Biologic&
(1991)19,171-175
Rapid leotation of Monoclonal Hybridoma Cu a ‘Fusion-cloning’ Method: the ReqWmmmt
* Department
for
R. N. Butcher,* T. U. Obi,*t and K. C. McCullough*$ of Immunology Animal Virus Research Institute (now institute for Animal Health), Pirbright, Waking, Surrey GU24 ONE lJ. K.
Abstract. Hybridomas were generated by fusing the Balb/c SP2/0 myeloma-like cell line with either: (i) splenocytes from Balb/c mice immunized with foot-and-mouth disease virus (FMDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), African swine fever virus (ASFV) or pig thymocytes; or (ii) lymph node cells from cattle immunized with FMDV. If the fusion mixtures were plated in cloning medium of methyl cellulose and HAT medium, small hybridoma colonies developed which rarely survived. Fusion mixtures were then plated in liquid HT medium on to 3T3/A31 feeder layers in 75 cm* flasks, incubated at 37°C for 24 h before adding aminopterin, and incubated for a further 2 to 4 days before cloning in methyl cellulose/HT medium. Without the aminopterin in the cloning medium, colonies of hybridomas, which could be cultured, developed from the majority of fusions. These colonies were isolated in HT medium over feeder layers and given two subcultures in HAT medium as a precaution against any reversion to aminopterin sensitivity during the cloning, No evidence of such reversions were seen, and recloning results suggested that the initial cloning was highly efficient at generating monoclonal cultures.
Introduction It has been reported that monoclonal hybridoma cultures can be isolated by plating cells in HAT medium containing methyl cellulose immediately after fusi0n.l Although this procedure would appear to be relatively simple, we have found that only those cultures which initially grow rapidly (often non-secretors of antigen-specific antibody) could be isolated with any success (Butcher and McCullough, unpublished data). A possible explanation may be that under such conditions, slower growing colonies would become sensitive to aminopterin which is metabolized at a slower rate than the hypoxanthine used by the cells to bypass the action of this antimetabolite. Consequently, we developed a modified ‘fusioncloning’ procedure in which the fusion mixture was treated with aminopterin before plating in a semit Permanent address: University of Ibadan, Ibadan, Nigeria.
$ Correspondence
to Dr K. C. McCullough,
present
address: Institut f%r Krnskrankheiten und Immnnoprophylaxe, Hagenaustrasse 74, CH-4025 Basel, Switzerland 1045-1056/91/030171+5$03.00/0
solid cloning medium 21 days (depending could be successfully thing which was not published methods. Materials
free of aminopterin. After 10 to on the hybridomas) colonies isolated or subcultured, somepossible using the previously
and methods
immunizations Female Balb/c mice, 6 to 8 weeks of age, were immunized with purified or semi-purified antigens of FMDV,2s3 rinderpest viru~,~ ASFV, PPRV or pig thymocytes. The FMDV was purified by sucrose density centrifugation, and the other viruses by differential centrifugation; the pig thymocytes were isolated on Ficoll-Hypaque gradients and fmed with 4% (w/v) p-formaldehyde in PBS. Mice were immunized with either 10 M FMDV, l/lOth of a 75 cma flask culture
of the other viruses or lo6 thymocytes
in
Freund’s complete adjuvant, boosted after 4 to 6 weeks in the absence of adjuvant, and again after a further 2 weeks. Three days tier the final boost, the animals were sacrificed and splenectomy performed. 0 1991The International Associationof BiologicalStandardization
172
R. N. Butcher
Fusions
Balb/c 3T3/A31 feeder layers at a density of 2 X lo4 cells/ml treated for 16 to 18 h at 37°C with 1 pg/ml mitomycin C (Sigma) were prepared as described.5,6 The Balblc hybridoma cell line SP2/0 which has myeloma cell-like characteristics was fused with the splenocytes obtained from the disruption of immune spleens at a ratio of 5 splenocytes: 1 SP2 cell, using 50% (v/v) PEG in Eagle’s medium, Glasgow modification (GMEM). The PEG was added over 1 to 2 min at 4°C the cells then left at 4°C for a further 2 min followed by dilution with 20 ml prewarmed (37°C) GMEM and incubation at 37°C for 15 min.6 The fusion mixture was then centrifuged and resuspended in medium according to the cloning procedure used. Preparation
of methyl cellulose
Methyl cellulose was prepared in 10 ml volumes as follows: 0.2 g methyl cellulose (Sigma, 4000 cp) was placed in a glass universal (20 ml) bottle; 5 ml of deionized water was carefully added so as not to disturb the methyl cellulose; the bottles were autoclaved (15 lbs in2/15 min), allowed to cool and 5 ml ‘2X’ GMEM (twice the normal concentration of GMEM) was added before storing at 4°C for at least 3 days (with several inversions of the bottle each day to ensure good mixing).6 Fusion/cloning
