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Improved methodology for the production of monoclonal antibodies against parasites
Immunology Letters, 9 (1985) 225-227 Elsevier lmlet 564
IMPROVED
METHODOLOGY FOR THE PRODUCTION ANTIBODIES AGAINST PARASITES
OF MONOCLONAL
P. APPLEBY I, S. HAWKINS l, D. I. P R I T C H A R D 2, L. YEPEZ-MULIA I and D. CATTY l I Department of hnmunology, University of Birmingham, and 2Parasitology Research Group, Department of Zoology, University of Nottingham, Nottingham. U.K.
(Received 13 November1984) (Modified version receivedand accepted30 November1984)
1. Summary
A modification of the standard fusion methodology is described which results in greatly increased yields of monoclonal antibodies against certain organ-specific parasites. Several fusions were carried out using mice infected with Schistosoma mansoni or Nematospiroides dubius, using B lymphocytes harvested from either the spleen or the mesenteric lymph nodes. Results indicated a greatly improved yield of positive clones using the lymph nodes as a source of B cells for fusion. A 7-fold increase in the number of positive clones was seen with N. dubius injections, while S. mansoni fusions showed a 2-fold increase. 2. Introduction
Monoclonal antibodies are of great potential value in immunoparasitology; many research groups have produced antibodies to a wide range of parasites [1, 2]. The methodology employed for the production of these monoclonal antibodies has been based largely on the original technique described by Galfr6 et al. [3]. This method does not take into account the organ specificity of many parasites, which results in stimulation and enlargement of local lymph nodes. Such is the case in murine infections of both S. mansoni, a blood fluke which resides in the mesenteric vessels of the host, and N. dubius, a helminth parasite used as a model for human hook-
worm, which lives in the small intestine. Both of these infections cause gross enlargement of the draining mesenteric lymph nodes, an observation that suggests the presence of large numbers of parasitestimulated B lymphocytes. It is logical to assume that such lymphocytes would be ideal donor cells for fusions and hybridoma production, as they have been extensively primed, in vivo, by released parasite antigens. We have investigated this hypothesis by taking lymphocytes from both the spleen and mesenteric lymph nodes of parasite-infected mice, and fusing these with the NS1 Ag4 myeloma line, and then comparing the numbers of positive clones obtained from fusions with cells of both origins. Results for both parasites showed a greatly increased number of positive clones in fusions using the lymph nodes. It is envisaged that such a methodology could be applied with advantage to any gut-associated infection in the murine host. 3. Materials and Methods
3.1. S. mansoni infections Three-month-old female BALB/c mice were infected by inoculation of 200 S. mansoni cercariae i.p. The S. mansoni Puerto Rican strain from the Liverpool School of Tropical Medicine and Hygiene (kindly provided by Dr. I. Marshall) was used. The life cycle is maintained in our laboratory using Biomphaleria glabrata as the intermediate host. Six to
Key words: monoclonalantibodies - lymph nodes - parasites 0165-2478 / 85 / $ 3.30 © 1985 ElsevierScience Publishers B.V.(BiomedicalDivision)
225
nine weeks after infection, when the worms have migrated to their final position in the mesenteric veins, the mice were killed by cervical dislocation, and the spleen or lymph nodes removed aseptically as a source of B cells. 3.2. N. dubius infections Three-month-old BALB/c mice were infected with N. dubius following the primary divided infection schedule of Behnke and Parish [4]. Seven days after the final infection the mice were sacrificed and the spleen or mesenteric nodes removed aseptically. 3.3. Fusion methodology Fusions were carried out following the methodology described by Lowe et al. [5], using the NSI Ag4 murine myeloma line and 40% (w/v) PEG 1500 (B.D.H.) containing 5% (v/v) D.M.S.O. (BDH). The lymphocyte: myeloma ratio in all fusions was 5: 1. Care was taken to adopt an exactly similar procedure for all fusions. Supernates from growing clones were screened by solid phase radioassay, using an affinity-purified 1251labelled rabbit anti-mouse immunoglobulin as the developing antibody. Anti-schistosome fusions were screened for reactivity against whole adult (male and female) worm homogenate (WWH) and cercarial homogenate (CH), prepared by homogenization of washed parasites followed by centrifugation at 1200×g, the supernate being retained as a crude antigen preparation. Anti-N. dubius clones were assayed against day 6 larvae and adult worm homogenate antigens, prepared as described by Pritchard et al. [6]. All antigens were used at a concentration of 10 ~tg/ml protein for sensitizing Linbro PVC disposable microagglutination trays. All fusions were screened twice, usually on days 10 and 14, and clones were considered as positive if they gave a c.p.m, reading 3-fold greater than background in both assays. The number of antigen positive clones per fusion was thus obtained, allowing a comparison between the different B cell sources.
