Journal of Immunological Methods, 153 (1092) 81-84
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0022-1759/92/$1)5.110
A simple standardised protocol for the production of monoclonai antibodies against viral and bacterial antigens P e t e r V, Coyle, D o r o t h y Wyatt, Conall M c C a u g h e y and H u g h J, O ' N e i l l Regional Vi~us Laboratory, Royal Victoria Hospital, Belfast BTI 2 6BN, UK
(Received9 Janua~ 1992, revised received30 March 1992,accepted 31 March 1992)
A simple standardised protocol for making monoclonal antibodies against a range of human,, bacteria and viruses is described, The protocol was designed to reduce the number of steps to a minimum. A one step footpad immunisation was followed by the fusion schedule 10-15 days later. A vital step in the technique was the use of the immunised mouse's spleen to provide a feeder layer post fusion. This simplified the protocol and more importantly greatly accelerated the growth of the hybridomas produced. Immunisation, fusion and clonal expansion of specific antibody secreting hybridomas was complete within 5 weeks. The percentage of hybridomas secreting specific antibody ranged from 6% to 28%, ihe majority of which were of the IgG isotypes. The method was economical in'the use of tissue culture medium and simple to perform. Key words: Monoclonalantibody; Hybridoma technique; Human respiratory virus; Helicobacter pylori
Introduction The ability to produce stable immunogiobulin secreting hybridomas was first described in 1975 (K6hler and Milstein, 1975). Since then the impact of monoclonal antibodies on the biological, biotechnological and clinical sciences has been immense.
Correspondence to: P.V. Coyle, RegionalVirus Laboratory, Royal Victoria Hospital, Belfast BTI2 6BN, UK. Tel.: (44) 232 240503; Fax: (44) 232 439181. Abbreciations: PBS, phosphate-bufferedsaline; FCS, foetal calf serum; PEGs0, 50% polyethylene glycol in serum-free growth medium; SFM, serum-free growth medium; HAT, hypoxanthine, aminopterine, thymidine; HT, hypoxanthine, thymidine; DMSO, dimethylsulphoxide; PN, popliteal node; BS, bacterial suspension.
lmmunogens employed in raising monoclonal antibodies against infectious agents range from crude homogenates or intact infected cells, to purified protein. The timing and routes of administration vary, and tend to be established and followed by individual laboratories, according to their work schedules and experience. Generally two or three doses are administered w i ~ adjuvanL separated by intervals of 2 - 3 weeks. A final intravenous booster dose complete~ the schedule approximately 3 days before a fusion is carried out. This allows a primary antibody response to he followed by class switching to a predo,~i~rantly lgG repertoire. The booster dose activates resting immune lymphocytes to ensure a r;~'~l of rapidly dividing cells. These are thought to participate preferentially in fusion reactions (Campbell, 1984). Feeder layers may be employed after the fusion step and at subsequent cloning stages.
Materials and methods
a Dounce homogeniser and the resulting cells resuspended in a Falcon tube made up to 40 ml with PBS. NSO cells and spleen cells were both pelleted by centrifuging at 200 × g for 10 min, after which the NSO cells were placed in 10 ml PBS, while the spleen cell pellet was kept until the fusion was carried out. Sufficient NSO cells were then added to the popliteal node lymphocytes in a ratio of 1:5 respectively. This was then made up to 40 ml with PBS and centrifuged at 200 × g for 10 min to pellet the cells, ready for fusion.
lmmunisation of mice
BALB/c mice were lightly anaesthetised using ether and inoculated into the hind footpad with a single dose of 50 ~i of antigen emulsified in incomplete Freund's adjuvant. The virus inocula were infected cell preparations, which had been frozen and thawed once only. The titre of each virus inoculum was determined by indirect immunofluorescence assay (O'Neill et al., 1988). A crude mixture of ten different clinical isolates of Helicobacter pylori in PBS was used as an inoculure for this bacteria. Hybridisation was carried out 10-15 days post inoculation.