under methyl cellulose/HAT
were incubated at 37°C for a further 2 to 4 days (this variation in time did not appear to influence the efficacy of the procedure). The cells were then resuspended and washed three-times using HT medium and centrifugation at 250 g/10 min before resuspending in half of the original volume of ‘X2’ HT medium (HT medium in which the concentration of hypoxanthine, thymidine, serum and glutamine had been doubled). This cell suspension was completely mixed with an equal volume of 2% (w/v) methyl cellulose in complete medium, and plated on to 3T3/A31 feeder layers5,6 in the wells of 12-well plates at 1.5 ml/well. These plates were incubated at 37°C for 7 days, and observations were then made for the development of colonies, visible either microscopically or by eye. Hybridoma colonies were isolated from the methyl cellulose overlay as described elsewhere.6 ELISA
The indirect and trapping ELISA (antigen-capture or indirect sandwich) ELISA were as described by McCullough et ~1.~ for the detection of antibody reactivity against FMDV antigen. A sandwich ELISA was also used to detect secreted murine immunoglobulin, without concern for the antigenspecificity.8
medium
The fusion mixture was resuspended in GMEM supplemented with 20% (v/v) foetal calf serum, 4 mu glutamine, 2 X 10m4 M hypoxanthine, 3.2 X 10-5~ thymidine and 8 X 10m7 M aminopterin to give 6 X 10” cells/ml. Aliquots of 10 ml of this cell suspension were mixed with 10 ml of the above 2% (w/v) methyl cellulose in GMEM, plated on to 2- to 5-day-old 3T3/A3 1 feeder layers5s6 in 12-well or 24-well plates and incubated at 37°C. Modified fusion/cloning cellulose/HT medium
et al.
method under methyl
The fusion mixture was resuspended in GMEM supplemented with 10% (v/v) foetal calf serum, 2 mu glutamine (complete medium), 1 X 10e4 M hypoxanthine and 1.6 X 10m5 M thymidine (HT medium) to give 3 X lo6 cells/ml. The fusion mixture was plated on to 2- to 5-day-old 3T3/A31 feeder layers5s6 in 75 cm2 flasks at 30 ml/flask, and incubated for 24 h at 37°C in the HT medium. Aminopterin was then added from a ‘x10' preparation in HT medium to give a final concentration of 4 X 10-7~. The cultures
Results Cloning of hybridoma fusion in the presence
cells immediately of aminopterin
after the
When fusion mixtures of SP2/0 cells and splenocytes from mice immunized with FMDV or pig thymocytes were cloned in methyl cellulose/HAT medium, over 3T3/A31 feeder cells, hybridomas could be found (Table 11, but rarely subcultured. In only one experiment (with pig thymocytes as the immunogen) were specific antibody-secreting hybridomas isolated and propagated. When the development of colonies was studied microscopically, it was noted that for the first 7 days cells could be seen growing and colony development appeared to be progressing. Sometimes, this ‘normal growth could be seen for 14 days (in one experiment, for 20 days). Generally, after 14 days, this growth ceased, and the microscopically visible colonies eventually regressed (Table 2). When attempts were made to isolate the colonies, the cells therein were found to be dead, or died soon afier.
Rapid isolation of monoclonal
hybridoma
1. Results from experiments in which hybridomas fusion in methyl cellulose/HAT medium.
Table
Expt. 177 209 192 194 203 179 182
No. wells seeded
Immunogen FMDV 01 Pacheco FMDVOl BFS 1860 FMDV A24 Cruzeiro FMDV A24 Cruzeiro FMDV A24 Cruzeiro Pig thymoqytes Pig thymocytes
No. wells with colonies (%o)
24 6 28 28 38 12 24
18 6 0 14 22
(75) (100) (0) (50) (86.8)
8 (67)
ll(45.8)
Cloning of hybridoma cells immediately after fusion in the absence of aminopterin
From the previous results it was construed that the presence of aminopterin in the semi-solid media might be producing a problem. Since the aminopterin was essential for the selection of hybridomas in preference to the continued survival of myeloma cells,g a compromise had to be reached. Thus, the fusion mixture was treated with aminopterin for 2 to 4 days before cloning in semisolid medium without the aminopterin as described in Materials and Methods. The results showed that there was considerable improvement in the efficiency of cloning (Table 3), and that the majority of clones could be isolated and subcultured with relative ease on 3T31A31 feeder layers. It was also possible for the first time to isolate by this fusion cloning method growing colonies of heterohybridomas between mouse myeloma cells and bovine lymph node cells from animals immunized with FMDV (Table 3).
