HYBRIDOMA Volume 11, Number 5, 1992 Mary Ann Liebert, Inc., Publishers
Negative Effect of Multiple Antigen Injections on
the Yield of Murine Monoclonal Antibodies Obtained by
Hybridoma Technology SONIA NÉRON and REAL LEMIEUX
C.P. 1033 Terminus, Québec, Québec, GIK 7B5, Canada Science Faculty, Laval University, Québec, Québec, GIK 7P4, Canada
Canadian Red Cross Society, Blood
Department of Biochemistry,
Transfusion Service,
ABSTRACT One of the critical steps in the preparation of monoclonal antibodies is the obtainment, by in vivo immunization and boost, of the maximal number of antigen-activated B lymphocytes. In hybridoma laboratories, the common procedure is to immunize a group of mice with several antigen injections and use the mouse showing the highest serum antibody titre for the fusion experiment. The observation that the use of mice hyperimmunized with human red blood cells failed to yield a high number of monoclonal antibodies, led us to study the effect of multiple antigen injection prior to the fusion experiment. The results obtained showed that the maximal yields of monoclonal antibodies were obtained using mice that had received only one or two antigen injections while the mice immunized with three antigen injections consistently yielded at least a three-fold reduction in the number of monoclonal antibodies. The negative effect could not be reversed by prolonged resting of the animals and suggests the induction of a tolerance/suppression state which can prevent the final activation step. These results point out to the importance of avoiding the hyperimmunization of mice for the preparation of a high number of monoclonal antibodies by standard hybridoma technology. INTRODUCTION
technology of somatic cell fusion now routinely used in the preparation of antibody (mAb) (1) requires many steps for the successful production of antibodysecreting hybridomas. One critical step is the obtainment by in vivo immunization of lymphoid organs containing antigen-specific B lymphocytes at the appropriate maturation stage. Although the exact differentiation stage at which B cells fuse efficiently with the myeloma partner is not yet identified (2,3,4), Andersson and Melchers (5) showed that only the large activated B-cells could fuse efficiently to yield antibody-secreting hybridomas. Previous studies have shown that the maximal number of splenic B lymphocytes suitable for cell fusion occurred three or four days after the last antigen injection (6,7,8,9). A generally accepted procedure is to immunize a group of mice until the obtainment of a high titre of specific antibody and uses in the fusion experiments the spleen cells obtained from the individual mouse showing the highest antigen-specific serum antibody concentration (7,9,10). In recent years we have often observed that fusion experiments done with spleen cells obtained from mice immunized with human red blood cells did not yield a high number of antibody-secreting hybridomas despite the presence of a high level of specific serum antibodies. One parameter that has not been extensively studied concerns the potential induction of a suppressive and/or tolerance state in the repeatedly immunized animal that would prevent the activation of the B The monoclonal
639
We have therefore performed a series of experiments study this phenomenon and to determine the relationship between the number of antigen injections, the resulting serum antibody titre and the efficiency of monoclonal antibody
lymphocytes after the last antigen boost.
to further
preparation.