The fusion was carried out on the N S O / lymphocyte cell pellet using a 50% polyethylene glycol (PEGs0) solution (Sigma, Poole, England), molecular weight 1300-1600, made up in serumfree growth medium (SFM). The PEGs0 was constituted in a glass bijou by mixing 0.6 ml PEG, 0.6 ml serum-free medium, and 10 p.I 0.1 M HCI together in a waterbath at 56°(2. This was transferred to a waterbath at 37°C at the beginning of the fusion. The Falcon tube containing the cell pellet was held in a beaker of warm water (37°C) during the fusion. All solutions were added in a steady drop-wise fashion while applying gentle agitation to the cell pellet. The serum-free medium used at this stage was prewarmed to 37°C. A single sterile disposable pastette (Alpha Laboratories Ltd., Eastleigh, Hampshire, England) was used to add all solutions during stages 1-5. The fusion procedure was as follows: 1 PEGs0, 1 ml, was added over 1 min. The mixture was gently aspirated up and down the pastette for a further 1 min. 2 SFM, 1 ml, was added over 1 rain; 3 SFM, 1 mi, was added over 1 min; 4 SFM, 2 ml, was added over 1 rain; 5 SFM, 7 mi, was added over 1 rain; 6 SFM was added to a final volume of 40 ml. The fusion products were pelleted by centrifuging at 200 × g for 10 min. The fused cell pellet and corresponding spleen cell pellet were resuspended in 160 ml of HAT medium (growth medium plus HAT supplement Sigma, Poole, England) prewarmed to 37°C, and added to eight flat bottomed microtitre plates (Nunc, Kamstrup,
A rapid technique for the production of monoclonal antibodies to mouse autoantigens using a footpad immunisation protocol has previously been described (Holmdahl et al., 1985). We describe a modification of this technique that we have developed and now use routinely. Our aim was to simplif3, the technical aspect~ of monoclonal antibody production while maintaining a high degree of consistency.
Hybridisation NSO murine myeloma cells were used as a fusion partner. They were grown in growth medium (RPMI 1640 (Gibco, Paisley, Scotland) supplemented with 10 mM L-glutamine, 100 I U / m l penicillin, 100 ~ g / m l streptomycin, 1% sodium pyruvate, and 20% Myoclone Plus FCS (Gibeo, Paisley, Scotland)). They were established in exponential phase growth before each fusion. On the day of hybridisation, the spleen and popliteai nodes were removed into sterile phosphate buffered saline (PBS). The popliteal nodes were then teased apart in 2 ml of fresh PBS in a 5 cm petfi dish, using two hypodermic needles, to allow the lymphocytes to flow into suspension. The PBS containing the lymphocytes, but not the remnants of node tissue, was removed to a 50 ml Falcon tube and made up to 10 ml and counted. At the same time the NSO cells were resuspended and placed in a 50 ml Falcon tube, made up to 40 mi with PBS and counted. Concurrently the spleen was disrupted in 5 ml sterile PBS using
Denmark) at 200 /zl per well. The plates were then incubated at 37°C in an atmosphere of 10% CO2.
lsotype determination Monoclonal antibodies were isotyped using a m o u s e m o n o c l o n a l a n t i b o d y isotyping kit (Amersham International, Amersham, UK).
Care of hybridomas The plates were checked beginning 7 days post fusion, and at 2 day intervals thereafter. Those wells with clones occupying 1 / 6 or greater of their surface area were screened by indirect immunofluorescence. Smaller clones were not screened until they had reached this size. Clones producing specific antibody on screening were immediately transferred to the well of a 24 well Linbro plate (Flow Laboratories, Irvine, Scotland), containing 1 ml of H T medium (growth medium plus H T supplement, Sigma, Poole, England). Each hybridoma transferred was checked daily and the growth recorded as the percentage of the bottom of the well covered by hybridoma cells. When 40% confluence was reached, the supernatant was replaced daily with fresh H T medium, until approximately 8 0 - 9 0 % confluence was attained. At this stage the supernatant medium was removed for storage at - 2 0 ° C and further testing, while the cells were resuspended in 2 ml of Freeze Down Medium (45% H T medium, 45% FCS, 10% DMSO), placed in a Nalgene Cryo I°C freezing container (Techmate, Old Wolverton, Milton Keynes, England) and left at - 7 0 ° C overnight. They were then transferred to liquid nitrogen.
Hybridoma subcloning This was carried out only on hybridomas after further testing of their respective s u p e m a t a n t medium showed them to be secreting antibody of interest. The frozen cells were removed from the liquid nitrogen container and rapidly thawed in a waterbath at 37°C. They were counted and resuspended in H T medium at concentrations of 100, 10 and 1 cell/ml, before being added to preformed monolayers of MRC-5 cells in three 96 well microtitre plates (Nunc). Each concentration of hybridoma cells was added at 200 #1 per well to one of the microtitre plates. Wells containing single clones were screened for antibody production. Subcloning was repeated if it was thought necessary.
