INFECITION AND IMMUNrTy, May 1975, p. 991-995 Copyright 0 1975 American Society for Microbiology
Vol. 11, No. 5 Printed in U.S.A.
Detection of Virus-Specific Antibody-Forming Cells of Mice Immunized with Newcastle Disease Virus YOUNG TAI KIM,' JANE AULT,' AND R. J. WODZINSKI3* International Minerals and Chemical Corporation, Libertyville, Illinois 60048 Received for publication 16 September 1974
The hemolytic plaque assay was adapted to the detection of antibodies to Newcastle disease virus (NDV) in an in vivo, an in vitro system, and a combined in vivo-in vitro system. Several conditions were tested for coupling of sheep erythrocytes to NDV and for the kinetics of plaque formation in the in vivo and in vitro systems. The one set of conditions which provided the best responses is presented. The effect of multiple injections of NDV into mice on plaque formation was optimized. Virus. NDV strain B, was used as the viral antigen. The virus was propagated in 9-day embryonated eggs for 48 h. The allantoic fluid was harvested, pooled, and centrifuged at 3,000 x g for 30 min. The supernatant was centrifuged at 30,000 x g for 1 h and the pellet was resuspended in cold phosphate-buffered saline (PBS) at pH 7.2. The suspension was centrifuged at 30,000 x g for 1 h and the pellet was resuspended in PBS. The final suspension was adjusted to 10' infectious particles as measured in a chicken embryo assay. This suspension contained 50 Mg of protein per ml as measured by the method of Lowry et al. (11). Sensitization of SRBC. Fresh sheep blood was centrifuged and the SRBC was washed three times with PBS. A 5% suspension of the washed SRBC in PBS was added to an equal volume of 1 mg of tannic acid per ml. After 15 min of incubation in a water bath at 37 C, the mixture was centrifuged for 5 min at 750 x g. The pellet containing the SRBC was washed once with 20 ml of PBS. The SRBC in the washed pellet were resuspended in PBS to achieve a 5% suspension. Ten milliliters of the SRBC suspension was added to 10 ml of PBS containing 100 gg of NDV on a protein basis. The NDV-SRBC mixture was incubated in a water bath for 30 min at 37 C. The SRBC were recovered by centrifugation at 750 x g for 5 min. The virus-sensitized SRBC were washed three times with 20 ml of PBS containing 1% inactivated agcmma-globulin (Pentex Inc. Kankakee, Ill.). The washed, sensitized SRBC were suspended in PBS at a 20% concentration and were used in the HPA. MATERIALS AND METHODS HPA. The HPA previously described (4) was emAnimals and immunization procedures. CFI F ployed with some modifications. Four days after the mice about 8 weeks old (Carworth, Inc., Portage, final immunization, mouse spleens were removed Mich.) were injected intravenously with 0.2 ml of aseptically and placed in 60-mm petri dishes (Falcon saline containing 10 ,g of NDV on the basis of protein Plastics, Los Angeles, Calif.) containing 10 ml of content. If secondary immunization was desired, the Eagle minimal essential medium (Flow Laboratories, Los Angeles, Calif.). The spleen cells were released by mice were reinjected as described above 3 weeks later. teasing with forceps. The resultant suspension of ' Present address: Department of Allergy and Immunology, dissociated cells was centrifuged for 10 min at 1,000 x g. The cell pellet was resuspended in Eagle medium Cornell Medical School, New York, N. Y. 10021. (Flow Laboratories, Los Angeles, Calif.) at the desired 2Present address: 20 Cedar St., Potsdam, N. Y. 13676. ' Present address: Department of Biological Sciences, concentration. For the HPA, 0.7 g of Noble agar (Difco LaboratoFlorida Technological University, Orlando, Fla. 32816. 391
The hemolytic plaque assay (HPA) in agar for the detection of antibody-forming cells to sheep erythrocytes (SRBC) was introduced by Jerne and Nordin (5). Ingraham and Bussard (4) reported a similar method for the detection of specific antibodies but utilized a slide technique. The method has been modified to detect the antibody-forming cells against bacterial polysaccharides (1, 8, 10), haptens (12, 15, 21), synthetic polypeptides (20), and proteins (3, 7, 9, 17). The basic modifications in each of the above methods is the coupling of the various antigens to SRBC. Johnson et al. (6) described a modification in which 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ECDI) is used to couple the antigen to SRBC. Further, SRBC can be sensitized with tannic acid and then coupled with various antigens (2, 18, 19). The HPA method was adapted to the detection of single cells releasing antibody to influenza A virus (16). The present paper reports a method for coupling Newcastle disease virus (NDV) to SRBC and for detecting the antibody-forming cells to NDV by using mouse spleen cells in a HPA. It also reports synthesis of antibody to NDV by mouse spleen cells in vitro.