4. Results
4.1. S. mansoni fusions The number of clones positive for crude S. man226
Table 1 Results from fusions using S. mansoni infections Fusion B cell no. source
Age of infection (wk)
No. of clones
No. of Isotype positive - clonesa G M
22 24 26 33
8 8 12 9
13 26 26 48
3 (22%) 7 (27%) 6 (22%) 8 (16%)
0 NT NT 8
7~/2
36
9 (25%)
NT 4
9
24 h
9 (36%)
9
35 36
spleen spleen spleen mesenteric lymph node mesenteric lymph node mesenteric lymph node
3 2 3 0
0
a Of 48 wells tested, combined total of cercarial homogenate and whole worm homogenate positive clones. Screened days 10 and 14 post-fusion. b Of 24 wells tested.
soni antigens obtained from 6 fusions, 3 using splenic lymphocytes and 3 using mesenteric lymph node cells, are shown in Table 1. Included in this Table are details of period of infection, total number of clones and the isotypes of the monoclonals, where tested. As the same fusion technique was employed for all 6 fusions, and no clones were lost due to infection etc., the figures represent differences due solely to the source of B lymphocytes. It can be seen that mesenteric lymph node cells give rise to a greater number of growing clones, resulting in more schistosome-positive clones per fusion, even though the number of positive clones as a percentage of total clones is approximately the same, at about 25%, for both cell sources. It is of interest to note that the majority of clones resulting from splenic fusions were of the lgM isotype, while mesenteric lymph node fusions gave a mixture of IgGl and IgM isotypes. It is clear that this methodology produces almost twice as many positive clones per fusion compared with the conventional spleen fusion technique.
4.2. N. dubiusfusions The results obtained from fusions using N. dub# us-infected mice, are presented in Table 2. Adults of N. dubius, unlike the blood fluke S. mansoni, reside in the small intestine, and it was anticipated that the mesenteric lymph nodes of infected mice may not be as actively stimulated as those exposed to S. manso-
Table 2 Results from fusions using N. dubius infections Fusion
B cella source
No. of clones
No. of positive clonesb
28 29
spleen mesenteric lymph node
10 48
6 45
a Immunisation by primary divided schedule of Behnke and Parish [4]. b Of 48 wells tested, combined total obtained with day 6 larval antigen and adult homogenate.
ni infections. The fusion results, however, proved this not to be so, with 45 of the 48 clones produced showing reactivity against the test antigens, a 7-fold greater success rate than that obtained with splenic cell fusions.
5. Discussion
The concept of taking lymphocytes for fusions from local lymph nodes draining areas of infection, is clearly valid in the case of the two parasites investigated in this study. The release of parasite antigens results in large numbers of stimulated lymphocytes, which, from these results, appear to be ideal for fusion and subsequent monoclonal antibody production. In both examples, there were increases in the number of both specific and non-specific clones when compared with the splenic lymphocyte fusions, and this was especially so in the case of N. dubius, where most clones produced antibody directed against the parasite. With S. rnansoni fusions, there were a large number of non-specific clones produced, which may reflect non-specific stimulation by the parasite, in a manner analogous to that seen in trypanosome infections [7] or, conversely, suppression of the local parasite specific response by released soluble factors from the adult worms [8]. Our results, in terms of yield of positive clones, compare favourably with those of Zodda et al. [2] even when comparing yields from spleen cell fusions
only, although this may partially reflect their choice of an assay system that could only detect IgG monoclonal antibodies. In our hands, fusions using splenocytes seem to favour the production of IgM antibodies. Results obtained by Taylor and Butterworth [9] using splenic lymphocytes for fusion, were similar to those we obtained using mesenteric node cells, although this may be partially explained by the high numbers of cercariae used to boost the mice prior to fusion and by the lower stringency of the screening radioassay. The fact that we could obtain a similar number of positive clones from a single challenge low level infection by using lymph node cells clearly underlines the efficiency of this alternative approach. Although we have only produced monoclonal antibodies against two parasites by this technique it is obvious that it may be applied, with advantage, to any gut-associated parasites or micro-organisms that can infect mice as the final host, e.g. Trichuris, Hymenolepis, Nippostronglyus, Trichinella, Giardia [10].
