Proc. Nat. Acad. Sci. USA Vol. 71, No. 4, pp. 1229-1233, April 1974

Antigen-Secreting Cells: Enumeration of Immunoglobulin-Allotype-Secreting Cells in Nonimmunized Rabbits by Means of Hybrid-Antibody-Coated Erythrocytes in a Reverse Hemolytic Plaque Assay* (lymphoid organs/light chain allotypes/rosette-forming cells/antigen-reactive erythrocytes) GIUSEPPE A. MOLINAROt, ELCHANAN MARONt, AND SHELDON DRAY Department of Microbiology, University of Illinois at the Medical Center, Chicago, Ill. 60612 Communicated by Herman N. Eiven, December 13, 1973

ABSTRACT A reverse hemolytic plaque-forming cell b4 or b5 Ig reacted with anti-b4 or anti-b5 antibody-coated (PFC) assay for the enumeration of immunoglobulin- erythrocytes. (Ig)secreting cells in nonimmunized rabbits was developed by using erythrocytes coated with anti-Ig antibody. These MATERIALS AND METHODS indicator cells lysed after reacting with the secreted Ig antigen and complement. The anti-Ig antibodies used Antisera and Purified Antibodies. Antisera to rabbit Ig were specific for the b4 or b5 allotypic specificities, which allotypes b4 or b5 were produced and characterized as pre- are antigenic determinants on the kappa chain of rabbit Ig and controlled by alleles at the b locus. We coated viously described (7). Anti-SRBC (sheep red blood cells) erythrocytes with anti-IA or anti-b5 antibody, using hy- antisera were elicited in b4b4 and b5bM homozygous rabbits by brid antibody with dual specificity to sheep red blood cells weekly intravenous injections of a 1-ml suspension of 2.5% and to the b4 or b5 kappa chain. The coated erythrocytes washed SRBC in saline for 2 months. Anti-b4 or anti-b5 were plated with cells from various lymphoid organs of im- nonimmunized rabbits. These lymphoid cells formed anti-allotype antibodies were isolated by elution from allotype-specific plaques, i.e., the cells of IA homozygous munosorbents prepared by coupling the b4 or b5 IgG to rabbits formed hemolytic plaques with anti-IA but not Sepharose 4B (8). The adsorbed antibodies were eluted with with anti-b5-coated erythrocytes and vice versa. In eight 0.2 M glycine sulfate buffer, pH 2.2, or with 1 M acetic acid nonimmunized rabbits, 0.06-0.3% of the spleen cells and the acidic eluates were immediately neutralized with 3 M (590-2900 PFC per 108 cells) secreted the b locus Ig allo- types. In three nonimmunized rabbits, 320-590 PFC per Tris base and dialyzed against saline. Anti-SRBC antibodies 106 cells were counted in the lymph node or bone marrow; were purified by elution from SRBC by a method similar to 26-40 PFC per 106 cells were detected in the thymus. Since that of Bratcher et al. (9). Essentially the SRBC were first the IA and b5 specificities are present on approximately incubated with the antisera at 20° for 2 hr and at 40 over- 90% of the serum Ig, our results indicate that less than 1% of the lymphoid cells in nonimmunized rabbits secrete night. The SRBC were washed and then lysed with digitonin Ig at any one time. By this reverse PFC assay, Ig-secreting (Nutritional Biochemical Corp., Cleveland, Ohio) (1 mg/ml cells were for the first time detected as antigen-secreting of a 10% SRBC suspension) at 40 overnight. The antibodies cells. This assay can be applied to the detection of cells were eluted from the washed stroma with 1 M propionic acid secreting other antigens. and the eluates were immediately neutralized with 3 M Tris Antibody-secreting cells can be identified by the plaque- base and dialyzed against saline. forming cell (PFC) assay (1). The PFC assays are based on Hybrid Antibodies (Anti-SRBC/Anti-allotype). Hybrid the hemolytic reaction of the secreted antibody with antigenic antibody molecules were formed by recombination of half determinants