992
KIM, AULT, AND WODZINSKI
ries, Inc., Detroit, Mich.) was added to 100 ml of Eagle minimal essential medium. The mixture was boiled for 30 min on a hot plate. The agar in Eagle minimal essential medium (2.0 ml) was preincubated at 46 C in a water bath. Washed, sensitized SRBC (0.1 ml) and 0.2 ml of the spleen suspension were added to the agar. The materials were mixed quickly and poured into 100-mm plastic petri dishes. After 1 h of incubation at 37 C, the plates were flooded with 1.5 ml of a 1: 10 dilution of guinea pig complement (Pentex Corp., Kankakee, Ill.) in PBS. After 1 h of incubation at 37 C, the complement was poured off and the number of hemolytic plaques was counted under a x 10 stereomicroscope. The results were expressed as number of plaqueforming cells per 106 viable cells.
INFECT. IMMUN.
incubated in vitro before assay. Plaque formation increased rapidly up to 4 days after inoculation (Fig. 1). It decreased thereafter and was barely measurable 7 days after inoculation. Effect of multiple injections of NDV on hemolytic plaque formation. Mice that were injected intravenously with a single dose of NDV were divided into two groups. One group received no further treatment. The other group was injected a second time with NDV 3 weeks after the first injection. Spleen cultures were prepared from each group 4 days after the last injection. After incubation of the cell cultures, the spleen cells were assayed by the hemolytic plaque assay. The spleens from the mice receiv-
RESULTS TABLE 1. Hemolytic plaque formation by spleen cells from mice immunized with NDV Effect of tannic acid on coupling of SRBC with NDV. SRBC must be coupled with NDV Hemolytic plaques if antibody-forming cells against NDV are to be per 106 cells SRBC Spleen observed in the HPA. Sensitization of SRBC cells with tannic acid often allows subsequent couExptl Expt2 pling with various antigens (2, 18, 19). The coupling of SRBC with NDV is measurable via Control 2 0 Control 1 3 Coupled with a passive hemagglutination test. NDV Serial twofold dilutions of NDV in saline were added to an equal volume of tanned SRBC. The 3 0 Control hemagglutinating titer of the NDV preparation From mice immunized was 64 when tannic acid-sensitized SRBC were against NDV used, whereas the titer was less than 2 when untreated SRBC were used. The above result 101 51 Coupled with indicates that tannic acid treatment of SRBC NDV permits the coupling of NDV to these treated cells. 100 Hemolytic plaque formation by spleen cells with The spleens immunized NDV. from mice of mice which were immunized once with NDV were removed after 4 days in two separate experiments. Spleen cell cultures (See Materials and Methods) were made from immunized and control mice and were measured by HPA. so Four hemolytic plaques per 106 cells were - 60 formed in the control cell cultures tested with NDV coupled with SRBC, whereas 152 hemo- 04 lytic plaques per 106 cells were found in the cell 40 cultures prepared from the NDV-immunized mice (Table 1). Cell cultures from both control 0 and immunized mice formed 4 or less hemolytic plaques per 106 cells when tested against non20 coupled SRBC. Although some variation in the response to immunization occurred between experiments, these results indicate that spleen cells prepared from mice immunized against NDV produce specific antibody-forming cells 10 9 5 6 8 7 4 3 1 2 against NDV. WITH N D V INOCULATION AFTER DAYS Kinetics of hemolytic plaque formation. Spleens from mice which received a single FIG. 1. Kinetics of antibody formation against injection of NDV were assayed daily for 10 days NDV in mouse spleen cells after a single injection of using coupled SRBC. The spleen cells were not antigen. v
v
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993
NDV HEMOLYTIC PLAQUE ASSAY
ing two injections produced four times as many plaques as those from the mice receiving one injection, when tested against SRBC coupled with NDV (Table 2). Approximately the same number of plaques was formed from the spleens of both groups of mice when they were tested against control SRBC. In vitro immunization of mouse spleen cells with NDV. Mishell and Dutton (13) reported that dissociated spleen cell suspensions from normal mice could be immunized in vitro to
heterologous erythrocytes. They obtained marked increases in plaque-forming cells when spleen culture cells obtained from in vivo immunized mice were challenged with additional antigen in vitro and then incubated in vitro (14). A similar experiment was performed to confirm these observations, using NDV as the antigen. One-milliliter aliquots of spleen cell suspensions, from mice immunized with NDV and from control mice, were added to NDV (1 gg/ml) in 35-mm petri dishes (Falcon plastic). The dishes were placed in boxes and incubated at 37 C in an atmosphere of 7% 02, 10% CO2, and 83% N2. The boxes were placed on a rocker platform set for 7 to 10 complete cycles per min. The cultures were fed daily with 0.1 ml of a nutrient mixture (13). The control spleen cell suspension was incubated in the petri dishes as described above. After 4 days of incubation, the cells were suspended by scraping them from the dish surface with a plastic policeman. The treated and untreated cell