References [I] Mitchell, G. F. (1981) Immunol. Today 2, 140-142. [2] Zodda, D. M., Abdel Hafez, S. K. and Phillips, S. M. (1983) Am. J. Trop. Med. Hyg. 32, 69-77. [3] Galfr6, G., Howe, S. C. and Milstein, C. (1977) Nature (London) 266, 550-552. [4] Behnke, J. M. and Parish, H. A. (1979) Parasite Immunol. 1,13. [5] Lowe, J., Hardie, D., Jefferis, R., Ling, N. R., Drysdale, P., Richardson, P., Raykundalia, C., Catty, D., Appleby, P., Drew, R. and MacLennan, 1. C. M. (1981) Immunology 42, 649-659. [6] Pritchard, D. 1., Maizels, R. M., Behnke, J. M. and Appleby, P. (1984) Immunology 53, 325-335. [7] Urquhart, G. M., Murray, M., Murray, P. K., Jennings, P. W. and Bate, E. (1973) Trans. R. Soc. Trop. Med. Hyg. 67, 528-535. [8] Ottesen, E. A. and Poindexter, R. W. (1980) Am. J. Trop. Med. Hyg. 29, 592-597. [9] Taylor, D. W. and Butterworth, A. E. (1982) Parasitology 84, 65. [10] Wakelin, D. (1978) Nature (London) 273,617 620.
227
IMPROVED
METHODOLOGY FOR THE PRODUCTION ANTIBODIES AGAINST PARASITES
OF MONOCLONAL
P. APPLEBY I, S. HAWKINS l, D. I. P R I T C H A R D 2, L. YEPEZ-MULIA I and D. CATTY l I Department of hnmunology, University of Birmingham, and 2Parasitology Research Group, Department of Zoology, University of Nottingham, Nottingham. U.K.
(Received 13 November1984) (Modified version receivedand accepted30 November1984)
1. Summary
A modification of the standard fusion methodology is described which results in greatly increased yields of monoclonal antibodies against certain organ-specific parasites. Several fusions were carried out using mice infected with Schistosoma mansoni or Nematospiroides dubius, using B lymphocytes harvested from either the spleen or the mesenteric lymph nodes. Results indicated a greatly improved yield of positive clones using the lymph nodes as a source of B cells for fusion. A 7-fold increase in the number of positive clones was seen with N. dubius injections, while S. mansoni fusions showed a 2-fold increase. 2. Introduction
Monoclonal antibodies are of great potential value in immunoparasitology; many research groups have produced antibodies to a wide range of parasites [1, 2]. The methodology employed for the production of these monoclonal antibodies has been based largely on the original technique described by Galfr6 et al. [3]. This method does not take into account the organ specificity of many parasites, which results in stimulation and enlargement of local lymph nodes. Such is the case in murine infections of both S. mansoni, a blood fluke which resides in the mesenteric vessels of the host, and N. dubius, a helminth parasite used as a model for human hook-
worm, which lives in the small intestine. Both of these infections cause gross enlargement of the draining mesenteric lymph nodes, an observation that suggests the presence of large numbers of parasitestimulated B lymphocytes. It is logical to assume that such lymphocytes would be ideal donor cells for fusions and hybridoma production, as they have been extensively primed, in vivo, by released parasite antigens. We have investigated this hypothesis by taking lymphocytes from both the spleen and mesenteric lymph nodes of parasite-infected mice, and fusing these with the NS1 Ag4 myeloma line, and then comparing the numbers of positive clones obtained from fusions with cells of both origins. Results for both parasites showed a greatly increased number of positive clones in fusions using the lymph nodes. It is envisaged that such a methodology could be applied with advantage to any gut-associated infection in the murine host. 3. Materials and Methods
3.1. S. mansoni infections Three-month-old female BALB/c mice were infected by inoculation of 200 S. mansoni cercariae i.p. The S. mansoni Puerto Rican strain from the Liverpool School of Tropical Medicine and Hygiene (kindly provided by Dr. I. Marshall) was used. The life cycle is maintained in our laboratory using Biomphaleria glabrata as the intermediate host. Six to