MATERIALS AND METHODS Mice and immunization
Freshly collected human red blood cells were obtained from our blood bank, washed three times and stored at 4°C in Alsever-adenine solution (11). Female Balb/c mice (5 to 7 weeks old) were obtained from Charles River Co. (St-Constant, Qué.) and were divided in four groups at the start of each experiment. Mice in each group were immunized by injections of 200 ul of human red cells (5% v/v) (corresponding to 108 red cells) following the schedule summarized in Table 1. The animals were rested for two weeks between antigen injections and three weeks between the last antigen injection and the boost injection which was given three days before cell fusion. The immunization protocol allowed us to perform all the cell fusions on the same day. Mice care was in accordance with the statements of the Canadian Council on Animal Care. Preparation
of
hybridomas
Spleens from immunized mice were aseptically removed and a cell suspension was prepared from pooled or individual spleens. Viable cells were counted using an hemacytometer in presence of trypan blue. SP2/0-Ag14 myeloma cells and splenocytes were fused at a 1:5 ratio in presence of PEG-4000 (Merck, Darmstadt, West-Germany) as previously described (7). The fused cells were suspended at a final concentration of 1 X 106 cells/mL in Iscove's medium (Sigma,St-Louis,MO) containing 20% Fetal Bovine Serum (ICN Flow, Costa Mesa.CA), antibiotics (50 U/mL penicillin and 50 ug/mL streptomycin) (Sigma), 100 \iM hypoxanthine, 0.4 uM aminopterin and 16 uM thymidine (Sigma). The medium was supplemented with P388D1 cell-derived hybridoma growth factor (containing interleukin-6 at 15 U/mL) (12). The cells were distributed in 96-well microplates ( 2 X 105 cells per well) and cultured for 12-14 days at 37°C in a humid atmosphere containing 10% CO2. Serological techniques Seven days after each antigen injection, samples of blood were obtained by cutting a small section of the mouse tail. Samples of 200 uL were collected and allowed to clot at room temperature for 30 min and maintained at 4°C overnight. Serum was separated by centrifugation and diluted (1:5) in 150 mM NaCI containing 3% Bovine serum albumin (Canadian Red Cross, Ottawa) and 0.1% NaN3 (Sigma). Titres of hemagglutinating antibodies were determined by mixing 25 uL of serial dilutions prepared in 3% BSA-150 mM NaCI and 25 uL of a 2% (v/v) suspension of human red blood cells treated with ficin in order to detect both IgM and IgG antibodies (13). Briefly packed red blood cells were incubated for 15 minutes at 37°C with 3 volumes of 0.1% ficin (Sigma) prepared in PBS. The red cells were washed 3 times with 150 mM NaCI and stored at 4°C in Alsever-adenine solution. The degree of agglutination was determined visually and the titre defined as the reciprocal of the last dilution giving a macroscopic agglutination. For the screening of the hybridoma cultures, 25 uL of culture supernatant and of red blood cell suspension (2% v/v prepared as described above) were mixed in 96-well U-shaped microplates and centrifuged at 500 g (Beckman TJ-6 centrifuge) for 30s. The red blood cell pellets were suspended with a microplate shaker (ICN Flow) and the degree of agglutination determined visually.
RESULTS The effect of the number of antigen injections on the serum antibody titre and on the yield of mAbs was determined in a series of four experiments (A,B,C and D) each one done using the immunization protocol outlined in Table 1. The results are listed in Table 2. When groups of mice were used (experiments A and B), the spleen cells were pooled before the fusion experiment. The results first showed, as expected, that repeated antigen injections led 640
TABLE 1
IMMUNIZATION PROTOCOL Schedule (days) Number of
antigen injections
antigen injection
0
serum
boost
sampling
injection
Cell Fusion
-14
-3
0
1
-24
-14
-3
0
2
-38,-24
-14
-3
0
3
-52,-38,-24
-14
-3
0
to a continuous increase in the serum anti-red blood cell titre, This increase was observed in the four experiments (A,B,C,D).
The effect of the number of antigen injections on the efficiency of the cell fusion was determined by counting the number of growth and antibody-positive wells two weeks after the cell fusion. We did not observe significant fluctuation in the number of growth positive wells within each experiment, indicating that the fusion efficiency did not vary much.