suspensions were centrifuged at 1,000 x g for 10 min at 8 C and were resuspended in culture media at a concentration of 2 x 106 viable cells. When spleen cells prepared from mice immunized with NDV were incubated with NDV in vitro, the number of antibody-forming cells was increased at least twofold over that of the culture without incubation in vitro (Table 3). Control spleen cell cultures incubated with NDV in vitro formed more hemolytic plaques than those to which no NDV was added in vitro. However, the great variation in the number of plaques from experiment to experiment makes it difficult to reach definite conclusions at this time. DISCUSSION The hemolytic plaque assay to detect virusspecific antibody by an in vivo, in vitro, or combined in vivo-in vitro system would enjoy widespread use if the technique yielded consistent results, measured small differences in the antibodies formed, and could be made routine. The following factors must be rigidly controlled if reproducible results are to be obtained with the HPA when NDV is used as antigen: (i)
TABLE 2. Effect of multiple injection of NDV into mice on hemolytic plaque formation Spleen cells
Control
From mice receiving 1 injection of NDV
Hemolytic plaques per 106 cells
SRBC
5
Control
Coupled with NDV
10
Control
25
Coupled with NDV From mice receiving 2 injections of NDV
130
Control
10
Coupled with NDV
515
TABLE 3. Effect of in vitro immunization of mouse spleen cells with NDV NDV added to
Spleen cells
Hemolytic plaques per per 106 cells
spleen cells in vitro
Control In vivo immunized
Expt 1
Expt 2
Yes
2 21
4 36
No
84 415
106 206
No
Yes
sensitization of SRBC to permit their coupling with NDV; (ii) prevention of hemolysis by the sensitizing agent; (iii) source of SRBC; (iv) kinetics of plaque formation; (v) the procedure for immunizing mice with antigen; and (vi) the elimination of nonspecific plaque formation. Since NDV is not extensively coupled to untreated SRBC (i), different sensitizing agents, ECDI and tannic acid, were used to enhance coupling of NDV to SRBC. Experiments not reported here showed that tannic acid-sensitized cells yielded higher hemagglutination titers than did ECDI-sensitized cells. However, it was necessary to adjust the tannic acid concentration to a level which would maximize coupling of SRBC to NDV but not cause hemolysis (ii). Although several conditions were tested to increase the sensitization, fewer plaques were formed when NDV was the antigen than if either SRBC or bovine serum albumin was used as the antigen. The number of antibody-forming cells against NDV was about one-tenth that obtained from mice in-
994
KIM, AULT, AND WODZINSKI
jected with SRBC and about half that seen in mice immunized with bovine serum albumin. When SRBC were used as antigen, the number of hemolytic plaques was about 103 to 104 per 106 cells, whereas about 5.0 x 102 to 1.0 x 103 plaques per 106 cells were counted when bovine serum albumin was the antigen. Another cause of variation in the assay is the source of sheep blood (iii). The blood of six different sheep tested in a single experiment in which all other conditions were constant yielded values ranging from 25 to 2,500 hemolytic plaques. The blood of a single sheep which showed higher titers was used in all experiments reported in this paper. When the single source of sheep blood was used, the variation in plaque count was 40 to 100 plaques per 106 spleen cells from experiment to experiment. However, the spleen cells from the control mice produced relatively consistent numbers of plaques. The sensitization of the SRBC would influence the number of hemolytic plaques that were formed from the spleens of control mice. The wide differences observed between control mice and those injected intravenously with NDV show that the mice did produce antibodies against the NDV preparation. The kinetics of antibody formation (iv) in spleen cells prepared from the mice immunized with NDV were similar to those observed in mice injected with SRBC. The number of antibody-forming cells rapidly increases up to the day 4 after immunization and decreases drastically with time. However, the kinetics of hemolytic plaque formation shows greater variation from experiment to experiment, roughly 100 to 500 at the peak. A secondary immunization of mice with NDV appears to stimulate the antibody-forming cells (v). Four times more hemolytic plaques were formed if mice were immunized twice than if only a single immunization was used. In vitro incubation of spleen cell cultures prepared from mice injected with NDV yields more antibodyforming cells than if no in vitro incubation is used. The increase in hemolytic plaques can be explained in two ways. One explanation is that the number of antibody-forming cells actually increases during incubation with NDV. The other explanation is that viable cells decrease during in vitro incubation and the number of hemolytic plaques per 106 viable cells is, therefore, increased proportionately. The viable cells do decrease by 50% during the day 4 in vitro incubation. However, it is difficult to envision that non-antibody-forming cells died discriminately and caused the ratio of antibody-forming cells to other cells to increase. The number of
INFECT. IMMUN.