either native to (1-3) or artificially coupled to molecules of purified anti-allotype and anti-SRBC antibody (4-6) erythrocytes. Thus, only cells secreting immunoglobulin preparations according to the method of Nisonoff and his co- (Ig) with known antibody specificity can be enumerated. workers (10, 11). The antibodies to be hybridized were Thus far, detection of cells secreting Ig with unknown anti- matched so as to have identical allotypic specificities on their body activity by a PFC assay has not been achieved. Here we light chain to prevent an antigen-antibody reaction between report the detection of such cells in rabbits by a PFC assay the anti-SRBC antibody and the anti-allotype antibody. based on the allotypic antigenic properties of the secreted Equal amounts of anti-allotype and anti-SRBC antibodies at Ig, i.e., the b4 and b5 specificities present on the kappa chain a concentration of 5-10 mg/ml were mixed and dialyzed of Ig and controlled by allelic genes. In the assay, the secreted against 0.1 M Tris-HCl buffer, pH 8.2. Reduction was per- formed with 0.01 M 2-mercaptoethanol at 200 for 1 hr. The reducing agent was removed by passage through a cation Abbreviations: Ig, immunoglobulin; PFC, plaque-forming cells; exchange resin (Dowex 50W-X4, Bio Rad Labs, Richmond, RFC, rosette-forming cells; SRBC, sheep red blood cells. Calif.) equilibrated with 0.1 M acetate buffer, pH 4.5. The * Presented in part at the 57th Annual Meeting of the Federation effluent from the cation exchange column was acidified to pH of American Societies for Experimental Biology, Atlantic City, 2.5 with 1 M HC1 and dialyzed against 0.025 M NaCl, pH N.J., April 15-20, 1973. 2.5, at 40 overnight. Sodium chloride was then added to the t On leave from CNEN, Rome, Italy. solution to a final concentration of 0.5 M, thus reducing the I Passed away suddenly at the age of 28 on August 8, 1973. internal electrostatic repulsions. Finally, the protein solution 1229 Downloaded by guest on September 30, 2021 1230 Immunology: Molinaro et al. Proc. Nat. Acad. Sci. USA 71 (1974)

was dialyzed against 0.01 M phosphate-buffered saline, pH were mixed and plated on a 60-mm Falcon plastic petri dish, 7.2. This method leads to a random recombination of half precoated with 3 ml of 1% agarose (Seakem, Marine Colloids, molecules so as to yield three populations of molecules in a Inc.) in Hanks' solution. After gelification of the top layer the 1:2:1 ratio as follows: bivalent anti-SRBC, hybrid anti- dishes were incubated at 370 for 2 hr in a humid, 5% CO2 SRBC/anti-allotype, and bivalent anti-allotype antibodies. atmosphere and then flooded with 2 ml of saline or an optimal The reformed bivalent anti-SRBC antibodies if left, would dilution (usually 1:200) of anti-b4 or anti-b5 antiserum agglutinate and lyse the SRBC. Therefore, the hybrid prepa- ("enhancing antiserum") in saline. The dishes were then kept rations were passed through a b4 or b5 immunosorbent. The at 40 overnight. The next morning, after decantation, 1 ml of effluents containing the unwanted bivalent anti-SRBC anti- complement solution (reconstituted guinea-pig serum) (Gibco, body were discarded. The eluates obtained by acidic elution Grand Island, N.Y.) diluted 1:10 in Hanks' solution was contained the desired hybrid antibodies as well as the bivalent added and the dishes were incubated at 37° for 1-2 hr. anti-allotype anti-bodies. After neutralization and dialysis In every experiment, serial concentrations of cells were against saline, the eluates were used for coating the SRBC tested in duplicate or triplicate, and in some experiments the without further purification since the reformed bivalent anti- plaques were counted by two or three individuals. Only dishes allotype