Key words: monoclonalantibodies - lymph nodes - parasites 0165-2478 / 85 / $ 3.30 © 1985 ElsevierScience Publishers B.V.(BiomedicalDivision)
225
nine weeks after infection, when the worms have migrated to their final position in the mesenteric veins, the mice were killed by cervical dislocation, and the spleen or lymph nodes removed aseptically as a source of B cells. 3.2. N. dubius infections Three-month-old BALB/c mice were infected with N. dubius following the primary divided infection schedule of Behnke and Parish [4]. Seven days after the final infection the mice were sacrificed and the spleen or mesenteric nodes removed aseptically. 3.3. Fusion methodology Fusions were carried out following the methodology described by Lowe et al. [5], using the NSI Ag4 murine myeloma line and 40% (w/v) PEG 1500 (B.D.H.) containing 5% (v/v) D.M.S.O. (BDH). The lymphocyte: myeloma ratio in all fusions was 5: 1. Care was taken to adopt an exactly similar procedure for all fusions. Supernates from growing clones were screened by solid phase radioassay, using an affinity-purified 1251labelled rabbit anti-mouse immunoglobulin as the developing antibody. Anti-schistosome fusions were screened for reactivity against whole adult (male and female) worm homogenate (WWH) and cercarial homogenate (CH), prepared by homogenization of washed parasites followed by centrifugation at 1200×g, the supernate being retained as a crude antigen preparation. Anti-N. dubius clones were assayed against day 6 larvae and adult worm homogenate antigens, prepared as described by Pritchard et al. [6]. All antigens were used at a concentration of 10 ~tg/ml protein for sensitizing Linbro PVC disposable microagglutination trays. All fusions were screened twice, usually on days 10 and 14, and clones were considered as positive if they gave a c.p.m, reading 3-fold greater than background in both assays. The number of antigen positive clones per fusion was thus obtained, allowing a comparison between the different B cell sources.
4. Results
4.1. S. mansoni fusions The number of clones positive for crude S. man226
Table 1 Results from fusions using S. mansoni infections Fusion B cell no. source
Age of infection (wk)
No. of clones
No. of Isotype positive - clonesa G M
22 24 26 33
8 8 12 9
13 26 26 48
3 (22%) 7 (27%) 6 (22%) 8 (16%)
0 NT NT 8
7~/2
36
9 (25%)
NT 4
9
24 h
9 (36%)
9
35 36
spleen spleen spleen mesenteric lymph node mesenteric lymph node mesenteric lymph node
3 2 3 0
0
a Of 48 wells tested, combined total of cercarial homogenate and whole worm homogenate positive clones. Screened days 10 and 14 post-fusion. b Of 24 wells tested.
soni antigens obtained from 6 fusions, 3 using splenic lymphocytes and 3 using mesenteric lymph node cells, are shown in Table 1. Included in this Table are details of period of infection, total number of clones and the isotypes of the monoclonals, where tested. As the same fusion technique was employed for all 6 fusions, and no clones were lost due to infection etc., the figures represent differences due solely to the source of B lymphocytes. It can be seen that mesenteric lymph node cells give rise to a greater number of growing clones, resulting in more schistosome-positive clones per fusion, even though the number of positive clones as a percentage of total clones is approximately the same, at about 25%, for both cell sources. It is of interest to note that the majority of clones resulting from splenic fusions were of the lgM isotype, while mesenteric lymph node fusions gave a mixture of IgGl and IgM isotypes. It is clear that this methodology produces almost twice as many positive clones per fusion compared with the conventional spleen fusion technique.
