and ST70 were purchased commer- STZ test. cially. In each PHA test, an ARF serum of known Trypsin digestion. A 20-M1 portion of ACF (2 mg/ titer was included as a positive control, and saline ml) was incubated with an equal volume of 1% trypwas included as a negative control. sin (Difco) in PBS (pH 7.8) for 2 h at 37 C. Lima bean Affinity chromatography. This technique was trypsin inhibitor (Difco) was then added, and the carried out as described by Mannik and Stage (11). antigenic activity of the mixture was assayed as Briefly, rabbit antiserum, obtained 7 days after im- described above. Controls containing PBS without munization with live group A streptococci, was trypsin were included. Polyacrylamide gel electrophoresis. Polyacrylbrought to 45% saturation with ammonium sulfate at 4 C. The resulting precipitate was redissolved in amide gel electrophoresis was done by the method of PBS, dialyzed against distilled water, and lyophi- Davis (4). A 50-,ul portion of ACF (1.5 mg/ml) or ECP lized. A 166-mg portion of the lyophilized material (200 mg/ml) from group A streptococcus was loaded was dissolved in PBS and applied to a Sepharose 4-B on the gels. After termination of electrophoresis, the cyanogen bromide-activated immunoadsorbent col- gels were cut into 3-mm sections; each section was umn (1 by 8 cm). The column was thoroughly extracted with 0.5 ml of saline for 24 h at 4 C. The washed, and 1 ml of 200 mg of ECP per ml from eluates were separated, dialyzed against PBS, and group A streptococcus was applied. The ECP prepa- tested for antigenic activity. ration employed contained streptolysin 0, 31,024 U/ ml; DNase B, ¢1,024 U/ml; NADase, streptokinase, RESULTS and hyaluronidase,
Vol. 13, No. 2 Printed in U-SA.
Copyright © 1976 American Society for Microbiology
Antigens of Group A Streptococci Involved in Passive Hemagglutination Reactions ALAN L. BISNO,* ITZHAK OFEK,
AND
EDWIN H. BEACHEY
Departments of Medicine and Microbiology, University of Tennessee Center for the Health Sciences,* and Memphis Veterans Administration Hospital, Memphis, Tennessee 38163 Received for publication 15 September 1975
Antibodies to streptococcal extracellular products were detected in rabbit sera by passive hemagglutination tests as early as 1 week after intravenous injection of live group A streptococci (strain C203S). Using these antibodies in immunoadsorbent columns, we prepared an antigenic fraction from crude concentrates of streptococcal extracellular products. The specific activity of this fraction in sensitizing glutaraldehyde-treated erythrocytes for passive hemagglutination tests and in absorbing passive hemagglutination antibodies from immune sera was increased by 80-fold and at least 24-fold, respectively, as compared with crude extracellular products. The fraction has been found to contain at least three serologically active antigens, which are of considerable interest because: (i) they gave rise to early and consistent immune responses both in humans and in experimental animals; (ii) they appear so far to be produced only by betahemolytic streptococci; and (iii) antibodies to these antigens are present in high titers in patients with acute rheumatic fever. Our data suggest that passive hemagglutination antigens may be distinct from those extracellular enzymes and hemolysins ordinarily employed in streptococcal serological studies.
During the course of human infection, group A streptococci elaborate a variety of extracellular products (ECP), many of which are antigenic (3, 5). Only a few of these ECP antigens are characterized precisely enough to allow their use in antibody tests for clinical and basic immunological purposes. Conventionally, the serodiagnosis of streptococcal infection is based upon titration of antibodies that neutralize the enzymatic or hemolytic activities of streptococcal extracellular antigens. Tests currently in use include: antistreptolysin 0 (ASO), anti-deoxyribonuclease B (ADNase B), antihyaluronidase, antistreptokinase and anti-nicotinamide adenine dinucleotidase (ANADase). Recently, considerable attention has been given to measurement of immune responses to streptococcal infection by means of an alternative assay procedure: passive hemagglutination (PHA) of erythrocytes sensitized with ECP of group A streptococci. Although this approach is currently being subjected to clinical evaluation (1, 2, 6, 9, 10), there is little available information regarding the identity of the antigens and antibodies involved. This study focuses upon streptococcal antigens that participate in PHA reactions. Some of the immunochemical properties of these antigens, as well as the kinetics of the immune response induced by them in rabbits, will be described.