antibodies do not react with SRBC. Approximately containing 20-200 plaques were counted. The average of all 30-40% of the antibodies initially mixed were recovered as a counts was expressed as number of PFC per 106 spleen cells. mixture of hybrid and bivalent anti-allotype antibodies. Individual counts did not usually vary by more than 10-20% from the average. Coating of SRBC with Anti-Allotype Antibody. The hybrid antibody preparations were used to coat SRBC through the Immunocytoadhesion Assay. Lymphoid cells bearing an Ig binding site directed against SRBC. SRBC (1010 cells) were allotype on their surface were enumerated by a modified im- incubated with either the anti-b4 or anti-b5 hybrid antibody munocytoadhesion assay derived from that described by preparation (usually 0.2-0.5 mg) in a final volume of 2 ml of Paraskevas et al. (13) for mouse Ig. Briefly, 0.1 ml of a suspen- saline at 200 for 30 min and then washed with saline. The sion of spleen cells (3 X 106 cells per ml) in saline was incu- anti-b4 and anti-b5 coated cells were tested for hemagglutina- bated with 0.1 ml of suspension of hybrid-antibody-coated tion and hemolysis with the respective b4 and b5 antigens SRBC (3 X 108 SRBC per ml) in saline containing 0.2% (12). gelatin and 0.05 M sodium azide at 40 overnight. Lympho- cytes bearing the allotype formed rosettes with the corre- Cell Suspension. Rabbit organs (spleen, popliteal lymph sponding antibody-coated erythrocyte; only rosettes consist- node, thymus, and bone marrow from the tibia) were removed ing of a cell with at least four erythrocytes were counted. and teased with needles in Hanks' solution at 4°. After re- moval of tissue fragments by settling, the cells were washed RESULTS in Hanks' solution at suitable two times at 40 and resuspended Formed Celis with the trypan blue exclusion method, the cell Hemolytic Plaques by Spleen Assayed concentrations. By SRBC. SRBC were coated with anti-b4 viability was greater than 85%. Anti-allotype-Coated or anti-b5 hybrid antibody molecules having one combining Plaque-Forming Cell Assay. The method described by Jerne site specific for an SRBC antigenic determinant and the other et al. (1) was followed with minor modifications. In this order, site specific for a b4 or b5 allotypic determinant. These anti- 0.1 ml of a 12% suspension of anti-allotype-coated SRBC and allotype-coated SRBC were agglutinated by the corresponding 0.1 ml of the cell suspensions at various cell concentrations Ig allotype, proving that the other binding site of the hybrid were pipetted in a 100 X 75-mm tube containing 1 ml of 0.8% antibody was free to react with the Ig allotype. When rabbit agarose (Sea Plaque, Marine Colloids, Inc., Rockland, Maine) spleen cells were plaque-assayed with the corresponding anti- in Hanks' solution pH 7.4 at 37°. The contents of the tube allotype-coated SRBC, complement-dependent hemolytic plaques were obtained (Fig. 1). The macroscopic and micro- scopic morphology of these hemolytic plaques was similar to that described by Jerne (1); i.e., they were circular, 0.1-1.0 mm in diameter, and cell-centered. The Jerne plaques are formed by cells secreting antibody; the plaques in our assay were formed by cells secreting the Ig allotype. Since the secre- * ,Eg1N L>'X1 sax|||_IN tion of Ig by cells is temperature-dependent (14), no hemolytic plaques were obtained when spleen cells were plaque-assayed at 40 rather than 37°. Moreover, at 370, the number of plaques increased with the time of incubation, reaching a maximum at 2 hr. Plaque Formation. Lymphoid cells from a b4b4 * I-u..He','"p Specificity of homozygote, bob' homozygote or b4b5 heterozygote were plaque-assayed with anti-b4- or anti-b5-coated SRBC. The b4b4 lymphoid cells formed