hemolytic plaques from the control spleen cell culture did increase when the culture was incubated with NDV in vitro. The increase in plaques is of the same magnitude that is often seen in the controls as variation from experiment to experiment. However, an increase of this magnitude within one experiment is real. Nonspecific plaque formation (vi) is minimized in the HPA assay by washing SRBC extensively with agamma calf serum (Pentex, Inc.). When the procedures for washing the SRBC described in this paper are followed, the number of nonspecific plaques formed (especially from experiment to experiment) is greatly reduced. It is probable that the increased number of plaques formed in the in vivo, in vitro, and combined in vivo-in vitro experiments reported here is a specific antibody-forming response to the presence of NDV. Specificity would definitely be demonstrated in a plaque inhibition assay in which free NDV was incorporated into the agar-inhibited hemolytic plaque formation. Also, viruses closely related to NDV paramyxoviruses and myxoviruses should be used to immunize mice and the spleen cells of the immunized mice, tested against NDV coupled to sheep erythrocytes, to determine whether cross-reactions between similar viruses occur in these assay systems. Although many factors influence the results obtained in the HPA, the HPA can yield significant data on antibody formation against NDV and probably other viruses if these factors are rigidly controlled. ACKNOWLEDGMENT We are grateful to Bemard Jaroslow, Argonne National Laboratory, for his guidance while leaming the intricacies of the hemolytic plaque assay.
LITERATURE CITED 1. Baker, P. J., and P. W. Stashak. 1969. Quantitative and qualitative studies on the primary antibody response to pneumococcal polysaccharides at the cellular level. J. Immunol. 103:1342-1348. 2. George, M., and J. H. Vaughan. 1962. In vitro cell migration as a model for delayed hypersensitivity. Proc. Soc. Exp. Biol. Med. 111:514-521. 3. Golub, E. S., R. J. Mishell, W. 0. Weigle, and R. W. Dutton. 1968. A modification of the hemolytic plague assay for use with protein antigens. J. Immunol. 100:133-137. 4. Ingraham, J. S., and A. Bussard. 1964. Application of a localized hemolysin reaction for specific detection of individual antibody-forming cells. J. Exp. Med.