Table 2. Microscopic
cellulose/HAT
Expt 177 209 192 194 203 179 182
medium,
cultures
were generated
No. hybridoma colonies per positive well 1 to 4 to 0 1 to 1 to 3 to 1
10 7
FMDV 01 Pacheco FMDV 01 BFS 1860 FMDV A24 Cruzeiro FMDV A24 Cruzeiro FMDV A24 Cruzeiro Pig thymocytes Pig thymocytes
by cloning
Per cent isolated colonies subcultured
immediately
after
Per cent isolated colonies secreting immunoglobulin
0 0 0 0 1.2 87.5 0
5 15 25
0 0 0 0 0 73.8 0
Characteristics of the monoclona hybridoma cultures cloned in the absence of aminopterin
Greater than 90% of the homohybridoma colonies shown in Table 3 secreted murine immunoglobulins (data not presented), the majority of which were found to be antigen-specific. This is in contrast to the colonies isolated using the methyl cellulose/HAT medium overlay (Table 1). After further subcultures, these hybridomas were seen to be relatively stable with respect to antibody production. Re-cloning of the hybridomas did not alter the characteristics of antibody production. It was standard procedure to isolate the colonies into HAT medium. In some experiments, the cells were isolated into HT medium and then divided into HAT medium-fed and HT medium-fed sister cultures. Aminopterin-sensitivity was not observed in any of the antibody-secreting hybridomas thus treated (data not presented).
observations on the development plated immediately after fusion
Immunogen
173
of hybridomas
under methyl
Time after plating of fusion products that developing hybridoma colonies were identified 20 10 7 7 14 7 14
days days days days days days days
R. N. Butcher et al.
174
Table 3. Comparison of the efficiency of cloning immediately after fusion in methyl or in methyl cellulose/HT medium (after 3 days in liquid HAT medium)
Expt
Immunogen
Cloning medium
177 209 192 194 203 204 179 182 207 219 224 235 237 238 251
FMDV FMDV FMDV FMDV FMDV FMDV Pig T/cytes Pig T/cytes FMDV MAb (anti-Id) ASFV FMDV (hetero)* FMDV (hetero) FMDV (hetero) FMDV(hetero)
+HAT +HAT +HAT +HAT +-HAT +HAT +HAT +HAT +HT +HT +HT +HT +HT +HT +HT
Per cent wells containing hybridoma colonies
* hetero = bovine/mouse heterohybridomas cattle immunized with FMDV 0, BFS 1860.
75 100 0 50 86.8 100 67 45.8 100 100 100 100 100 100 100
Average number of hybridomas per positive well 5.1 5.0 0 o-9 6.3 5.4 9.3 1.0 4.5 19.1 29.0 12.4 32.5 6.0 5.6
cellulose/HAT
medium
Per cent isolated colonies subcultured 0 0 0 0 1.2 0 87.5 0 90 100 80 63.1 36.1 25 18.7
produced by fusing Balb/c mouse SP2/0 cells, with lymph node cells from
Discussion The cloning of hybridoma cell lines9 has generally favoured the methods of limiting dilution passage (see the revieh by Fazekas de St Groth”‘) and the colony isolation under semi-solid medium (for example, see Sharon et al.“). There are certain arguments for and against each procedure, but it would appear that the latter method may prove to be both more reliable and easier to perform (for example, see McCullough et ~1.~). Nevertheless, the major limiting factor has been the selection and growth of the hybridomas before cloning, which can result in a delay of up to 3 months before cloning. Daviset al.’ described a method whereby the initial culturing of hybridomas prior to cloning could be avoided. This entailed resuspending the fused products in a semi-sold medium (based on methyl cellulose) supplemented with hypoxanthine, aminopterin and thymidine (HAT) to select for hybridomas. Although their rate of success was high, our attempts to repeat this procedure resulted in a low and variable success rate. Few hybridoma colonies could be isolated and subcultured, and then only those which did not produce mouse immunoglobulins. This is not surprising since those hybridomas which do not secrete immunoglobulin often grow
more efficiently than the antibody-producing cells. Taken together, the experiments with methyl cellulose/HAT medium overlay suggested that there was a selection for the faster growing clones of hybridoma cells which did not produce antigenspecific antibody. When viewed microscopically, a number of clones which were initially seen to be growing soon ceased to expand and the cells died. These cells would have used anabolically hypoxanthine and thymidine in order to bypass the enzyme inhibition by aminopterin in the nucleoside biosynthesis pathways. Thus, it was considered possible that such growing cells could deplete the medium in their vicinity more quickly of hypoxanthine than of aminopterin. In a liquid medium this would not normally present a problem, but in the semi-solid conditions of methyl cellulose, hypoxanthine would diffuse from areas of high concentrations to areas of low concentrations more slowly. Since the hypoxanthine is essential for the hybridomas to bypass the aminopterin block, a selective depletion of the hypoxanthine could result in the concentration around the hybridoma cells falling to a level which would be growth limiting; at this point the aminopterin could become toxic.
