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Enhancement of rosette formation between sheep erythrocytes and bovine T-lymphocytes by 2-aminoethylisothiouronium bromide and dextran
Immunology Letters, 1 (1979) 93-96
© Elsevier/North-HollandBiomedicalPress
ENHANCEMENT OF ROSETTE FORMATION BETWEEN SHEEP ERYTHROCYTES AND B O V I N E T - L Y M P H O C Y T E S BY 2 - A M I N O E T H Y L I S O T H I O U R O N I U M B R O M I D E AND DEXTRAN P. S. PAUL, T. T. BROWN, Jr., J. M. MILLER and M. J. Van Der MAATEN National Animal Disease Center, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Ames, IA 50010, USA
(Accepted 28 May 1979)
I. Summary A method for the detection of bovine T-lymphocytes involves rosette formation between lymphocytes and sheep erythrocytes treated with both 2-aminoethylisothiouronium bromide (AET) and dextran. The method detects approximately 70% of peripheral blood leukocytes as rosette-forming cells. Evidence that the rosette-forming cells are T-lymphocytes is also presented.
2. Introduction In man and animals, lymphocytes can be classified as thymus-derived (T) and bone marrow-derived (B) and can be distinguished from each other by surface membrane markers. Thymus-derived lymphocytes can be identified by the rosette formation with heterologous erythrocytes [1], whereas, B-lymphocytes lack this property but are identified by presence of surface membrane immunoglobulins (slg) and receptors for the third component of complement [2]. In cattle, the methods for B-lymphocyte identification have been used extensively and successfully [3-5]; however, a rosette assay for the detection of T-lymphocytes has not been satisfactorily developed. Grewal et al. [6] reported that untreated sheep erythrocytes (SRBC) form rosettes with a very small percentage (4-6%) of bovine peripheral blood leukocytes (PBL). Dextran treatment of SRBC enhances rosette formation in bovine PBL but the
extent of enhancement is variable [7-9]. Neuraminidase treatment of SRBC also enhances rosette formation resulting in the detection of approximately 38% of bovine PBL as rosette forming cells (RFC) [6]. More recemly Paul et al. found that 2-aminoethylisothiouronium bromide (AET) enhances rosette formarion with approximately 50-60% of bovine PBL and 86% fetal thymocytes forming rosettes using this technique [9]. In the present study, the effect of treating SRBC with either AET or dextran alone or both on the rosette formation by bovine PBL was investigated. Our data indicates that a combined treatment of SRBC with AET and dextran detects an additional 20% RFC which are not detected by the AET or dextran treatment alone.
3. Materials and methods Heparinized peripheral blood was obtained from normal adult cattle and the mononuclear cells were separated on Ficoll-Hypaque gradients [ 10]. The isolated cells were washed with Hanks' balanced salt solution (HBSS) 3 times and resuspended in RPMI1640 culture medium supplemented with 25 mM Hepes buffer and 10% fetal calf serum, hereafter referred to as cell maintenance medium (CMM), at a concentration of 107 cells/ml. The mononuclear cells were comprised of approximately 80% lymphocytes and 20% monocytes as judged by latex phagocytosis and were referred to as PBL. Dextrans 10, 40, 80, and 500 with molecular 93
weights of 10,000, 40,000, 80,000, and 500,000, respectively, were obtained from Pharmacia, Uppsala, Sweden and 6% solutions (w/v) were prepared in physiological saline. Dextran 150 of 150,000 molecular weight was obtained from Pharmacia, Uppsala, Sweden, and from Fisons, England, and 2 , 4 , 6, 8 and 10% solutions (w/v) o f dextran 150 were prepared in saline. Sheep erythrocytes preserved in Alsever's solution were washed 3 times with phosphate buffered saline, pH 7.2, and a portion of cells were treated with AET as previously described [9]. Four vols. of freshly prepared 0.1 M AET solution, pH 9.0, were added to 1 vol. of packed SRBC and incubated at 37°C for 20 min. After incubation, the cells were washed 3 times with cold (4°C) physiological saline followed by 2 washes with fetal calf serum-containing medium. Then untreated and AET-treated SRBC were resuspended in CMM at 4% cell concentration, stored at 4°C and used within 5 days. Before use, 1 ml o f 4% SRBC stock was washed with 10 ml o f CMM and resuspended in CMM or dextran to 1% cell concentration. The rosette assay was performed as described previously [9]. One-hundred ~tl o f PBL resuspended in CMM medium at a concentration o f 107 cells/ml were mixed with 200/al of 1% SRBC. After incubation for I0 min in a 37°C water bath, the cells were centrifuged at 200 g for 5 min and incubated overnight at 4°C. The cells were gently resuspended and the percentage of RFC with 3 or more adherent SRBC was determined by counting a minimum of 200 live cells. B-lymphocytes were identified by staining for the presence o f slg and monocytes were identified by latex ingestion [9]. For simultaneous detection of monocytes, B-cells and RFC, PBL were first incubated with latex particles, stained for the presence o f slg, washed 3 times with HBSS and then processed in the rosette assay.