hemolytic plaques with anti-b4- coated SRBC but not with anti-b5-coated SRBC, and the b5b5 cells formed plaques with anti-b5- but not with anti-b4- FIG. 1. Hemolytic plaques formed by b4-allotype secreting coated SRBC (Table 1). As expected, the b4b5 cells formed spleen cells taken from a nonimmunized rabbit and assayed with plaques with the anti-b4- and with the anti-b5-coated SRBC; SRBC coated with hybrid antibody (anti-b4/anti-SRBC). the combined number of b4 and b5 plaques was approxi- Downloaded by guest on September 30, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Enumeration of Antigen-Secreting Cells 1231

TABLE 1. Enumeration of b4- and b5-allotype-secreting cells as PFC in various lymphoid organs of homozygous and heterozygous nonimmunized rabbits 800 Plaque-forming cells/106 cells Normal Serum SRBC Bone coated mar- Lymph Thy- , 600 Rabbit Genotype with* Spleen row node mus '0 K1 15-7 b4b4 anti-b4 640 510 320 40 U 400 anti-b5 3 2 2 0 K139-6 b5b5 anti-b4 5 3 3 1 anti-b5 720 560 430 35 Anti-b4 K13-5 b4bO anti-b4 320 300 190 14 200 anti-b5 310 290 170 12

* Anti-SRBC/anti-allotype hybrid antibody. 10 20 30 40 mately the same as that found in the b4b4 or blb5 homozygotes. ANTISERUM ADDED (uL) The number of plaques formed by homozygous cells with the FIG. 3. Inhibition of plaque formation by specific antiserum noncorresponding anti-allotype-coated SRBC was similar embedded in the gel. Spleen cells (SpC) from a b4 homozygous (0-5 PFC per 106 cells) to the background number of plaques rabbit were plaque-assayed with hybrid antibody-(anti-b4/ with uncoated SRBC (not shown). Thus, the anti-SRBC)coated SRBC, along with increasing amounts of formed plaques serum in 0.1 ml of Hanks' were formed cells. anti-b4 antiserum or normal rabbit in our assay by sp)ecific-allotype-secreting solution. Dependency of Plaque Formation on the Degree of Coating of the SRBC. SRBC (1010 cells) treated with increasing amounts (0-0.6 mg) of the hybrid anti-b4 antibody preparation were a maximum (roughly 700 PFC per 106 cells) obtained with tested with b4 spleen cells. The number of plaques progres- optimally coated SRBC (Fig. 2, lower curve). A similar de- sively increased from the background (5 PFC per 106 cells) pendency of plaque formation on the degree of coating was obtained with uncoated or very "lightly" coated SRBC to found with anti-b5-coated SRBC. Enhancement of Plaque Formation by Anti-Allotype Anti- serum Added after Incubation. The possibility was considered that the addition of anti-b4 or ainti-b5 antibodies ("enhancing antibodies") after incubation would crosslitik the secreted b4 or b5 allotype molecules bound by the coated SRBC and that the formation of these immune complexes, by enhancing the fixation of complement, would facilitate the lysis of the indicator cells. Thus, dishes containing b4 spleen cells plaque- assayed with anti-b4 coated SRBC, after incubation at 370 for 2 hr, were treated with anti-b4 antiserum at a dilution of *(, 1: 200 in 2 ml of saline. Indeed, the enhancing antiserum in- creased the number of PFC per 106 cells above that obtained 400 antiserum (Fig. 2). This enhancing effect "I without enhancing was greater with SRBC coated with smaller amounts of hy- a- brid antibody. Moreover, the "enhanced" lplaques were some- what more distinct. Inhibition of the Formation of Hemolytic Plaques by Anti- Allotype Antiserum Added before Incubation. The formation of the plaques was tested in the presence of various amounts of anti-allotype antibody. Spleeni cells from a b4 homozygote did not form plaques with anti-b4 coated SRBC when anti-b4 antiserum was incorporated into the gel, whereas normal .15 .30 .45 .60 serum had no inhibitory effect (Fig. 3). This inhibition was ANTI BODY