Res~dts and discussion We have carried out a consecutive scr!es of hybridisations using respiratory viruses as detailed in Table 1. The average yield was 24.6 x 106 lymphocytes from each pair of activated popliteai lymph nodes, which produced an average of 272 hybridomas at a single fusion. The yield of iymphocytes from a pair of nodes following :,~munisation with incomplete Freund's adjuvant alone was 6 x 106 cells in total. This base-line value is important since the technique does not permit measurement of serum antibody as an indicator of the success of immunisation. Thus the yield of
TABLE 1 FUSION RESULTS FOR NINE BACTERIAL AND VIRAL HYBRID1SATIONS Microbial iuoculum
Titre ~ (per ml)
Respiratory syncytialvirus 103 Parainfluenza virus type 2 102 Parainfluenza virus type 3 10 6 Influenza A virus 106 Influenza B virus I0 2 Adenovirus type 2 104 Hazara virus 106
Helicobacter pylori Herpesvirus type6
PN cells ( x 106)
~' Titre represents the infectiousvirus content of the inocula determined from their respective highest Iog,~ dilutions producing specific fluorescence in an indirect immunofluorescence assay. b Not titrated.
84 cells from the nodes indicates whether the immunisation has worked. The percentage of hybridomas secreting specific antibody ranged from 6.3% for respiratory syncytial virus to 28% for Helicobacter pylori. If non-producing hybridomas were excluded, the percentage of specific secretors would be higher. We have also successfully used this technique on a wider range of viruses ano bacteria including Coxsackieviruses, hepatitis B virus and My-
coplasma pneumoniae. The majority of monoclonal antibodies have been of the lgG subclass including IgG1, IgG2a and lgG2b, with a small number of lgM antibodies also produced. Although we found the low level of IgM secreting hybridomas surprising, it accords with previous observations using a similar immunisation protocol (Holmdahl et al., 1985). The technique was efficient, simple to perform and consistently reliable. It was economic in the number of mice needed per antigen. The use of the same mouse to provide a spleen as the source of the feeder layer, simplified the protocol and more importantly greatly accelerated the growth of the hybridomas produced. This allowed screening for specific antibody production to begin as early as 7 days post fusion. It also allowed us to screen selectively, and consecutively without having to feed non-productive wells. The results were the reduction in attention the fusion demanded and the early movement to liquid nitrogen of positive hybridomas. The outlined procedure was highly economical in the quantity of tissue culture medium needed. Screening and initial expansion of positive hybridomas was complete for most fusions within 19 days of the fusion and 29 days of immunisation. A major advantage of the accelerated growth of the clones is the lack of time for the overgrowth of hybridomas by non-producing hybrids. Under most circumstances only a minority of hybridomas will prove useful fo~' a predetermined purpose. Thus only subcloning when the respective antibody has shown itself sufficiently interesting, negates unnecessary effort for little practical use. The use of a pre-formed feeder layer at this stage, combines its use simultaneously for both cloning and thawing from frozen storage, two of
the areas where it has been recognised to be beneficial (Coffino et al., 1972). The strong adherence of the MRC-5 cell line to plastic tissue culture surfaces, when used as a feeder layer, obviates the need for treatment to inhibit its growth (Harlow et al., 1988). Immunisation with antigen emulsified in complete and incomplete Freund's adjuvant worked equally well. However, the use of incomplete adjuvant is preferable for reasons of animal welfare. The omission of adjuvant resulted in the failure to adequately stimulate the popliteal nodes. We believe the immunisation and fusion protocol described here is the simplest yet reported. Every aspect except the exact yield of lymphocytes from the popliteal lymph nodes can be predicted for any fusion and thus prepared in advance. In simplifying and standardising the technique, and by using the mouse's spleen to provide a feeder layer post fusion, we believe we have enhanced the chances of carrying out a successful fusion. This protocol should facilitate the production of monoclonal antibodies in most laboratories.
References Campbell, A.M. (1984) in: R.H. Burdon and P.H. Van Knippenberg (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Monoclonal Antibody Technology. Elsevier,Amsterdam, p. 88. Coffino, P., Baumal, R., Laskov, R. and Scharff, M.D. (1972) Cloning of mouse myeloma cells and detection of rare variants. J. Cell Physiol. 79, 429. Harlow, E. and Lane, D. (1988) Antibodies, A Laboratory Manual. Cold Spring Harbour Laboratory, New York, p. 221. Holmdahl, R., Moran, T. and Anderson, M. (1985) A rapid and efficient immunisation protocol for production of monoclonal antibodies reactive with autoantigens. J. Immunol. Methods 83, 379. K6hler, G. and Milstein, C. (1975) Continuous culture of fused cells secreting antibodies of predefined specificities. Nature 256, 495. O'Neill,H.J., Shirodaria, P.V., Connolly,J.H., Simpson,D.I.H. and McGeown, M.G. (1988) Cytomegalovirus-specificantibody responses in renal transplant patients with primary and recurrent cytomegalovirusinfection.J. Med. Virol. 24, 461.