Increased yield of antibody-producing murine spleen cell hybridomas from fusions cultured in medium supplemented with mouse serum

Journal of Immunological Methods, 127 (1990) 39-42 Elsevier

39

JIM05457

Increased yield of antibody-producing murine spleen cell hybridomas from fusions cultured in medium supplemented with mouse serum Mats A.A. Persson and Richard A. Lerner Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA, U.S.A. (Received 8 August 1989, revised received 12 October 1989, accepted 13 October 1989)

The number of monoclonal antibody-producing murine hybridomas were increased 7-10-fold in three separate fusions when supplementing the FBS-contalning, or serum-free, growth factor-containing, Culture medium with 5% normal mouse serum. The beneficial effect was seen in fusions of A / J and 129GIX ÷ spleen cells, from primary and secondary responses, and when using SP2/0 or P3X63Ag8U.1 as the fusion partner. Key words: Spleen cell hybridoma; Mouse serum; Tissue culture; Monoclonal antibody

Introduction

The powerful monoclonal antibody technique invented by KShler and Milstein (1975) has been performed in a large number of methodological variants. To increase the number of established antibody-producing hybridomas, use of different myeloma fusion partners, modifications of immunization procedures and various culture conditions has been tried (for a review see Goding, 1986). In the present report, we describe a sub-

Correspondence to: M.A.A. Persson, Department of Molecular Biology (MBll), Research Institute of Scripps Clinic, 10666 North Torrey Pines Rd., La Jolla, CA 92037, U.S.A. Abbreviations: ATCC, American Type Culture Collection; BSA, bovine serum albumin; CFA, complete Freund's adjuvant; DMEM, Dulbecco's modified Eagle medium; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; HAT, hypoxanthine-aminopterin-thymidine; HCF, hybridoma cloning factor; KLH, keyhole limpet hemocyanin; NMS, normal mouse serum; PBS, phosphate-buffered saline.

stantially increased amount of antibody-producing murine hybridomas when supplementing the medium with 5% normal mouse serum.

Materials and methods Immunogens, mouse strains and media

Organic haptens synthesized by Dr. K.D. Janda (hapten (1_) in Janda et al. (1989)) and Dr. B.L. Iverson (unpublished), both at Research Institute of Scripps Clinic, La JoUa, CA, were coupled to keyhole limpet hemocyanin (KLH) under aqueous condition, pH 7, utilizing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) (Pierce, Rockford, IL, U.S.A.); the haptens had a glutaric acid tail to provide a 5 carbon linker and a carboxyl group for linking to free amine groups. Strain A / J and 129GIX + mice were obtained from inhouse breeding facilities. The culture medium used was Dulbecco's modified Eagle

0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

40 medium (DMEM) with 1 mM sodium pyruvate, 100 /~M nonessential amino acids, 4 mM Lglutamine, 1 mM vitamins, penicillin (100 U/ml), streptomycin (100 #g/ml), gentamicin (80/~g/ml), 100 /~M hypoxanthine and 32 /tM thymidine (complete DMEM-HT). Media reagents were from Whittaker Bioproducts, Walkersville, MD, U.S.A. For HAT selection, 500 ng/ml methotrexate was added to the media used the first 7 days after fusion. Serum or cloning factor supplement to the medium were in the proportions described below. Fetal bovine serum (FBS) was purchased from Gibco, Grand Island, NY, U.S.A.; normal mouse serum from Bioproducts for Science, Indianapolis, IN, U.S.A. and 'hybridoma cloning factor' (HCF) from Igen, Rockville, MD, U.S.A. The serum products were heat inactivated and sterile filtered before use. Myeloma cells SP2/0 and P3X63Ag8U.1 were originally from ATCC, Atlanta, GA, U.S.A., but have been subject to several subclonings in our laboratory. Immunization procedures 6-8-week-old mice were given a first injection i.p. with the KLH-hapten conjugate (100 /~g) in complete Freund's adjuvant (CFA) (incomplete Freund's adjuvant from Sigma Chemical Co, killed M. butyricum from Difco, Detroit, MI, U.S.A.). Subsequent injections were given intraperitoneally every three weeks with the KLH-hapten conjugate in alum (5 mg/ml, Sigma). 3 days before fusion of the secondary repsonse hybridomas (fusions no. 2 and no. 3) the immunogens were dissolved in PBS and given i.v. (50/~g protein). Fusion and establishment of antibody-producing hybridomas Spleen cells were fused with hypoxanthine guanine phosphoribosyl transferase negative myeloma cells by mixing cells in proportion 5:1 (spleen cells/myeloma) in the presence of 0.5 mg/ml polyethylene glycol (Boehringer Mannheim, Indianapolis, IN, U.S.A.) according to established procedures. All cells in one fusion were handled in a uniform fashion until the last step when they were diluted in their respective media and plated out in 96 well culture plates, 40,000 cells/well in 150 /~1 medium in fusion 1 and 2, 25,000 cells/well in fusion 3. Fusions 1 and

2 were propagated in complete DMEM-HT medium supplemented with 10% FBS or 5% FBS and 5% NMS, fusion no. 3 in complete DMEM-HT with 1% Nutridoma, 2% BSA (both from Boehringer Mannheim) and 2% cloning factor (HCF) or 5% NMS, 1% Nutridoma, 2% BSA and 2% HCF. The cultures were incubated at 37°C humidity and 7% CO 2. Growth positive cultures were tested for specific antibody production as determined in ELISA after 10-20 days. Selected, antibody positive cell cultures were propagated and subsequently cloned, at least twice, by limiting dilution. Cell lines for which supernatants showed an antibody titer of > 1/16 in ELISA and which tolerated freezing and thawing were considered established, monoclonal antibody-producing hybridomas.