USED (mig) dose-dependent, i.e., the number of plaques decreased with FIG. 2. Dependency of plaque formation on the amount of increasing amounts of antiserum. In a similar experiment, hybrid antibody used to coat the 1010 SRBC and enhancement of anti-b5 antiserum blocked the appearance of b5 plaques. plaque formation by specific antiserum. Spleen cells (b4b4, ab- Thus, the anti-b4 or anti-b5 antiserum competed with the breviated SpC) were plaque-assayed with SRBC coated with antibody bound to SRBC for the secreted b4 or b5 allotype. increasing amount of hybrid antibody (anti-b4/anti-SRBC). Dishes were incubated with enhancing anti-b4 antiserum at Enumeration of Allotype-Secreting Cells in Various Lymphoid 1:200 dilution (0) or saline (0) before the incubation with com- Organs. In the following experiments, optimally coated SRBC plement. (0.2 mg of antibody per 1010 cells) and an optimal dilution of Downloaded by guest on September 30, 2021 1232 Immunology: Molinaro et al. Proc. Nat. Acad. Sci. USA 71 (1974)

TABLE 2. Enumeration of b4-allotype-secreting cells type secreted by the cells with the anti-allotype-coated SRBC as plaque-forming cells (PFC) and allotype-bearing was an absolute requirement for plaque formation, hemolytic cells as rosette-forming cells (RFC) among spleen cells plaques formed by rabbit spleen cells with anti-allotype anti- of b4 homozygous nonimmunized rabbits body-coated SRBC were allotype-specific. The allotypic specificity of these hemolytic plaques was Rabbit PFC/106 cells RFC/105 cells confirmed by the finding that anti-b4 antibody present in the EM-1 750 260,000 gel during the secretion period inhibited the formation of b4 EM-2 1660 320,000 allotype plaques by competing for the secreted b4 allotype L324-1 2900 160,000 with the anti-b4 antibody bound to SRBC. Similar inhibition K31-2 1030 130,000 of b5 plaques was found by incorporation of anti-b5 antibody K65-3 590 210,000 in the gel. Moreover, anti-b4 antibody added after the secre- tion, i.e., when the secreted b4 Ig had already been bound by anti-b4-coated SRBC, enhanced the formation of plaques, by enhancing the fixation of complement. The enhancing antiserum (1: 200) were used. Allotype-secreting presumably by Jerne et al. (1) detects lymphoid cells were enumerated in spleens of five b4b4 homozygotes method first described cells secreting antibody (mainly IgM) with high hemolytic (table 2) and in other lymphoid organs of a b4b4 homozygote, efficiency and thus forming "direct plaques" upon addition a b5b5 homozy~ote and a b4b5 heterozygote (Table 1). In of complement. Cells secreting other classes of antibody with spleen, lymph node, and bone marrow, of the cells 0.03-0.3% form formed allotype plaques; in the thymus, 0.003-0.004% of the low hemolytic efficiency (mainly IgG) do not hemolytic cells formed plaques. plaques unless an anti-Ig antibody (a developing antiserum), reactive with antigenic determinants on the secreted anti- Enumeration of Spleen Cells Bearing Surface Ig Allotypes. body, is added (2, 3); presumably the formation of immune For comparison with the number of PFC, cells bearing Ig complexes on the erythrocyte surface enhances fixation of allotype molecules on their surface were also enumerated as complement; thus, "indirect plaques" are formed by cells rosette-forming cells (RFC) by an immunocytoadhesion secreting antibody with low hemolytic efficiency. Similarly, assay, making use of another application of antibody-coated in the reverse PFC assay the treatment with anti-allotype erythrocytes (12). Spleen cells were mixed with hybrid anti- antiserum reactive with the secreted Ig increased the number allotype-coated SRBC and observed under the microscope. of hemolytic plaques. However, it is