4. Results
4.1. Effect of molecular weight and concentration of
dextran on rosette formation by bovine lymphocytes Untreated sheep erythrocytes formed a small 94
Table 1 Enhancement of rosette formation by bovine peripheral blood leukocytes with sheep erythrocytes using increased molecular weight dextrans Average molecular weight of dextran in rosetting medium
% RFC* (mean 5 S.D.)
-
6.4 16.6 ± 4.9 29.0 5 8.5 26.4 ± 7.6 48.0 5 8.3 49.0 5 10.8 23.0 5 7.1 10.5 ±
10,000 40,000 80,000 150,000"* 150,000"** 500,000
* Percentage estimated from the mean value for 3 animals after counting a minimum of 200 cells from each sample. ** Pharmacla, Uppsala, Sweden. * ** Fison, England.
percentage o f rosettes with peripheral blood leukocytes from 3 normal cattle (table 1). Dextrans of molecular weights between 10,000 and 500,000 varied in their ability to enhance rosette formation (table 1). Dextran 150 with a molecular weight o f 150,000 gave the maximum enhancement of rosette formation (table 1). The concentration of dextran also affected rosette formation (table 2). Sheep erythrocytes resuspended in a 6% solution of dextran 150 formed the highest percentage of rosettes with bovine lymphocytes (table 2) and were used routinely in all tests with dextran. Table 2 Effect of dextran-150 concentration on rosette formation by bovine lymphocytes with sheep erythrocytes % Dextran
% RFC* (mean ± S.D.)
0 2 4 6 8 10
9.0±1.0 22.0±2.4 32.0±4.0 51.0±9.0 39.351.2 38.550.5
* Percentage estimated from the mean value for 3 normal animals after counting a minimum of 200 live cells from each sample.
4.2.Enhancement of rosette formation by bovine lymphocytes with dextran and AET treatment of SRBC As shown in table 3, untreated SRBC formed a small percentage o f rosettes with PBL from 12 normal cattle. Separately, dextran and AET treatment o f SRBC enhanced rosette formation significantly. The difference between AET and dextran treatment of SRBC was not statistically significant (P > 0.05). Combined treatment Of SRBC with AET and dextran detected a significantly higher percentage o f RFC in bovine blood than by either treatment alone (P < 0.01).
4.3. Characterization of the rosette-forming cell population Peripheral blood leukocytes from 5 cattle were examined simultaneously for the presence o f sIg, latex ingestion and rosette formation. Cells with 1 or more markers were identified. As shown in table 4, none o f the RFC were positive for sIg or had ingested latex particles, which indicates that SRBC treated with AET and dextran do not form rosettes with B-lymphocytes or monocytes but, presumably with Tolymphocytes.
5. D i s c u s s i o n
Enhancement o f rosette formation between bovine l y m p h o c y t e s and SRBC by the addition o f dextran [ 7 - 9 , 1 1 ] is well documented. Considerable disagreem e n t exists, however, on the percentage o f T-lymphocytes detected by these techniques. Wardley [7] reported that 70% o f bovine PBL form rosettes with SRBC in the presence o f dextran. Other investigators obtained much lower percentages o f 21 [11], 31 [9] and 37 [8] o f RFC in bovine blood with dextrantreated SRBC. The possible reason for these discrepancies is that dextrans o f different molecular weights, which may vary in their ability to enhance rosette formation, were used. In the present report, we have presented evidence that the enhancement o f rosette formation by dextran is indeed dependent upon its molecular weight and concentration. Dextran o f 150,000 molecular weight at 6% concentration was found to give maximum enhancement o f rosette formation detecting 50% RFC in bovine PBL. The high percentage (70%) o f RFC observed by Wardley could not be confirmed in our laboratory. Recently, Pltul et al. [9] described a modified rosette
Table 3 Enhanced rosette formation between bovine peripheral blood leukocytes and AET-treated sheep erythrocytes in the presence of dextran Animal no.