Rapid
isolation
of monoclonal
For these reasons, hybridomas were plated in methyl cellulose based cloning medium free of aminopterin. Under these conditions, the development of colonies which could be isolated and successfully subcultured was most efficient. Although it has been reported that the ideal method is to leave fusion products undisturbed for 7 days after the addition of aminopterin, 3,12the early manipulation of the fusion products in the above procedure did not prevent the generation of hybridoma colonies. Furthermore, the removal of aminopterin from the cultures after only 2 to 4 days did not produce problems with aminopterin sensitive cells arising; nevertheless, our preference is to isolate the hybridomas colonies into a HAT medium. In conclusion, we define in this paper the conditions for the successful isolation of monoclonal hybridoma colonies rapidly after fusion. The method consistently produces monoclonal hydridoma colonies generated from splenocytes immunized with any one of a wide range of antigens. In addition, specific antibody-secreting bovine/mouse heterohybridomas have also been isolated, demonstrating both the applicability and efficacy of the method. References 1. Davis JM, Pennington JE, Kubler AM, Conscience JF. A simple single step technique for selecting and cloning hybridomas for the production of monoclonal antibodies. J Immunol Meth 1982; 50: 161-171. 2. McCullough KC, Butcher RN. Monoclonal antibodies against foot-and-mouth disease virus 146s and 12s particles. Arch Viral 1982; 74: l-36
hybridoma
cultures
175
3. McCullough KC, Butcher RN, Parkinson D. Hybridoma cell lines secreting monoclonal antibodies against foot-and-mouth disease virus. I. Cell culturing requirements. J Biol Stand 1983; 11: 171-181. 4. McCullough KC, Sheshberadaran H, Norrby E, Obi TU, Crowther JR. Monoclonal antibodies against morbilliviruses. Rev Sci Tech Off Int Epiz 1986; 5: 411-427. 5. Butcher RN, McCullough KC, Jarry C, Bryant J. Mitomycin C-treated 3T3/A31 cell feeder layers in hybridoma technology J Immunol Meth 1988; 107: 245-251. 6. McCullough KC, Spier RE. Monoclonal Antibodies in Biology and Biotechnology; in Theory and in Practice. Cambridge: Cambridge University Press, 1990. 7. McCullough KC, Crowther JR, Butcher RN. Alteration in antibody reactivity with foot-and-mouth disease virus (FMDV) 146s antigen before and after binding to a solid phase or complex& with specific antibody. J Immunol Metb 1985; 82: 91-100. 8. Voller A, Bidwell DE, Bartlet A. Enzyme immunoassays in diagnostic medicine: theory and practice. Bull WHO 1976; 53: 55-65. 9. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497. 10. Fazekas de St. Groth S. The evaluation of limiting dilution assays. J Immunol Meth 1982; 49: Rll-R23. 11. Sharon J, Morrison SL, Kabat EA. Detection of specific hybridoma clones by replica plate immunoadsorption of their secreted antibodies. Proc Nat1 Acad Sci USA, 1979; 76: 1420-1424. 12. Fazekas de St. Groth S, Scheiddegger D. Production of monoclonal antibodies: strategy and tactics. J. Immunol Meth 1980; 35: 1-21. Received for publication 26 April accepted 24 May 1991.