technique
-
TABLE 2 EFFECT OF THE NUMBER OF ANTIGEN INJECTIONS ON SERUM ANTIBODY TITRE AND YIELD OF MONOCLONAL ANTIBODIES CELL FUSION
Experiment
Number of Number of mice per antigen injection group
0 1 2 3
Serum anti-RBC Titer (range)
(1/dilution)
Number of wells seeded
Number of wells
positive for growth (%)
474
(82) (98) (84) (99)
8 94 34 34
(1.6) (20.2) (7.1) (7.1)
864 576 672 672
661 456 487 431
(77) (79) (72) (64)
0 15 21 4
(0) (2.6) (3.1)
480 864 1056 480
122 467 579 206
(25) (54) (55) (43)
0 39 39
(0)
384 576
208 293 234 462
(54) (51)
0 11 20 10
Negative Effect of Multiple Antigen Injections on
the Yield of Murine Monoclonal Antibodies Obtained by
Hybridoma Technology SONIA NÉRON and REAL LEMIEUX
C.P. 1033 Terminus, Québec, Québec, GIK 7B5, Canada Science Faculty, Laval University, Québec, Québec, GIK 7P4, Canada
Canadian Red Cross Society, Blood
Department of Biochemistry,
Transfusion Service,
ABSTRACT One of the critical steps in the preparation of monoclonal antibodies is the obtainment, by in vivo immunization and boost, of the maximal number of antigen-activated B lymphocytes. In hybridoma laboratories, the common procedure is to immunize a group of mice with several antigen injections and use the mouse showing the highest serum antibody titre for the fusion experiment. The observation that the use of mice hyperimmunized with human red blood cells failed to yield a high number of monoclonal antibodies, led us to study the effect of multiple antigen injection prior to the fusion experiment. The results obtained showed that the maximal yields of monoclonal antibodies were obtained using mice that had received only one or two antigen injections while the mice immunized with three antigen injections consistently yielded at least a three-fold reduction in the number of monoclonal antibodies. The negative effect could not be reversed by prolonged resting of the animals and suggests the induction of a tolerance/suppression state which can prevent the final activation step. These results point out to the importance of avoiding the hyperimmunization of mice for the preparation of a high number of monoclonal antibodies by standard hybridoma technology. INTRODUCTION
technology of somatic cell fusion now routinely used in the preparation of antibody (mAb) (1) requires many steps for the successful production of antibodysecreting hybridomas. One critical step is the obtainment by in vivo immunization of lymphoid organs containing antigen-specific B lymphocytes at the appropriate maturation stage. Although the exact differentiation stage at which B cells fuse efficiently with the myeloma partner is not yet identified (2,3,4), Andersson and Melchers (5) showed that only the large activated B-cells could fuse efficiently to yield antibody-secreting hybridomas. Previous studies have shown that the maximal number of splenic B lymphocytes suitable for cell fusion occurred three or four days after the last antigen injection (6,7,8,9). A generally accepted procedure is to immunize a group of mice until the obtainment of a high titre of specific antibody and uses in the fusion experiments the spleen cells obtained from the individual mouse showing the highest antigen-specific serum antibody concentration (7,9,10). In recent years we have often observed that fusion experiments done with spleen cells obtained from mice immunized with human red blood cells did not yield a high number of antibody-secreting hybridomas despite the presence of a high level of specific serum antibodies. One parameter that has not been extensively studied concerns the potential induction of a suppressive and/or tolerance state in the repeatedly immunized animal that would prevent the activation of the B The monoclonal
639
We have therefore performed a series of experiments study this phenomenon and to determine the relationship between the number of antigen injections, the resulting serum antibody titre and the efficiency of monoclonal antibody
lymphocytes after the last antigen boost.
to further
preparation.