% RFC with Untreated SRBC
AET-treated SRBC
in CMM
in dextran
in CMM
in dextran
50 52 36 50 43 58 49 54 57 69 47 52
85 70 61 74 73 72 76 70 75 80 64 69
51.4±8.14 (36-69)
72.5±6.5 (61-85)
8
6
9 10
15 21
11
12
12
11
38 63 35 55 33 42 60 33 56 60 37 53
Mean ± S.D. Range
8.5±5.8 (0-21)
47.0±11.7 (33-63)
1
5
2 3 4 5 6 7
5 0 7 2 10 8
RFC = rosette forming cell; SRBC = sheep erythrocytes; CMM = cell maintenance medium consisting of RPMI 1640 cultures medium and 10% fetal calf serum. 95
Table 4 Simultaneous detection of slg-hearing and rosette-forming cells in bovine peripheral blood leukocytes Animal no.
Percentage of cells RFC+ slg-
RFC÷ slg+
RFCslg+
RFCslg-
1 2 3 4 5
73.7* 72.6 84.6 70.4 61.0
0 0 0 0 0
10.1 15.0 4.8 16.7 17.0
16.2 12.4 10.6 12.9 22.0
Mean _+S.D.
72.46 +-8.43
0
12.72 +-5.22
14.82 -+4.49
* None of the rosette-forming cells ingested latex particles. RFC = rosette-forming cell with 3 or more adherent SRBC; RFC+ = positive for rosette formation; RFC- = negative for rosette formation; slg- = surface membrane immunoglobulinnegative; slg÷= surface membrane immunoglobulinpositive. assay using AET-treated SRBC. Approximately 5 0 - 6 0 % of PBL formed rosettes with this method. In the present study we have confirmed the enhancing effect ofAET-treatment of SRBC on rosette formation with bovine lymphocytes. Additionally, we have shown that SRBC treated with both 0.1 M AET and 6% dextran of 150,000 molecular weight provides an improved rosette assay allowing the detection of approximately 70% PBL as RFC. The procedure described here is similar to that reported by Grewal and Babiuk [ 11 ] which utilizes SRBC treated with 0.143 M AET and suspended in 6% dextran of 264,000 molecular weight for the rosette assay. However, our procedure is slightly more sensitive forming rosettes with 70% while that of Grewal and Babiuk [ 11 ] forms rosettes with approximately 60% of PBL. Although the exact mechanism of rosette enhancement is u n k n o w n , our data show that AET and dextran may act through different mechanisms, and detect in part a different subpopulation of T-lymphocytes, since the combined treatment of SRBC with both AET and dextran increases the percentage of RFC detected over single treatment with AET or dextran alone. Thus, the routine use of SRBC treated with AET, dextran and a combination of A E T dextran for rosette assays may reveal abnormalities in specific subpopulations of T-lymphocytes which would go undetected if only AET-dextran-treated SRBC are used. 96
Acknowledgements The authors wish to thank Dr. Gordon Booth for statistical analysis of the data and Ms. Joanne Stevenson for her technical assistance.
References [ 1] Jondal, M., Holm, G. and WigzeU,H. (1972) J. Exp. Med. 136,207-215. [2] Froland, S. S. and Natvig, J. B. (1973)Transplant Rev. 16, 114-162. [3] Muscoplat, C. C., Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L., Stevens, J. B. and Sorensen, D. K. (1974) Am. J. Vet. Res. 35,593-595. [4] Higgins, D. A. and Stack, M. J. (1978) J. Immunol. Methods 20, 211-224. [5] Kumar, S. P., Paul, P. S., Pomeroy, K. A., Johnson, D. W., Muscoplat, C. C., Van Der Maaten, M. J. and Miller, J. M. (1978) Am. J. Vet. Res. 39, 45-49. [6] Grewal, A. S., Rouse, B. T. and Babiuk, L. A. (1976) Can. J. Comp. Med. 40, 298-305. [7] Wardley, R. (1977) Br. Vet. J. 133,432-434. [8] Binns, R. M. (1978) J. lmmunol. Methods 21,197-210. [9] Paul, P. S., Senogles, D. R., Muscoplat, C. C. and Johnson, D. W. (1979) Clin. Exp. Immunol. 35,306-316. [10] Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77-89. [11] Grewal, A. S. and Babiuk, L. A. (1978) J. Immunol. Methods 24, 355-361.