1990;
(1991)19,171-175
Rapid leotation of Monoclonal Hybridoma Cu a ‘Fusion-cloning’ Method: the ReqWmmmt
* Department
for
R. N. Butcher,* T. U. Obi,*t and K. C. McCullough*$ of Immunology Animal Virus Research Institute (now institute for Animal Health), Pirbright, Waking, Surrey GU24 ONE lJ. K.
Abstract. Hybridomas were generated by fusing the Balb/c SP2/0 myeloma-like cell line with either: (i) splenocytes from Balb/c mice immunized with foot-and-mouth disease virus (FMDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), African swine fever virus (ASFV) or pig thymocytes; or (ii) lymph node cells from cattle immunized with FMDV. If the fusion mixtures were plated in cloning medium of methyl cellulose and HAT medium, small hybridoma colonies developed which rarely survived. Fusion mixtures were then plated in liquid HT medium on to 3T3/A31 feeder layers in 75 cm* flasks, incubated at 37°C for 24 h before adding aminopterin, and incubated for a further 2 to 4 days before cloning in methyl cellulose/HT medium. Without the aminopterin in the cloning medium, colonies of hybridomas, which could be cultured, developed from the majority of fusions. These colonies were isolated in HT medium over feeder layers and given two subcultures in HAT medium as a precaution against any reversion to aminopterin sensitivity during the cloning, No evidence of such reversions were seen, and recloning results suggested that the initial cloning was highly efficient at generating monoclonal cultures.
Introduction It has been reported that monoclonal hybridoma cultures can be isolated by plating cells in HAT medium containing methyl cellulose immediately after fusi0n.l Although this procedure would appear to be relatively simple, we have found that only those cultures which initially grow rapidly (often non-secretors of antigen-specific antibody) could be isolated with any success (Butcher and McCullough, unpublished data). A possible explanation may be that under such conditions, slower growing colonies would become sensitive to aminopterin which is metabolized at a slower rate than the hypoxanthine used by the cells to bypass the action of this antimetabolite. Consequently, we developed a modified ‘fusioncloning’ procedure in which the fusion mixture was treated with aminopterin before plating in a semit Permanent address: University of Ibadan, Ibadan, Nigeria.
$ Correspondence
to Dr K. C. McCullough,
present
address: Institut f%r Krnskrankheiten und Immnnoprophylaxe, Hagenaustrasse 74, CH-4025 Basel, Switzerland 1045-1056/91/030171+5$03.00/0
solid cloning medium 21 days (depending could be successfully thing which was not published methods. Materials
free of aminopterin. After 10 to on the hybridomas) colonies isolated or subcultured, somepossible using the previously
and methods
immunizations Female Balb/c mice, 6 to 8 weeks of age, were immunized with purified or semi-purified antigens of FMDV,2s3 rinderpest viru~,~ ASFV, PPRV or pig thymocytes. The FMDV was purified by sucrose density centrifugation, and the other viruses by differential centrifugation; the pig thymocytes were isolated on Ficoll-Hypaque gradients and fmed with 4% (w/v) p-formaldehyde in PBS. Mice were immunized with either 10 M FMDV, l/lOth of a 75 cma flask culture
of the other viruses or lo6 thymocytes
in
Freund’s complete adjuvant, boosted after 4 to 6 weeks in the absence of adjuvant, and again after a further 2 weeks. Three days tier the final boost, the animals were sacrificed and splenectomy performed. 0 1991The International Associationof BiologicalStandardization
172
R. N. Butcher
Fusions
Balb/c 3T3/A31 feeder layers at a density of 2 X lo4 cells/ml treated for 16 to 18 h at 37°C with 1 pg/ml mitomycin C (Sigma) were prepared as described.5,6 The Balblc hybridoma cell line SP2/0 which has myeloma cell-like characteristics was fused with the splenocytes obtained from the disruption of immune spleens at a ratio of 5 splenocytes: 1 SP2 cell, using 50% (v/v) PEG in Eagle’s medium, Glasgow modification (GMEM). The PEG was added over 1 to 2 min at 4°C the cells then left at 4°C for a further 2 min followed by dilution with 20 ml prewarmed (37°C) GMEM and incubation at 37°C for 15 min.6 The fusion mixture was then centrifuged and resuspended in medium according to the cloning procedure used. Preparation
of methyl cellulose
Methyl cellulose was prepared in 10 ml volumes as follows: 0.2 g methyl cellulose (Sigma, 4000 cp) was placed in a glass universal (20 ml) bottle; 5 ml of deionized water was carefully added so as not to disturb the methyl cellulose; the bottles were autoclaved (15 lbs in2/15 min), allowed to cool and 5 ml ‘2X’ GMEM (twice the normal concentration of GMEM) was added before storing at 4°C for at least 3 days (with several inversions of the bottle each day to ensure good mixing).6 Fusion/cloning