MATERIALS AND METHODS Mice and immunization
Freshly collected human red blood cells were obtained from our blood bank, washed three times and stored at 4°C in Alsever-adenine solution (11). Female Balb/c mice (5 to 7 weeks old) were obtained from Charles River Co. (St-Constant, Qué.) and were divided in four groups at the start of each experiment. Mice in each group were immunized by injections of 200 ul of human red cells (5% v/v) (corresponding to 108 red cells) following the schedule summarized in Table 1. The animals were rested for two weeks between antigen injections and three weeks between the last antigen injection and the boost injection which was given three days before cell fusion. The immunization protocol allowed us to perform all the cell fusions on the same day. Mice care was in accordance with the statements of the Canadian Council on Animal Care. Preparation
of
hybridomas
Spleens from immunized mice were aseptically removed and a cell suspension was prepared from pooled or individual spleens. Viable cells were counted using an hemacytometer in presence of trypan blue. SP2/0-Ag14 myeloma cells and splenocytes were fused at a 1:5 ratio in presence of PEG-4000 (Merck, Darmstadt, West-Germany) as previously described (7). The fused cells were suspended at a final concentration of 1 X 106 cells/mL in Iscove's medium (Sigma,St-Louis,MO) containing 20% Fetal Bovine Serum (ICN Flow, Costa Mesa.CA), antibiotics (50 U/mL penicillin and 50 ug/mL streptomycin) (Sigma), 100 \iM hypoxanthine, 0.4 uM aminopterin and 16 uM thymidine (Sigma). The medium was supplemented with P388D1 cell-derived hybridoma growth factor (containing interleukin-6 at 15 U/mL) (12). The cells were distributed in 96-well microplates ( 2 X 105 cells per well) and cultured for 12-14 days at 37°C in a humid atmosphere containing 10% CO2. Serological techniques Seven days after each antigen injection, samples of blood were obtained by cutting a small section of the mouse tail. Samples of 200 uL were collected and allowed to clot at room temperature for 30 min and maintained at 4°C overnight. Serum was separated by centrifugation and diluted (1:5) in 150 mM NaCI containing 3% Bovine serum albumin (Canadian Red Cross, Ottawa) and 0.1% NaN3 (Sigma). Titres of hemagglutinating antibodies were determined by mixing 25 uL of serial dilutions prepared in 3% BSA-150 mM NaCI and 25 uL of a 2% (v/v) suspension of human red blood cells treated with ficin in order to detect both IgM and IgG antibodies (13). Briefly packed red blood cells were incubated for 15 minutes at 37°C with 3 volumes of 0.1% ficin (Sigma) prepared in PBS. The red cells were washed 3 times with 150 mM NaCI and stored at 4°C in Alsever-adenine solution. The degree of agglutination was determined visually and the titre defined as the reciprocal of the last dilution giving a macroscopic agglutination. For the screening of the hybridoma cultures, 25 uL of culture supernatant and of red blood cell suspension (2% v/v prepared as described above) were mixed in 96-well U-shaped microplates and centrifuged at 500 g (Beckman TJ-6 centrifuge) for 30s. The red blood cell pellets were suspended with a microplate shaker (ICN Flow) and the degree of agglutination determined visually.
RESULTS The effect of the number of antigen injections on the serum antibody titre and on the yield of mAbs was determined in a series of four experiments (A,B,C and D) each one done using the immunization protocol outlined in Table 1. The results are listed in Table 2. When groups of mice were used (experiments A and B), the spleen cells were pooled before the fusion experiment. The results first showed, as expected, that repeated antigen injections led 640
TABLE 1
IMMUNIZATION PROTOCOL Schedule (days) Number of
antigen injections
antigen injection
0
serum
boost
sampling
injection
Cell Fusion
-14
-3
0
1
-24
-14
-3
0
2
-38,-24
-14
-3
0
3
-52,-38,-24
-14
-3
0
to a continuous increase in the serum anti-red blood cell titre, This increase was observed in the four experiments (A,B,C,D).
The effect of the number of antigen injections on the efficiency of the cell fusion was determined by counting the number of growth and antibody-positive wells two weeks after the cell fusion. We did not observe significant fluctuation in the number of growth positive wells within each experiment, indicating that the fusion efficiency did not vary much.
technique
-
TABLE 2 EFFECT OF THE NUMBER OF ANTIGEN INJECTIONS ON SERUM ANTIBODY TITRE AND YIELD OF MONOCLONAL ANTIBODIES CELL FUSION
Experiment
Number of Number of mice per antigen injection group
0 1 2 3
Serum anti-RBC Titer (range)
(1/dilution)
Number of wells seeded
Number of wells
positive for growth (%)
474
(82) (98) (84) (99)
8 94 34 34
(1.6) (20.2) (7.1) (7.1)
864 576 672 672
661 456 487 431
(77) (79) (72) (64)
0 15 21 4
(0) (2.6) (3.1)
480 864 1056 480
122 467 579 206
(25) (54) (55) (43)
0 39 39
(0)
384 576
208 293 234 462
(54) (51)
0 11 20 10