119:667-684. 5. Jerne, N. K., and A. A. Nordin. 1963. Plague formation in agar by single antibody producing cells. Science 140:405. 6. Johnson, H. M., K. Brenner, and H. E. Hall. 1966. The use of water-soluble carbodiimide as a coupling reagent in the passive hemagglutination test. J. Immunol. 97:791-796. 7. Kaplan, A. M., and M. J. Freeman. 1967. Enumeration of
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cells synthesizing antiprotein by a modified hemolytic plague assay. Proc. Soc. Exp. Biol. Med. 127:574-576. 8. Kerman, R., and D. Segre. 1970. Anti-pneumococcal polysaccharide type m hemolytic plagues in mice: cellular response in immunity and immunologic paralysis. J. Immunol. 104:1262-1266. 9. Kishimoto, S., I. Tsuyuguchi, and Y. Yamamura. 1968. The immune response to hapten azo bovine serum albumin as directed by hemolytic plague technique. Int. Arch. Allergy Appl. Immunol. 34:544-555. 10. Landy, M., R. P. Sanderson, and A. L. Jackson. 1965. Humoral and cellular aspects of the immune response to the somatic antigen of Salmonella enteritidis. J. Exp. Med. 122:483-504. 11. Lowry, 0. H., N. J. Rosebrough, A. L. Faff, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 12. Merchant, B., and T. Hraba. 1966. Lymphoid cells producing antibody against simple haptens: detection and enumeration. Science 152:1378-1379. 13. Mishell, R J., and R W. Dutton. 1966. Immunization of normal mouse spleen cell suspension in vitro. Science 153:1004-1005. 14. Mishell, R. J., and R. W. Dutton. 1967. Immunization of dissociated spleen cultures from normal mice. J. Exp. Med. 126:423-442. 15. Rittenberg, M. B., and K. L. Pratt. 1969. Antitrinitro-
995
phenyl (TNP) plague assay. Primary response of Balb/c mice to soluble and particulate immunogen. Proc. Soc. Exp. Biol. Med. 132:575-581. 16. Schwenk, H. U., and F. Lehmann-Grube. 1970. The localized hemolysis-in-gel method adapted to the detection of spleen cells releasing virus-specific antibodies. J. Immunol. 104:1184-1186. 17. Segre, M., and D. Segre. 1967. Hemolytic plague formation by mouse spleen cells producing antibodies against rabbit immunoglobulin G. J. Immunol. 99:867-875. 18. Shiori, K. 1964. The observation of proteins adsorbed on erythrocytes treated with tannic acid in passive hemagglutination tests (Boyden's method). Jpn. J. Exp. Med. 34:345-359. 19. Stein, B., and K. S. Desowitz. 1964. The measurement of antibody in human malaria by a formolized tanned sheep cell hemagglutination test. Bull. W. H. 0. 30:45-49. 20. Walsh, P., P. Maurer, and M. Egan. 1967. Detection of immune response against synthetic polymers of amino acids employing the plague-forming all cell system. J. Immunol. 98:344-350. 21. Yamada, H., A. Yamada, and V. P. Hollander. 1970. 2,4Dinitrophenyl-hapten specific hemolytic plague-in-gel formation by mouse myeloma (mopc-315) cells. J. Immunol. 104:251-255.
Vol. 11, No. 5 Printed in U.S.A.
Detection of Virus-Specific Antibody-Forming Cells of Mice Immunized with Newcastle Disease Virus YOUNG TAI KIM,' JANE AULT,' AND R. J. WODZINSKI3* International Minerals and Chemical Corporation, Libertyville, Illinois 60048 Received for publication 16 September 1974
The hemolytic plaque assay was adapted to the detection of antibodies to Newcastle disease virus (NDV) in an in vivo, an in vitro system, and a combined in vivo-in vitro system. Several conditions were tested for coupling of sheep erythrocytes to NDV and for the kinetics of plaque formation in the in vivo and in vitro systems. The one set of conditions which provided the best responses is presented. The effect of multiple injections of NDV into mice on plaque formation was optimized. Virus. NDV strain B, was used as the viral antigen. The virus was propagated in 9-day embryonated eggs for 48 h. The allantoic fluid was harvested, pooled, and centrifuged at 3,000 x g for 30 min. The supernatant was centrifuged at 30,000 x g for 1 h and the pellet was resuspended in cold phosphate-buffered saline (PBS) at pH 7.2. The suspension was centrifuged at 30,000 x g for 1 h and the pellet was resuspended in PBS. The final suspension was adjusted to 10' infectious particles as measured in a chicken embryo assay. This suspension contained 50 Mg of