unlikely that these A relatively high percentage of lymphoid cells rosetted, i.e., "enhanced plaques" were formed by cells secreting Ig with a single cell was surrounded by SRBC, firmly bound. How- low hemolytic efficiency; the hemolytic efficiency of the se- ever, no RFC were found with the unrelated anti-allotype- creted Ig is probably irrelevant for the formation of plaques, coated SRBC or uncoated SRBC. Among spleen cells of five since the complement should be fixed by the hybrid antibody homozygous rabbits, although 13-32% of the cells formed rather than by the secreted Ig which behaves as antigen. rosettes, only 0.06-0.3% formed hemolytic plaques (Table 2). This hypothesis is testable with the described assay and with Also, there was no correlation between the percentages of other methods. PFC and RFC, i.e., the number of PFC was not predictable The efficiency of plaque formation was dependent on the from the number of RFC and vice versa (Table 2). densitv of antibodies on the SRBC surface. As the antibody density on the SRBC increased, the number of plaques per DISCUSSION 106 cells progressively increased from the background to a A unique plaque-forming cell (PFC) assay for the enumeration maximum value. This finding is in agreement with previous of immunoglobulin-(Ig)secreting cells in nonimmunized reports relating the density of erythrocyte surface antigenic rabbits was developed by using sheep erythrocytes (SRBC) determinants to the efficiency of complement-dependent coated with anti-Ig antibody. In principle, the Ig secreted by hemolysis (15, 16). a single cell reacted with the anti-Ig antibody-coated SRBC In preliminary experiments not reported here, hemolytic and upon addition of complement formed a hemolytic plaque. plaques were also obtained with SRBC coated with anti-allo- More specifically, SRBC were coated with hybrid antibody type antibody by the chrominum chloride method (4). Thus having one specificity directed against SRBC and the other far, the efficiency and reproducibility of plaque formation specificity against an Ig antigen (i.e., the b4 or b5 kappa chain with the hybrid-antibody-coated SRBC was greater than with allotype). These antibody-coated SRBC become reactive with chromium-chloride-coated SRBC. the corresponding antigen, e.g., the anti-b4-coated SRBC The antibody-coated erythrocytes can be used for the enu- were agglutinated by b4 IgG but not by b5 IgG and vice meration of lymphoid cells having Ig molecules on their sur- versa. Spleen cells from nonimmunized rabbits when plaque- face as rosette-forming cells (RFC) by an immunocytoadhe- assayed with the corresponding anti-allotype anti-body-coated sion method (12). Thus, the same preparation of antibody- SRBC formed hemolytic plaques. These plaques were cell- coated SRBC was used for the enumeration of cells secreting centered, suggesting that they were formed by cells secreting the Ig allotype as PFC and cells bearing the allotype on their Ig allotype molecules. Lymphoid cells of b4 homozygous surface as RFC. As expected from the work of others (17, 18), rabbits formed hemolytic plaques with anti-b4-coated SRBC a relatively high percentage of the spleen cells formed rosettes but not with anti-b5-coated SRBC, whereas b5 lymphoid (13-32% RFC). However, relatively few cells formed plaques cells formed hemolytic plaques with anti-b5- but not with (0.06-0.3% PFC). Since the b4 and b5 allotypic specificities anti-b4-coated SRBC. Moreover, b4b5 heterozygous lymn- are present on 90% of the serum Ig (19) and thus presumably phoid cells formed hemolytic plaques with anti-b4- and with in 90% of Ig-secreting cells, our data indicate that much less anti-b5-coated SRBC. Since the correct matching of the allo- than 1% of the lymphoid cells secrete Ig at any one time in Downloaded by guest on September 30, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Enumeration of Antigen-Secreting Cells 1233