Results and discussion

Prefusion characteristics The serum antibody responses for the mice subjected to repeated immunizations were of similar magnitude and IgM/IgG distribution as our current experience of a large number of immunizations in these mouse strains with hapten immunizations (data not shown). For the mouse only given one immunization 14 days prior to fusion (no. 1), a substantial IgM and a minor IgG response in the serum was noted at day 12 (data not shown). For fusion 1, 30 x 10 6 cells were isolated from the spleen, for fusion 2 100 X 106 and for fusion 3 90 × 106. Comparison of yields from hybridomas propagated in media with or without mouse serum supplement The overall growth of hybridomas was increased in NMS-propagated cultures (62-92% compared to 30-44% for F B S / H C F cultures), their number of cultures with cells producing specific antibody was increased and the success rate of subcloning in NMS medium was higher than compared to F B S / H C F cultures. Taken together, the number of eventually established monoclonal antibody-producing hybridomas was 7-10 times higher for fusions involving NMS cultures (Table I). In particular, IgM-secreting cul-

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TABLE I N U M B E R O F H Y B R I D O M A S ESTABLISHED F R O M P R I M A R Y A N D S E C O N D A R Y I M M U N E RESPONSES, C U L T U R E D IN T H E PRESENCE O F M O U S E S E R U M O F F B S / H C F - S U P P L E M E N T E D M E D I A Yield of the fusions (last three columns) based on initial n u m b e r of cells in each fusion, respectively. Fusion no.

Strain × fusion partner

Type of immune response

medium supplement

1

A/J×

Primary a

FBS e NMS-FBS f

no. of plates a

%growthb

no. of wells with antibody activity c per 106 cells

no. of cultures for subcloning per 106 cells

no. of stable clones per 106 cells

IgG

IgM

4

44

0

0.26

0.13

0.065 g

5

65

0.16

3.85

3.44

0.677 s

16

36

0.21

0.06

0.05

0

5

92

0.57

2.92

0.99

0.312 g

HCF k

25

30

1.27 n

0.65

0.067 p

NMS-HCF m

22

62

2.01 n

1.78

0.492 p

P3X63

2

A/J × P3X63

Secondary h

FBS e NMS-FBS f

126GIX + × SP2/O

a b c d e f g

h J k m

n P

Secondary j

96 well tissue culture plates used for initial plating of fusion. % of microwells with visible cell cultures after 2 - 3 weeks. Determined by immunogen-specific ELISA of supernatants. 200 /~g K L H - h a p t e n in PBS i.p. 4 days prior to fusion. 10% FBS in complete D M E M - H T (see materials and methods section). 5% normal mouse serum, 5% FBS in complete D M E M - H T . All IgM. One boost i.p. 4 days prior to fusion. Boosted twice i.p. and finally once i.v. 4 days prior to fusion. 2% hybridoma cloning factor (HCF), 2% BSA, 1% Nutridoma in complete D M E M - H T . 5% normal mouse serum, 2% HCF, 2% BSA, 1~ Nutridoma in complete D M E M - H T . Not isotyped at this stage. All IgG.

tures in fusions 1 and 2, established from a primary and secondary immune response, respectively, were considerably increased in the NMS-containing cultures (14.8/plate compared to 1/plate in fusion 1, 8.8/plate (NMS) and 0.25/plate (FBS) in fusion 2). For these two fusions, it must be noted that we selected cultures for subsequent cloning that produced antibodies with cross-reactivity to a hapten similar to the immunogen. Thus the data should be treated with some caution since our selection criteria were different from fusions where high avidity antibodies to the immunogen is selected for. In fusion 3, however, we only selected cultures with high titer, anti-immunogen-specific antibodies. Here, NMS-supplemented medium resulted in

a 7-fold increase of established, monoclonal antibody-producing hybrids (Table I). The present report agrees well with the findings of Raybould et al. (1988), who were able to establish antibody-producing rabbit-murine hybridomas only when using rabbit serum-supplemented medium (FBS supplement brought no antibodyproducing hybrids). The principle of using serum supplement of the same species as the cell origin has to our knowledge not been reported for antibody-producing mufine hybridomas. The cause for the beneficial effect noted in the present report is unknown. However, this modification of culture conditions may be applied to hybridoma production for antigens where monoclonal antibodies have been difficult to establish or where a larger

42 s a m p l e of a n t i b o d y specificities occurring after i m m u n i z a t i o n is of p a r t i c u l a r interest.

Acknowledgements W e are i n d e b t e d to K i m D. J a n d a a n d Brent L. I v e r s o n for use of their haptens. T h e skillful technical assistance of K a t e C a m e r o n a n d D i a n e Schloeder is gratefully a c k n o w l e d g e d . This w o r k was s u p p o r t e d b y the Sven a n d D a g m a r Sal6n F o u n d a t i o n .

References Goding, J.W. (1986) Monoclonal Antibodies: Principles and Practice, 2nd ed. Academic Press, London, p. 59. Janda, K.D., Benkovic, S.J. and Lemer, R.A. (1989) Catalytic antibodies with lipase activity and R or S substrate selectivity. Science 244, 437. Ki)hler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 459. Raybould, T.J. and Takahashi, M. (1988) Production of stable rabbit-mouse hybridomas that secrete rabbit mAb of defined specificity. Science 240, 1788.