protein per ml as measured by the method of Lowry et al. (11). Sensitization of SRBC. Fresh sheep blood was centrifuged and the SRBC was washed three times with PBS. A 5% suspension of the washed SRBC in PBS was added to an equal volume of 1 mg of tannic acid per ml. After 15 min of incubation in a water bath at 37 C, the mixture was centrifuged for 5 min at 750 x g. The pellet containing the SRBC was washed once with 20 ml of PBS. The SRBC in the washed pellet were resuspended in PBS to achieve a 5% suspension. Ten milliliters of the SRBC suspension was added to 10 ml of PBS containing 100 gg of NDV on a protein basis. The NDV-SRBC mixture was incubated in a water bath for 30 min at 37 C. The SRBC were recovered by centrifugation at 750 x g for 5 min. The virus-sensitized SRBC were washed three times with 20 ml of PBS containing 1% inactivated agcmma-globulin (Pentex Inc. Kankakee, Ill.). The washed, sensitized SRBC were suspended in PBS at a 20% concentration and were used in the HPA. MATERIALS AND METHODS HPA. The HPA previously described (4) was emAnimals and immunization procedures. CFI F ployed with some modifications. Four days after the mice about 8 weeks old (Carworth, Inc., Portage, final immunization, mouse spleens were removed Mich.) were injected intravenously with 0.2 ml of aseptically and placed in 60-mm petri dishes (Falcon saline containing 10 ,g of NDV on the basis of protein Plastics, Los Angeles, Calif.) containing 10 ml of content. If secondary immunization was desired, the Eagle minimal essential medium (Flow Laboratories, Los Angeles, Calif.). The spleen cells were released by mice were reinjected as described above 3 weeks later. teasing with forceps. The resultant suspension of ' Present address: Department of Allergy and Immunology, dissociated cells was centrifuged for 10 min at 1,000 x g. The cell pellet was resuspended in Eagle medium Cornell Medical School, New York, N. Y. 10021. (Flow Laboratories, Los Angeles, Calif.) at the desired 2Present address: 20 Cedar St., Potsdam, N. Y. 13676. ' Present address: Department of Biological Sciences, concentration. For the HPA, 0.7 g of Noble agar (Difco LaboratoFlorida Technological University, Orlando, Fla. 32816. 391
The hemolytic plaque assay (HPA) in agar for the detection of antibody-forming cells to sheep erythrocytes (SRBC) was introduced by Jerne and Nordin (5). Ingraham and Bussard (4) reported a similar method for the detection of specific antibodies but utilized a slide technique. The method has been modified to detect the antibody-forming cells against bacterial polysaccharides (1, 8, 10), haptens (12, 15, 21), synthetic polypeptides (20), and proteins (3, 7, 9, 17). The basic modifications in each of the above methods is the coupling of the various antigens to SRBC. Johnson et al. (6) described a modification in which 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ECDI) is used to couple the antigen to SRBC. Further, SRBC can be sensitized with tannic acid and then coupled with various antigens (2, 18, 19). The HPA method was adapted to the detection of single cells releasing antibody to influenza A virus (16). The present paper reports a method for coupling Newcastle disease virus (NDV) to SRBC and for detecting the antibody-forming cells to NDV by using mouse spleen cells in a HPA. It also reports synthesis of antibody to NDV by mouse spleen cells in vitro.
992
KIM, AULT, AND WODZINSKI
ries, Inc., Detroit, Mich.) was added to 100 ml of Eagle minimal essential medium. The mixture was boiled for 30 min on a hot plate. The agar in Eagle minimal essential medium (2.0 ml) was preincubated at 46 C in a water bath. Washed, sensitized SRBC (0.1 ml) and 0.2 ml of the spleen suspension were added to the agar. The materials were mixed quickly and poured into 100-mm plastic petri dishes. After 1 h of incubation at 37 C, the plates were flooded with 1.5 ml of a 1: 10 dilution of guinea pig complement (Pentex Corp., Kankakee, Ill.) in PBS. After 1 h of incubation at 37 C, the complement was poured off and the number of hemolytic plaques was counted under a x 10 stereomicroscope. The results were expressed as number of plaqueforming cells per 106 viable cells.
INFECT. IMMUN.