under methyl cellulose/HAT
were incubated at 37°C for a further 2 to 4 days (this variation in time did not appear to influence the efficacy of the procedure). The cells were then resuspended and washed three-times using HT medium and centrifugation at 250 g/10 min before resuspending in half of the original volume of ‘X2’ HT medium (HT medium in which the concentration of hypoxanthine, thymidine, serum and glutamine had been doubled). This cell suspension was completely mixed with an equal volume of 2% (w/v) methyl cellulose in complete medium, and plated on to 3T3/A31 feeder layers5,6 in the wells of 12-well plates at 1.5 ml/well. These plates were incubated at 37°C for 7 days, and observations were then made for the development of colonies, visible either microscopically or by eye. Hybridoma colonies were isolated from the methyl cellulose overlay as described elsewhere.6 ELISA
The indirect and trapping ELISA (antigen-capture or indirect sandwich) ELISA were as described by McCullough et ~1.~ for the detection of antibody reactivity against FMDV antigen. A sandwich ELISA was also used to detect secreted murine immunoglobulin, without concern for the antigenspecificity.8
medium
The fusion mixture was resuspended in GMEM supplemented with 20% (v/v) foetal calf serum, 4 mu glutamine, 2 X 10m4 M hypoxanthine, 3.2 X 10-5~ thymidine and 8 X 10m7 M aminopterin to give 6 X 10” cells/ml. Aliquots of 10 ml of this cell suspension were mixed with 10 ml of the above 2% (w/v) methyl cellulose in GMEM, plated on to 2- to 5-day-old 3T3/A3 1 feeder layers5s6 in 12-well or 24-well plates and incubated at 37°C. Modified fusion/cloning cellulose/HT medium
et al.
method under methyl
The fusion mixture was resuspended in GMEM supplemented with 10% (v/v) foetal calf serum, 2 mu glutamine (complete medium), 1 X 10e4 M hypoxanthine and 1.6 X 10m5 M thymidine (HT medium) to give 3 X lo6 cells/ml. The fusion mixture was plated on to 2- to 5-day-old 3T3/A31 feeder layers5s6 in 75 cm2 flasks at 30 ml/flask, and incubated for 24 h at 37°C in the HT medium. Aminopterin was then added from a ‘x10' preparation in HT medium to give a final concentration of 4 X 10-7~. The cultures
Results Cloning of hybridoma fusion in the presence
cells immediately of aminopterin
after the
When fusion mixtures of SP2/0 cells and splenocytes from mice immunized with FMDV or pig thymocytes were cloned in methyl cellulose/HAT medium, over 3T3/A31 feeder cells, hybridomas could be found (Table 11, but rarely subcultured. In only one experiment (with pig thymocytes as the immunogen) were specific antibody-secreting hybridomas isolated and propagated. When the development of colonies was studied microscopically, it was noted that for the first 7 days cells could be seen growing and colony development appeared to be progressing. Sometimes, this ‘normal growth could be seen for 14 days (in one experiment, for 20 days). Generally, after 14 days, this growth ceased, and the microscopically visible colonies eventually regressed (Table 2). When attempts were made to isolate the colonies, the cells therein were found to be dead, or died soon afier.
Rapid isolation of monoclonal
hybridoma
1. Results from experiments in which hybridomas fusion in methyl cellulose/HAT medium.
Table
Expt. 177 209 192 194 203 179 182
No. wells seeded
Immunogen FMDV 01 Pacheco FMDVOl BFS 1860 FMDV A24 Cruzeiro FMDV A24 Cruzeiro FMDV A24 Cruzeiro Pig thymoqytes Pig thymocytes
No. wells with colonies (%o)
24 6 28 28 38 12 24
18 6 0 14 22
(75) (100) (0) (50) (86.8)
8 (67)
ll(45.8)
Cloning of hybridoma cells immediately after fusion in the absence of aminopterin
From the previous results it was construed that the presence of aminopterin in the semi-solid media might be producing a problem. Since the aminopterin was essential for the selection of hybridomas in preference to the continued survival of myeloma cells,g a compromise had to be reached. Thus, the fusion mixture was treated with aminopterin for 2 to 4 days before cloning in semisolid medium without the aminopterin as described in Materials and Methods. The results showed that there was considerable improvement in the efficiency of cloning (Table 3), and that the majority of clones could be isolated and subcultured with relative ease on 3T31A31 feeder layers. It was also possible for the first time to isolate by this fusion cloning method growing colonies of heterohybridomas between mouse myeloma cells and bovine lymph node cells from animals immunized with FMDV (Table 3).