MATERIALS AND METHODS Bacterial strains. Group A streptococcus, strain C203S, has been used extensively in many laboratories and is known to be a good producer of streptolysin 0, DNase B, NADase, and streptokinase (3). Strains B.P. (group C) and E.W. (group G) were isolated from the throats of patients with pharyngitis. Diplococcus pneumoniae, type 24, is a laboratory stock strain. The Streptococcus viridans strain used in this study was isolated from the blood of a patient with subacute bacterial endocarditis. Rabbit antisera. Bacteria were harvested after 18 h of growth in Todd-Hewitt broth at 37 C, washed twice with 0.9% NaCl, and adjusted to a turbidity of 100 U on a Klett-Summerson colorimeter (green filter). A 0.5-ml portion of the suspension of live organisms was injected intravenously into rabbits at zero time, and 1 ml was injected 3 days later. In some experiments a 1-ml booster injection was given during week 5. Bleedings were obtained from all rabbits before immunization and thrice weekly thereafter. The sera were separated, heated at 56 C for 30 min, and stored at -20 C until assayed for antibody. Human sera. Sera were collected from patients hospitalized at City of Memphis Hospitals with acute rheumatic fever (ARF) and acute glomerulonephritis (AGN). ECP. ECP were obtained from broth filtrates (5 to 10 liters) of 18-h growth of the various bacteria. The filtrates were dialyzed against distilled water and then lyophilized. In some experiments, the filtrates of group A strain C203S were precipitated with 60%
407
408
BISNO, OFEK, AND BEACHEY
INFECT. IMMUN.
ammonium sulfate, redissolved in .0O2 M phos- serum was absorbed with packed RBC obtained by phate-O.i5 M NaCl, pH 7.4 (PBS), dialyzed against centrifuging 1 ml of STZ reagent. Controls consisted distilled water, concentrated, and lyophilized (3). of unsensitized, glutaraldehyde-fixed sheep RBC. Antibody assays. The conventional streptococcal After incubation at 37 C for 30 min, the mixture was antibody tests were ASO, antihyaluronidase, ADN- centrifuged and the supernatant was separated for ase B, antistreptokinase, and ANADase. Detailed antibody assay. protocols for the performance of these tests have Antigenic activity. Serial dilutions of ECP or been described elsewhere (3). Assays for PHA anti- ACF were used to absorb A-STZ antibodies from an bodies were performed using streptozyme (STZ) re- ARF serum. Conditions of absorption were identical agent (Wampole Laboratories, Stamford, Conn.). to those described above. The ARF serum had an AThe reagent consists of aldehyde-stabilized sheep STZ titer of 1:1,600 and was employed at a dilution of red blood cells (RBC) sensitized with streptococcal 1:100 in these experiments. Antigenic activity was ECP. ECP were obtained from modified Todd-Hew- defined as the minimum amount of antigen (in miitt broth media by precipitation with ammonium crograms) that completely inhibited agglutination. sulfate at 60% saturation (information supplied by 2-ME. A 20-,ul portion of 0.03 M 2-mercaptoethathe manufacturer). The procedure for performance nol (2-ME) was mixed with an equal volume of rabof this test in our laboratory has been published (2). bit antiserum for 30 min at 37 C to inactivate immuLots of STZ reagent employed were as follows: 2574, nogobulin M (IgM) antibody. The mixture was then 121373, and 12573 were gifts of Wampole Laborato- further diluted to determine antibody titer in the ries; ST92, ST71, and ST70 were purchased commer- STZ test. cially. In each PHA test, an ARF serum of known Trypsin digestion. A 20-M1 portion of ACF (2 mg/ titer was included as a positive control, and saline ml) was incubated with an equal volume of 1% trypwas included as a negative control. sin (Difco) in PBS (pH 7.8) for 2 h at 37 C. Lima bean Affinity chromatography. This technique was trypsin inhibitor (Difco) was then added, and the carried out as described by Mannik and Stage (11). antigenic activity of the mixture was assayed as Briefly, rabbit antiserum, obtained 7 days after im- described above. Controls containing PBS without munization with live group A streptococci, was trypsin were included. Polyacrylamide gel electrophoresis. Polyacrylbrought to 45% saturation with ammonium sulfate at 4 C. The resulting precipitate was redissolved in amide gel electrophoresis was done by the method of PBS, dialyzed against distilled water, and lyophi- Davis (4). A 50-,ul portion of ACF (1.5 mg/ml) or ECP lized. A 166-mg portion of the lyophilized material (200 mg/ml) from group A streptococcus was loaded was dissolved in PBS and applied to a Sepharose 4-B on the gels. After termination of electrophoresis, the cyanogen bromide-activated immunoadsorbent col- gels were cut into 3-mm sections; each section was umn (1 by 8 cm). The column was thoroughly extracted with 0.5 ml of saline for 24 h at 4 C. The washed, and 1 ml of 200 mg of ECP per ml from eluates were separated, dialyzed against PBS, and group A streptococcus was applied. The ECP prepa- tested for antigenic activity. ration employed contained streptolysin 0, 31,024 U/ ml; DNase B, ¢1,024 U/ml; NADase, streptokinase, RESULTS and hyaluronidase,