incubated in vitro before assay. Plaque formation increased rapidly up to 4 days after inoculation (Fig. 1). It decreased thereafter and was barely measurable 7 days after inoculation. Effect of multiple injections of NDV on hemolytic plaque formation. Mice that were injected intravenously with a single dose of NDV were divided into two groups. One group received no further treatment. The other group was injected a second time with NDV 3 weeks after the first injection. Spleen cultures were prepared from each group 4 days after the last injection. After incubation of the cell cultures, the spleen cells were assayed by the hemolytic plaque assay. The spleens from the mice receiv-
RESULTS TABLE 1. Hemolytic plaque formation by spleen cells from mice immunized with NDV Effect of tannic acid on coupling of SRBC with NDV. SRBC must be coupled with NDV Hemolytic plaques if antibody-forming cells against NDV are to be per 106 cells SRBC Spleen observed in the HPA. Sensitization of SRBC cells with tannic acid often allows subsequent couExptl Expt2 pling with various antigens (2, 18, 19). The coupling of SRBC with NDV is measurable via Control 2 0 Control 1 3 Coupled with a passive hemagglutination test. NDV Serial twofold dilutions of NDV in saline were added to an equal volume of tanned SRBC. The 3 0 Control hemagglutinating titer of the NDV preparation From mice immunized was 64 when tannic acid-sensitized SRBC were against NDV used, whereas the titer was less than 2 when untreated SRBC were used. The above result 101 51 Coupled with indicates that tannic acid treatment of SRBC NDV permits the coupling of NDV to these treated cells. 100 Hemolytic plaque formation by spleen cells with The spleens immunized NDV. from mice of mice which were immunized once with NDV were removed after 4 days in two separate experiments. Spleen cell cultures (See Materials and Methods) were made from immunized and control mice and were measured by HPA. so Four hemolytic plaques per 106 cells were - 60 formed in the control cell cultures tested with NDV coupled with SRBC, whereas 152 hemo- 04 lytic plaques per 106 cells were found in the cell 40 cultures prepared from the NDV-immunized mice (Table 1). Cell cultures from both control 0 and immunized mice formed 4 or less hemolytic plaques per 106 cells when tested against non20 coupled SRBC. Although some variation in the response to immunization occurred between experiments, these results indicate that spleen cells prepared from mice immunized against NDV produce specific antibody-forming cells 10 9 5 6 8 7 4 3 1 2 against NDV. WITH N D V INOCULATION AFTER DAYS Kinetics of hemolytic plaque formation. Spleens from mice which received a single FIG. 1. Kinetics of antibody formation against injection of NDV were assayed daily for 10 days NDV in mouse spleen cells after a single injection of using coupled SRBC. The spleen cells were not antigen. v
v
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993
NDV HEMOLYTIC PLAQUE ASSAY
ing two injections produced four times as many plaques as those from the mice receiving one injection, when tested against SRBC coupled with NDV (Table 2). Approximately the same number of plaques was formed from the spleens of both groups of mice when they were tested against control SRBC. In vitro immunization of mouse spleen cells with NDV. Mishell and Dutton (13) reported that dissociated spleen cell suspensions from normal mice could be immunized in vitro to
heterologous erythrocytes. They obtained marked increases in plaque-forming cells when spleen culture cells obtained from in vivo immunized mice were challenged with additional antigen in vitro and then incubated in vitro (14). A similar experiment was performed to confirm these observations, using NDV as the antigen. One-milliliter aliquots of spleen cell suspensions, from mice immunized with NDV and from control mice, were added to NDV (1 gg/ml) in 35-mm petri dishes (Falcon plastic). The dishes were placed in boxes and incubated at 37 C in an atmosphere of 7% 02, 10% CO2, and 83% N2. The boxes were placed on a rocker platform set for 7 to 10 complete cycles per min. The cultures were fed daily with 0.1 ml of a nutrient mixture (13). The control spleen cell suspension was incubated in the petri dishes as described above. After 4 days of incubation, the cells were suspended by scraping them from the dish surface with a plastic policeman. The treated and untreated cell suspensions were centrifuged at 1,000 x g for 10 min at 8 C and were resuspended in culture media at a concentration of 2 x 106 viable cells. When spleen cells prepared from mice immunized with NDV were incubated with NDV in vitro, the number of antibody-forming cells was increased at least twofold over that of the culture without incubation in vitro (Table 3). Control spleen cell cultures incubated with NDV in vitro formed more hemolytic plaques than those to which no NDV was added in vitro. However, the great variation in the number of plaques from experiment to experiment makes it difficult to reach definite conclusions at this time. DISCUSSION The hemolytic plaque assay to detect virusspecific antibody by an in vivo, in vitro, or combined in vivo-in vitro system would enjoy widespread use if the technique yielded consistent results, measured small differences in the antibodies formed, and could be made routine. The following factors must be rigidly controlled if reproducible results are to be obtained with the HPA when NDV is used as antigen: (i)
TABLE 2. Effect of multiple injection of NDV into mice on hemolytic plaque formation Spleen cells
Control
From mice receiving 1 injection of NDV
Hemolytic plaques per 106 cells
SRBC
5
Control