© Elsevier/North-HollandBiomedicalPress
ENHANCEMENT OF ROSETTE FORMATION BETWEEN SHEEP ERYTHROCYTES AND B O V I N E T - L Y M P H O C Y T E S BY 2 - A M I N O E T H Y L I S O T H I O U R O N I U M B R O M I D E AND DEXTRAN P. S. PAUL, T. T. BROWN, Jr., J. M. MILLER and M. J. Van Der MAATEN National Animal Disease Center, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Ames, IA 50010, USA
(Accepted 28 May 1979)
I. Summary A method for the detection of bovine T-lymphocytes involves rosette formation between lymphocytes and sheep erythrocytes treated with both 2-aminoethylisothiouronium bromide (AET) and dextran. The method detects approximately 70% of peripheral blood leukocytes as rosette-forming cells. Evidence that the rosette-forming cells are T-lymphocytes is also presented.
2. Introduction In man and animals, lymphocytes can be classified as thymus-derived (T) and bone marrow-derived (B) and can be distinguished from each other by surface membrane markers. Thymus-derived lymphocytes can be identified by the rosette formation with heterologous erythrocytes [1], whereas, B-lymphocytes lack this property but are identified by presence of surface membrane immunoglobulins (slg) and receptors for the third component of complement [2]. In cattle, the methods for B-lymphocyte identification have been used extensively and successfully [3-5]; however, a rosette assay for the detection of T-lymphocytes has not been satisfactorily developed. Grewal et al. [6] reported that untreated sheep erythrocytes (SRBC) form rosettes with a very small percentage (4-6%) of bovine peripheral blood leukocytes (PBL). Dextran treatment of SRBC enhances rosette formation in bovine PBL but the
extent of enhancement is variable [7-9]. Neuraminidase treatment of SRBC also enhances rosette formation resulting in the detection of approximately 38% of bovine PBL as rosette forming cells (RFC) [6]. More recemly Paul et al. found that 2-aminoethylisothiouronium bromide (AET) enhances rosette formarion with approximately 50-60% of bovine PBL and 86% fetal thymocytes forming rosettes using this technique [9]. In the present study, the effect of treating SRBC with either AET or dextran alone or both on the rosette formation by bovine PBL was investigated. Our data indicates that a combined treatment of SRBC with AET and dextran detects an additional 20% RFC which are not detected by the AET or dextran treatment alone.
3. Materials and methods Heparinized peripheral blood was obtained from normal adult cattle and the mononuclear cells were separated on Ficoll-Hypaque gradients [ 10]. The isolated cells were washed with Hanks' balanced salt solution (HBSS) 3 times and resuspended in RPMI1640 culture medium supplemented with 25 mM Hepes buffer and 10% fetal calf serum, hereafter referred to as cell maintenance medium (CMM), at a concentration of 107 cells/ml. The mononuclear cells were comprised of approximately 80% lymphocytes and 20% monocytes as judged by latex phagocytosis and were referred to as PBL. Dextrans 10, 40, 80, and 500 with molecular 93
weights of 10,000, 40,000, 80,000, and 500,000, respectively, were obtained from Pharmacia, Uppsala, Sweden and 6% solutions (w/v) were prepared in physiological saline. Dextran 150 of 150,000 molecular weight was obtained from Pharmacia, Uppsala, Sweden, and from Fisons, England, and 2 , 4 , 6, 8 and 10% solutions (w/v) o f dextran 150 were prepared in saline. Sheep erythrocytes preserved in Alsever's solution were washed 3 times with phosphate buffered saline, pH 7.2, and a portion of cells were treated with AET as previously described [9]. Four vols. of freshly prepared 0.1 M AET solution, pH 9.0, were added to 1 vol. of packed SRBC and incubated at 37°C for 20 min. After incubation, the cells were washed 3 times with cold (4°C) physiological saline followed by 2 washes with fetal calf serum-containing medium. Then untreated and AET-treated SRBC were resuspended in CMM at 4% cell concentration, stored at 4°C and used within 5 days. Before use, 1 ml o f 4% SRBC stock was washed with 10 ml o f CMM and resuspended in CMM or dextran to 1% cell concentration. The rosette assay was performed as described previously [9]. One-hundred ~tl o f PBL resuspended in CMM medium at a concentration o f 107 cells/ml were mixed with 200/al of 1% SRBC. After incubation for I0 min in a 37°C water bath, the cells were centrifuged at 200 g for 5 min and incubated overnight at 4°C. The cells were gently resuspended and the percentage of RFC with 3 or more adherent SRBC was determined by counting a minimum of 200 live cells. B-lymphocytes were identified by staining for the presence o f slg and monocytes were identified by latex ingestion [9]. For simultaneous detection of monocytes, B-cells and RFC, PBL were first incubated with latex particles, stained for the presence o f slg, washed 3 times with HBSS and then processed in the rosette assay.