Table 2. Microscopic
cellulose/HAT
Expt 177 209 192 194 203 179 182
medium,
cultures
were generated
No. hybridoma colonies per positive well 1 to 4 to 0 1 to 1 to 3 to 1
10 7
FMDV 01 Pacheco FMDV 01 BFS 1860 FMDV A24 Cruzeiro FMDV A24 Cruzeiro FMDV A24 Cruzeiro Pig thymocytes Pig thymocytes
by cloning
Per cent isolated colonies subcultured
immediately
after
Per cent isolated colonies secreting immunoglobulin
0 0 0 0 1.2 87.5 0
5 15 25
0 0 0 0 0 73.8 0
Characteristics of the monoclona hybridoma cultures cloned in the absence of aminopterin
Greater than 90% of the homohybridoma colonies shown in Table 3 secreted murine immunoglobulins (data not presented), the majority of which were found to be antigen-specific. This is in contrast to the colonies isolated using the methyl cellulose/HAT medium overlay (Table 1). After further subcultures, these hybridomas were seen to be relatively stable with respect to antibody production. Re-cloning of the hybridomas did not alter the characteristics of antibody production. It was standard procedure to isolate the colonies into HAT medium. In some experiments, the cells were isolated into HT medium and then divided into HAT medium-fed and HT medium-fed sister cultures. Aminopterin-sensitivity was not observed in any of the antibody-secreting hybridomas thus treated (data not presented).
observations on the development plated immediately after fusion
Immunogen
173
of hybridomas
under methyl
Time after plating of fusion products that developing hybridoma colonies were identified 20 10 7 7 14 7 14
days days days days days days days
R. N. Butcher et al.
174
Table 3. Comparison of the efficiency of cloning immediately after fusion in methyl or in methyl cellulose/HT medium (after 3 days in liquid HAT medium)
Expt
Immunogen
Cloning medium
177 209 192 194 203 204 179 182 207 219 224 235 237 238 251
FMDV FMDV FMDV FMDV FMDV FMDV Pig T/cytes Pig T/cytes FMDV MAb (anti-Id) ASFV FMDV (hetero)* FMDV (hetero) FMDV (hetero) FMDV(hetero)
+HAT +HAT +HAT +HAT +-HAT +HAT +HAT +HAT +HT +HT +HT +HT +HT +HT +HT
Per cent wells containing hybridoma colonies
* hetero = bovine/mouse heterohybridomas cattle immunized with FMDV 0, BFS 1860.
75 100 0 50 86.8 100 67 45.8 100 100 100 100 100 100 100
Average number of hybridomas per positive well 5.1 5.0 0 o-9 6.3 5.4 9.3 1.0 4.5 19.1 29.0 12.4 32.5 6.0 5.6
cellulose/HAT
medium
Per cent isolated colonies subcultured 0 0 0 0 1.2 0 87.5 0 90 100 80 63.1 36.1 25 18.7
produced by fusing Balb/c mouse SP2/0 cells, with lymph node cells from
Discussion The cloning of hybridoma cell lines9 has generally favoured the methods of limiting dilution passage (see the revieh by Fazekas de St Groth”‘) and the colony isolation under semi-solid medium (for example, see Sharon et al.“). There are certain arguments for and against each procedure, but it would appear that the latter method may prove to be both more reliable and easier to perform (for example, see McCullough et ~1.~). Nevertheless, the major limiting factor has been the selection and growth of the hybridomas before cloning, which can result in a delay of up to 3 months before cloning. Daviset al.’ described a method whereby the initial culturing of hybridomas prior to cloning could be avoided. This entailed resuspending the fused products in a semi-sold medium (based on methyl cellulose) supplemented with hypoxanthine, aminopterin and thymidine (HAT) to select for hybridomas. Although their rate of success was high, our attempts to repeat this procedure resulted in a low and variable success rate. Few hybridoma colonies could be isolated and subcultured, and then only those which did not produce mouse immunoglobulins. This is not surprising since those hybridomas which do not secrete immunoglobulin often grow
more efficiently than the antibody-producing cells. Taken together, the experiments with methyl cellulose/HAT medium overlay suggested that there was a selection for the faster growing clones of hybridoma cells which did not produce antigenspecific antibody. When viewed microscopically, a number of clones which were initially seen to be growing soon ceased to expand and the cells died. These cells would have used anabolically hypoxanthine and thymidine in order to bypass the enzyme inhibition by aminopterin in the nucleoside biosynthesis pathways. Thus, it was considered possible that such growing cells could deplete the medium in their vicinity more quickly of hypoxanthine than of aminopterin. In a liquid medium this would not normally present a problem, but in the semi-solid conditions of methyl cellulose, hypoxanthine would diffuse from areas of high concentrations to areas of low concentrations more slowly. Since the hypoxanthine is essential for the hybridomas to bypass the aminopterin block, a selective depletion of the hypoxanthine could result in the concentration around the hybridoma cells falling to a level which would be growth limiting; at this point the aminopterin could become toxic.