4.2. N. dubiusfusions The results obtained from fusions using N. dub# us-infected mice, are presented in Table 2. Adults of N. dubius, unlike the blood fluke S. mansoni, reside in the small intestine, and it was anticipated that the mesenteric lymph nodes of infected mice may not be as actively stimulated as those exposed to S. manso-
Table 2 Results from fusions using N. dubius infections Fusion
B cella source
No. of clones
No. of positive clonesb
28 29
spleen mesenteric lymph node
10 48
6 45
a Immunisation by primary divided schedule of Behnke and Parish [4]. b Of 48 wells tested, combined total obtained with day 6 larval antigen and adult homogenate.
ni infections. The fusion results, however, proved this not to be so, with 45 of the 48 clones produced showing reactivity against the test antigens, a 7-fold greater success rate than that obtained with splenic cell fusions.
5. Discussion
The concept of taking lymphocytes for fusions from local lymph nodes draining areas of infection, is clearly valid in the case of the two parasites investigated in this study. The release of parasite antigens results in large numbers of stimulated lymphocytes, which, from these results, appear to be ideal for fusion and subsequent monoclonal antibody production. In both examples, there were increases in the number of both specific and non-specific clones when compared with the splenic lymphocyte fusions, and this was especially so in the case of N. dubius, where most clones produced antibody directed against the parasite. With S. rnansoni fusions, there were a large number of non-specific clones produced, which may reflect non-specific stimulation by the parasite, in a manner analogous to that seen in trypanosome infections [7] or, conversely, suppression of the local parasite specific response by released soluble factors from the adult worms [8]. Our results, in terms of yield of positive clones, compare favourably with those of Zodda et al. [2] even when comparing yields from spleen cell fusions
only, although this may partially reflect their choice of an assay system that could only detect IgG monoclonal antibodies. In our hands, fusions using splenocytes seem to favour the production of IgM antibodies. Results obtained by Taylor and Butterworth [9] using splenic lymphocytes for fusion, were similar to those we obtained using mesenteric node cells, although this may be partially explained by the high numbers of cercariae used to boost the mice prior to fusion and by the lower stringency of the screening radioassay. The fact that we could obtain a similar number of positive clones from a single challenge low level infection by using lymph node cells clearly underlines the efficiency of this alternative approach. Although we have only produced monoclonal antibodies against two parasites by this technique it is obvious that it may be applied, with advantage, to any gut-associated parasites or micro-organisms that can infect mice as the final host, e.g. Trichuris, Hymenolepis, Nippostronglyus, Trichinella, Giardia [10].
References [I] Mitchell, G. F. (1981) Immunol. Today 2, 140-142. [2] Zodda, D. M., Abdel Hafez, S. K. and Phillips, S. M. (1983) Am. J. Trop. Med. Hyg. 32, 69-77. [3] Galfr6, G., Howe, S. C. and Milstein, C. (1977) Nature (London) 266, 550-552. [4] Behnke, J. M. and Parish, H. A. (1979) Parasite Immunol. 1,13. [5] Lowe, J., Hardie, D., Jefferis, R., Ling, N. R., Drysdale, P., Richardson, P., Raykundalia, C., Catty, D., Appleby, P., Drew, R. and MacLennan, 1. C. M. (1981) Immunology 42, 649-659. [6] Pritchard, D. 1., Maizels, R. M., Behnke, J. M. and Appleby, P. (1984) Immunology 53, 325-335. [7] Urquhart, G. M., Murray, M., Murray, P. K., Jennings, P. W. and Bate, E. (1973) Trans. R. Soc. Trop. Med. Hyg. 67, 528-535. [8] Ottesen, E. A. and Poindexter, R. W. (1980) Am. J. Trop. Med. Hyg. 29, 592-597. [9] Taylor, D. W. and Butterworth, A. E. (1982) Parasitology 84, 65. [10] Wakelin, D. (1978) Nature (London) 273,617 620.
227