Coupled with NDV
10
Control
25
Coupled with NDV From mice receiving 2 injections of NDV
130
Control
10
Coupled with NDV
515
TABLE 3. Effect of in vitro immunization of mouse spleen cells with NDV NDV added to
Spleen cells
Hemolytic plaques per per 106 cells
spleen cells in vitro
Control In vivo immunized
Expt 1
Expt 2
Yes
2 21
4 36
No
84 415
106 206
No
Yes
sensitization of SRBC to permit their coupling with NDV; (ii) prevention of hemolysis by the sensitizing agent; (iii) source of SRBC; (iv) kinetics of plaque formation; (v) the procedure for immunizing mice with antigen; and (vi) the elimination of nonspecific plaque formation. Since NDV is not extensively coupled to untreated SRBC (i), different sensitizing agents, ECDI and tannic acid, were used to enhance coupling of NDV to SRBC. Experiments not reported here showed that tannic acid-sensitized cells yielded higher hemagglutination titers than did ECDI-sensitized cells. However, it was necessary to adjust the tannic acid concentration to a level which would maximize coupling of SRBC to NDV but not cause hemolysis (ii). Although several conditions were tested to increase the sensitization, fewer plaques were formed when NDV was the antigen than if either SRBC or bovine serum albumin was used as the antigen. The number of antibody-forming cells against NDV was about one-tenth that obtained from mice in-
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jected with SRBC and about half that seen in mice immunized with bovine serum albumin. When SRBC were used as antigen, the number of hemolytic plaques was about 103 to 104 per 106 cells, whereas about 5.0 x 102 to 1.0 x 103 plaques per 106 cells were counted when bovine serum albumin was the antigen. Another cause of variation in the assay is the source of sheep blood (iii). The blood of six different sheep tested in a single experiment in which all other conditions were constant yielded values ranging from 25 to 2,500 hemolytic plaques. The blood of a single sheep which showed higher titers was used in all experiments reported in this paper. When the single source of sheep blood was used, the variation in plaque count was 40 to 100 plaques per 106 spleen cells from experiment to experiment. However, the spleen cells from the control mice produced relatively consistent numbers of plaques. The sensitization of the SRBC would influence the number of hemolytic plaques that were formed from the spleens of control mice. The wide differences observed between control mice and those injected intravenously with NDV show that the mice did produce antibodies against the NDV preparation. The kinetics of antibody formation (iv) in spleen cells prepared from the mice immunized with NDV were similar to those observed in mice injected with SRBC. The number of antibody-forming cells rapidly increases up to the day 4 after immunization and decreases drastically with time. However, the kinetics of hemolytic plaque formation shows greater variation from experiment to experiment, roughly 100 to 500 at the peak. A secondary immunization of mice with NDV appears to stimulate the antibody-forming cells (v). Four times more hemolytic plaques were formed if mice were immunized twice than if only a single immunization was used. In vitro incubation of spleen cell cultures prepared from mice injected with NDV yields more antibodyforming cells than if no in vitro incubation is used. The increase in hemolytic plaques can be explained in two ways. One explanation is that the number of antibody-forming cells actually increases during incubation with NDV. The other explanation is that viable cells decrease during in vitro incubation and the number of hemolytic plaques per 106 viable cells is, therefore, increased proportionately. The viable cells do decrease by 50% during the day 4 in vitro incubation. However, it is difficult to envision that non-antibody-forming cells died discriminately and caused the ratio of antibody-forming cells to other cells to increase. The number of
INFECT. IMMUN.
hemolytic plaques from the control spleen cell culture did increase when the culture was incubated with NDV in vitro. The increase in plaques is of the same magnitude that is often seen in the controls as variation from experiment to experiment. However, an increase of this magnitude within one experiment is real. Nonspecific plaque formation (vi) is minimized in the HPA assay by washing SRBC extensively with agamma calf serum (Pentex, Inc.). When the procedures for washing the SRBC described in this paper are followed, the number of nonspecific plaques formed (especially from experiment to experiment) is greatly reduced. It is probable that the increased number of plaques formed in the in vivo, in vitro, and combined in vivo-in vitro experiments reported here is a specific antibody-forming response to the presence of NDV. Specificity would definitely be demonstrated in a plaque inhibition assay in which free NDV was incorporated into the agar-inhibited hemolytic plaque formation. Also, viruses closely related to NDV paramyxoviruses and myxoviruses should be used to immunize mice and the spleen cells of the immunized mice, tested against NDV coupled to sheep erythrocytes, to determine whether cross-reactions between similar viruses occur in these assay systems. Although many factors influence the results obtained in the HPA, the HPA can yield significant data on antibody formation against NDV and probably other viruses if these factors are rigidly controlled. ACKNOWLEDGMENT We are grateful to Bemard Jaroslow, Argonne National Laboratory, for his guidance while leaming the intricacies of the hemolytic plaque assay.
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