nonimmunized animals. The reverse PFC assay may not de- 1. Jerne, N. K., Nordin, A. A. & Henry, C. (1963) in Cell Bound Antibodies, eds. Amos, B. & Koprowski, H. (Wistar tect cells secreting Ig below a threshold level; the same ques- Institute Press, Philadelphia, Pa.), pp. 109-116. tion was considered in the formation of plaques by antibody- 2. Dresser, D. W. & Wortis, H. H. (1965) Nature 208, 859- secreting cells. A comparison of the number of Ig-antigen- 861. and Ig-antibody-secreting cells in hyperimmunized animals 3. Sterzl, J. & Riha, I. (1965) Nature 208, 858-859. would test the comparative efficiency of plaque formation in 4. Sweet, G. H. & Welborn, F. L. (1971) J. Immunol. 106, 1407- the reverse PFC assay and the conventional PFC assay. A 1410. comparison with the data obtained with immunofluorescent 5. Golub, E. S., Mishell, R. I., Weigle, W. 0. & Dutton, R. W. (1968) J. Immunol. 100, 133-137. methods is not meaningful because our method detects Ig- 6. Inman, J. K., Merchant, B., Claflin, L. & Tacey, S. E. secreting cells only, whereas immunofluorescent methods do (1973) Immunochemistry 10, 165-174. not discriminate between Ig-secreting and Ig-containing cells. 7. Dray, S., Young, G. 0. & Gerald, L. (1963) J. Immunol. 91, In this work Ig-secreting cells were identified on the basis 403-415. of antigenic determinants characteristic of kappa light chain 8. Axen, R. & Ernback, S. (1971) Eur. J. Biochem. 18, 351- allotypes present on all classes of Ig. However, any other anti- 360. genic determinants (isotypic, allotypic, or idiotypic) of Ig 9. Bratcher, R. L., Chong, C. A. & Dray, S. (1974) J. Immunol. molecules might be the basis for the detection of subpopula- 112, in press. tions of Ig-secreting cells. For example, erythrocytes coated 10. Hong, R. & Nisonoff, A. (1965) J. Biol. Chem. 240, 3883- with antibodies specific for the Fc fragment of IgG or IgM 3891. should identify cells secreting IgG or IgM. 11. Nisonoff, A. & Palmer, J. L. (1964) Science 143, 376-379. In conclusion, the reverse hemolytic plaque assay provides, 12. Molinaro, G. A. & Dray, S. (1974) Nature 247, in press. for the first time, a means for studying at the cellular level the 13. Paraskevas, F., Lee, S. T. & Israels, L. G. (1971) J. Immunol. secretion of Ig molecules independent of their antibody speci- 106, 160-170. 14. Helinreich, E., Kern, M. & Eisen, H. N. (1962) J. Biol. ficity. The technique has a much wider scope since it can be Chem. 237, 1925-1931. applied to other antigens secreted by lymphoid cells or to 15. Pasanen, V. J. & Makela, 0. (1969) Immunology 16, 399- antigens secreted by nonlymphoid cells, e.g., albumin secreted 407. by liver cells (G. A. Molinaro, E. Maron, and S. Dray, manu- 16. Rouques, R., Inman, J. K. & Merchant, B. (1972) Int. script in preparation). Arch. Allergy Appl. Immunol. 42, 852-870. 17. Davie, J. M., Paul, W. E., Mage, R. G. & Goldman, M. B. We are grateful to Donna Buchholz for her excellent technical assistance and to Dr. Leon LeBeau for taking photographs of the (1971) Proc. Nat. Acad. Sci. USA 68, 430-434. plaques. We also thank Alice GilmanSachs for providing the 18. Wolf, B., Janeway, C. A., Coombs, R. R. A., Catty, D., anti-allotype antisera generously. E. M. was a recipient of a Gell, P. G. H. & Kelus, A. S. (1971) Immunology 20, 931- Damon Runyon Cancer Research Fellowship. This investigation 944. was supported in part by the United States Public Health Service 19. Dray, S. & Nisonoff, A. (1963) Proc. Soc. Exp. Biol. Med. Grant PHS AI 07043. 113, 20-26. Downloaded by guest on September 30, 2021