4. Results
4.1. Effect of molecular weight and concentration of
dextran on rosette formation by bovine lymphocytes Untreated sheep erythrocytes formed a small 94
Table 1 Enhancement of rosette formation by bovine peripheral blood leukocytes with sheep erythrocytes using increased molecular weight dextrans Average molecular weight of dextran in rosetting medium
% RFC* (mean 5 S.D.)
-
6.4 16.6 ± 4.9 29.0 5 8.5 26.4 ± 7.6 48.0 5 8.3 49.0 5 10.8 23.0 5 7.1 10.5 ±
10,000 40,000 80,000 150,000"* 150,000"** 500,000
* Percentage estimated from the mean value for 3 animals after counting a minimum of 200 cells from each sample. ** Pharmacla, Uppsala, Sweden. * ** Fison, England.
percentage o f rosettes with peripheral blood leukocytes from 3 normal cattle (table 1). Dextrans of molecular weights between 10,000 and 500,000 varied in their ability to enhance rosette formation (table 1). Dextran 150 with a molecular weight o f 150,000 gave the maximum enhancement of rosette formation (table 1). The concentration of dextran also affected rosette formation (table 2). Sheep erythrocytes resuspended in a 6% solution of dextran 150 formed the highest percentage of rosettes with bovine lymphocytes (table 2) and were used routinely in all tests with dextran. Table 2 Effect of dextran-150 concentration on rosette formation by bovine lymphocytes with sheep erythrocytes % Dextran
% RFC* (mean ± S.D.)
0 2 4 6 8 10
9.0±1.0 22.0±2.4 32.0±4.0 51.0±9.0 39.351.2 38.550.5
* Percentage estimated from the mean value for 3 normal animals after counting a minimum of 200 live cells from each sample.
4.2.Enhancement of rosette formation by bovine lymphocytes with dextran and AET treatment of SRBC As shown in table 3, untreated SRBC formed a small percentage o f rosettes with PBL from 12 normal cattle. Separately, dextran and AET treatment o f SRBC enhanced rosette formation significantly. The difference between AET and dextran treatment of SRBC was not statistically significant (P > 0.05). Combined treatment Of SRBC with AET and dextran detected a significantly higher percentage o f RFC in bovine blood than by either treatment alone (P < 0.01).
4.3. Characterization of the rosette-forming cell population Peripheral blood leukocytes from 5 cattle were examined simultaneously for the presence o f sIg, latex ingestion and rosette formation. Cells with 1 or more markers were identified. As shown in table 4, none o f the RFC were positive for sIg or had ingested latex particles, which indicates that SRBC treated with AET and dextran do not form rosettes with B-lymphocytes or monocytes but, presumably with Tolymphocytes.
5. D i s c u s s i o n
Enhancement o f rosette formation between bovine l y m p h o c y t e s and SRBC by the addition o f dextran [ 7 - 9 , 1 1 ] is well documented. Considerable disagreem e n t exists, however, on the percentage o f T-lymphocytes detected by these techniques. Wardley [7] reported that 70% o f bovine PBL form rosettes with SRBC in the presence o f dextran. Other investigators obtained much lower percentages o f 21 [11], 31 [9] and 37 [8] o f RFC in bovine blood with dextrantreated SRBC. The possible reason for these discrepancies is that dextrans o f different molecular weights, which may vary in their ability to enhance rosette formation, were used. In the present report, we have presented evidence that the enhancement o f rosette formation by dextran is indeed dependent upon its molecular weight and concentration. Dextran o f 150,000 molecular weight at 6% concentration was found to give maximum enhancement o f rosette formation detecting 50% RFC in bovine PBL. The high percentage (70%) o f RFC observed by Wardley could not be confirmed in our laboratory. Recently, Pltul et al. [9] described a modified rosette
Table 3 Enhanced rosette formation between bovine peripheral blood leukocytes and AET-treated sheep erythrocytes in the presence of dextran Animal no.