Rapid
isolation
of monoclonal
For these reasons, hybridomas were plated in methyl cellulose based cloning medium free of aminopterin. Under these conditions, the development of colonies which could be isolated and successfully subcultured was most efficient. Although it has been reported that the ideal method is to leave fusion products undisturbed for 7 days after the addition of aminopterin, 3,12the early manipulation of the fusion products in the above procedure did not prevent the generation of hybridoma colonies. Furthermore, the removal of aminopterin from the cultures after only 2 to 4 days did not produce problems with aminopterin sensitive cells arising; nevertheless, our preference is to isolate the hybridomas colonies into a HAT medium. In conclusion, we define in this paper the conditions for the successful isolation of monoclonal hybridoma colonies rapidly after fusion. The method consistently produces monoclonal hydridoma colonies generated from splenocytes immunized with any one of a wide range of antigens. In addition, specific antibody-secreting bovine/mouse heterohybridomas have also been isolated, demonstrating both the applicability and efficacy of the method. References 1. Davis JM, Pennington JE, Kubler AM, Conscience JF. A simple single step technique for selecting and cloning hybridomas for the production of monoclonal antibodies. J Immunol Meth 1982; 50: 161-171. 2. McCullough KC, Butcher RN. Monoclonal antibodies against foot-and-mouth disease virus 146s and 12s particles. Arch Viral 1982; 74: l-36
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cultures
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3. McCullough KC, Butcher RN, Parkinson D. Hybridoma cell lines secreting monoclonal antibodies against foot-and-mouth disease virus. I. Cell culturing requirements. J Biol Stand 1983; 11: 171-181. 4. McCullough KC, Sheshberadaran H, Norrby E, Obi TU, Crowther JR. Monoclonal antibodies against morbilliviruses. Rev Sci Tech Off Int Epiz 1986; 5: 411-427. 5. Butcher RN, McCullough KC, Jarry C, Bryant J. Mitomycin C-treated 3T3/A31 cell feeder layers in hybridoma technology J Immunol Meth 1988; 107: 245-251. 6. McCullough KC, Spier RE. Monoclonal Antibodies in Biology and Biotechnology; in Theory and in Practice. Cambridge: Cambridge University Press, 1990. 7. McCullough KC, Crowther JR, Butcher RN. Alteration in antibody reactivity with foot-and-mouth disease virus (FMDV) 146s antigen before and after binding to a solid phase or complex& with specific antibody. J Immunol Metb 1985; 82: 91-100. 8. Voller A, Bidwell DE, Bartlet A. Enzyme immunoassays in diagnostic medicine: theory and practice. Bull WHO 1976; 53: 55-65. 9. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497. 10. Fazekas de St. Groth S. The evaluation of limiting dilution assays. J Immunol Meth 1982; 49: Rll-R23. 11. Sharon J, Morrison SL, Kabat EA. Detection of specific hybridoma clones by replica plate immunoadsorption of their secreted antibodies. Proc Nat1 Acad Sci USA, 1979; 76: 1420-1424. 12. Fazekas de St. Groth S, Scheiddegger D. Production of monoclonal antibodies: strategy and tactics. J. Immunol Meth 1980; 35: 1-21. Received for publication 26 April accepted 24 May 1991.
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