% RFC with Untreated SRBC
AET-treated SRBC
in CMM
in dextran
in CMM
in dextran
50 52 36 50 43 58 49 54 57 69 47 52
85 70 61 74 73 72 76 70 75 80 64 69
51.4±8.14 (36-69)
72.5±6.5 (61-85)
8
6
9 10
15 21
11
12
12
11
38 63 35 55 33 42 60 33 56 60 37 53
Mean ± S.D. Range
8.5±5.8 (0-21)
47.0±11.7 (33-63)
1
5
2 3 4 5 6 7
5 0 7 2 10 8
RFC = rosette forming cell; SRBC = sheep erythrocytes; CMM = cell maintenance medium consisting of RPMI 1640 cultures medium and 10% fetal calf serum. 95
Table 4 Simultaneous detection of slg-hearing and rosette-forming cells in bovine peripheral blood leukocytes Animal no.
Percentage of cells RFC+ slg-
RFC÷ slg+
RFCslg+
RFCslg-
1 2 3 4 5
73.7* 72.6 84.6 70.4 61.0
0 0 0 0 0
10.1 15.0 4.8 16.7 17.0
16.2 12.4 10.6 12.9 22.0
Mean _+S.D.
72.46 +-8.43
0
12.72 +-5.22
14.82 -+4.49
* None of the rosette-forming cells ingested latex particles. RFC = rosette-forming cell with 3 or more adherent SRBC; RFC+ = positive for rosette formation; RFC- = negative for rosette formation; slg- = surface membrane immunoglobulinnegative; slg÷= surface membrane immunoglobulinpositive. assay using AET-treated SRBC. Approximately 5 0 - 6 0 % of PBL formed rosettes with this method. In the present study we have confirmed the enhancing effect ofAET-treatment of SRBC on rosette formation with bovine lymphocytes. Additionally, we have shown that SRBC treated with both 0.1 M AET and 6% dextran of 150,000 molecular weight provides an improved rosette assay allowing the detection of approximately 70% PBL as RFC. The procedure described here is similar to that reported by Grewal and Babiuk [ 11 ] which utilizes SRBC treated with 0.143 M AET and suspended in 6% dextran of 264,000 molecular weight for the rosette assay. However, our procedure is slightly more sensitive forming rosettes with 70% while that of Grewal and Babiuk [ 11 ] forms rosettes with approximately 60% of PBL. Although the exact mechanism of rosette enhancement is u n k n o w n , our data show that AET and dextran may act through different mechanisms, and detect in part a different subpopulation of T-lymphocytes, since the combined treatment of SRBC with both AET and dextran increases the percentage of RFC detected over single treatment with AET or dextran alone. Thus, the routine use of SRBC treated with AET, dextran and a combination of A E T dextran for rosette assays may reveal abnormalities in specific subpopulations of T-lymphocytes which would go undetected if only AET-dextran-treated SRBC are used. 96
Acknowledgements The authors wish to thank Dr. Gordon Booth for statistical analysis of the data and Ms. Joanne Stevenson for her technical assistance.
References [ 1] Jondal, M., Holm, G. and WigzeU,H. (1972) J. Exp. Med. 136,207-215. [2] Froland, S. S. and Natvig, J. B. (1973)Transplant Rev. 16, 114-162. [3] Muscoplat, C. C., Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L., Stevens, J. B. and Sorensen, D. K. (1974) Am. J. Vet. Res. 35,593-595. [4] Higgins, D. A. and Stack, M. J. (1978) J. Immunol. Methods 20, 211-224. [5] Kumar, S. P., Paul, P. S., Pomeroy, K. A., Johnson, D. W., Muscoplat, C. C., Van Der Maaten, M. J. and Miller, J. M. (1978) Am. J. Vet. Res. 39, 45-49. [6] Grewal, A. S., Rouse, B. T. and Babiuk, L. A. (1976) Can. J. Comp. Med. 40, 298-305. [7] Wardley, R. (1977) Br. Vet. J. 133,432-434. [8] Binns, R. M. (1978) J. lmmunol. Methods 21,197-210. [9] Paul, P. S., Senogles, D. R., Muscoplat, C. C. and Johnson, D. W. (1979) Clin. Exp. Immunol. 35,306-316. [10] Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77-89. [11] Grewal, A. S. and Babiuk, L. A. (1978) J. Immunol. Methods 24, 355-361.