Nature Vol. 276 16 November 1978
`
`269
`
`strains, small but significant interstrain differences in lactate
`dehydrogenase activity were observed“. Therefore, it seems
`that biochemical data on the ova of random bred Swiss mice may
`not be generally representative.
`We conclude that
`the absolute level of ova GPI activity,
`although strictly regulated within each strain, can vary over a
`considerable range and yet permit successful reproduction. This
`situation now provides a unique opportunity for
`further
`exploration
`into
`developmental gene
`regulation during
`oogenesis and also during preimplantation development”'”.
`This study was supported by the MRC of Canada (the MRC
`Group in Developmental Neurobiology). We thank R. Camp-
`bell for technical assistance.
`
`A. C. PETERSON
`G. G. WONG
`
`Department of Neurosciences.
`McMaster University,
`1200 Main Street West,
`Hamilton, Ontario, Canada LSS 419
`
`pwsaaewwr
`
`Received 12 June: accepted 23 August 1978.
`Carter, N. D. & Parr, C. W. Nature 216, 511 (1967).
`DcLorenzo. R. J. & Ruddle. F. H. Biochem. Gcnel. 3, 151 (1969).
`Carter, N. D. & Yoshida. A. Biochi'm. biophys. Asia 181, 468—470 (1969).
`Peterson, A. C.. Frair. P. M. & Wong. G. Biachem. Genet. (in the press).
`Paigen. K , Swank, R T., Tomino. S. & Ganschow, R. E. I. CellPltysiol 85, 379 (1975).
`Sorensen, R. A. & Wasserman. P. M. Dev] Biol. 50, 531436 (1976).
`Stevens, W. L. J. Gum. 43. 301407 (1942).
`Padua, R. A., Bulfield. G. & Peters, J. Biochem. Genet. 16, 127—143 (1978).
`Epstein. C. L. Biol. Reprad. 12. 827105 (1975).
`10. Epstein, C J & Smith, S. A. Deal Bin]. 33, 1717184 (1973).
`11. Brinster, R. L. J'. Reprod. Fen. 17, 139446 (1968).
`12. Chapman, V. M.. Whitten. W. K. & Ruddle. F. H. Dev! Biol. M. 153—158 (1971).
`13. Brinster, R. L. Biochem. Genet. 9, 1877191 (1973).
`
`
`A better cell line for making
`hybridomas secreting specific antibodies
`
`FUSION of myeloma cells which grow in tissue culture with
`spleen cells from an immunised mouse provides a general
`method for obtaining cell lines (hybridomas) which make anti-
`body of the desired specificity"? Hybrids derived from these
`myelomas make the immunoglobulin (lg) heavy and light chains
`of the myeloma parent as well as the antigen—specific heavy and
`light chains of the spleen cell parent. In conditions in which the
`two heavy and two light chains associate randomly, a hybridoma
`would make 10 distinct Ig molecules, and the specific antibody
`would comprise only 1/ 16 of the total 1g“. To obtain hybrid-
`omas making only the specific antibodies requires a tumour cell
`fusion partner that itself makes no Ig but which can nevertheless
`be fused with spleen cells to obtain hybrids secreting only the
`specific antibody. We report here the identification of such a cell
`line, SpZ/O-Ag14.
`Sp2/0-Agl4 was isolated as a re-clone of SpZ/HL-Ag, itself
`derived in several steps from Sp2/HLGK, a hybrid between a
`BALB/c spleen cell contributing a y2b (H) and K (L) chain with
`anti—sheep red blood cell activity and the myeloma cell line
`X63—Ag8 (11 (G) and K (K))2. Sp2/0-Ag14 is resistant to 20 pg
`ml’l 8—azaguanine, dies in HAT supplemented medium and
`synthesises no lg chains. It has about 73 chromosomes which is
`only eight more than the chromosome number of X63-Ag8. a
`cell line commonly used to generate hybridomas.
`Sp2/0—Ag14 was fused with spleen cells of mice immunised
`with trinitrophenyl derivatised keyhole limpet haemocyanin
`(TNP—KLH)
`to generate hybridomas making TNP-specific
`immunoglobulin (Fig. 1). Fusions were done with either poly-
`ethylene glycol (PEG) 1500 or Sendai virus, and transferred to
`medium either containing or lacking mouse peritoneal exudate
`cells. Growth was observed in only eight culture Wells, all from
`the set that were fused with PEG and grown with peritoneal
`exudate cells.
`
`002870836/7 8111276~0269lé01 .01)
`
`
`
`lmmunoglobulin from TNP-specific hybridomas analysed
`Fig. l
`by SDS—polyacrylamide gel electrophoresis. To obtain hybridomas
`making lg specific for the hapten trinitrophenyl, BALB/c mice
`were immunised i.p. with trinitrophenyl-derivitised keyhold limpet
`haemocyanin (TNP-KLH) (from H. Kiefer), first with 100 ug
`TNP-KLH contained in 0.2 ml emulsion of 50% phosphate-
`bufiered saline (PBS), 50% complete Freund‘s adjuvant (Difco).
`35 days later a second injection of 100 pg TNP-KLH in 0.2 ml PBS
`was made i.p., and 5 days later the spleens of these mice were
`prepared for fusion. Fusion with PEG 1500 was as described by
`Galfre et (11.9, except that the medium used was R medium, RPMI
`1640 medium (Gibco) supplemented with 30mM HEPES (Flow).
`Here 3 x 107 Sp2/0-Ag14 and 5 X 107 spleen cells were centrifuged
`together at 200 g for 5 min and resuspended slowly in 0.6 ml 50%
`PEG in Dulbecco’s modified Eagle‘s medium (Flow). After 1 min
`at 37 °C, 20 m1 of R medium was added slowly. The cells were then
`centifuged and resuspended in 20 ml of R medium supplemented
`with 10% fetal calf serum (Gibco) (RF medium) and 0.2 ml of this
`suspension was then distributed to each of 200 wells containing
`0.8 ml RF medium. One hundred of these wells also contained
`2 x to5 mouse peritoneal exudate cells. After 24 h incubation, 1 ml
`RF supplemented with hypoxanthine, aminopterin and thymidine
`(HAT) was added to each well. Every 2—3 days, 1 ml of the medium
`was replaced with fresh RF+HAT. After 2 weeks, growth was
`observed in eight wells of the peritoneal exudate supplemented
`wells and the cultures were tested for immunoglobulin production
`by testing the culture medium for lysis of protein A coated sheep
`red blood cells (SRC) for nonspecific lgm and of TNP coated SRC
`(for TNP specific activity). After 1—2 months the cells from TNP-
`specific wells were cloned by plating in soft agar11 or in methyl
`cellulose”. The soft agar clones were overlaid with TNP coated
`SRC, and clones lysing the TNP coated SRC were transferred to
`liquid medium. The cells were grown in the presence of MC leucine
`to label secreted lg which was analysed by SDS polyacrylamide gel
`electrophoresis as described“. The hybridoma cell lines analysed
`were hy5.19 (a), hy2.15 (b), hyl.2 (c) and hy3.3 (d).
`
`Supernatant of four of the eight growth-positive wells lysed
`protein A but not TNP-coated sheep red blood cells (SRC)
`suggesting that the Ig made by these cells did not have TNP
`specificity. The Ig secreted by these cells was analysed by
`SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
`found to be lgM (data not shown). The remaining four growth
`positive wells lysed both TNP and protein A SRC and cells from
`these wells were cloned and analysed further, as described
`below. SDS-PAGE analysis suggested that
`these cell
`lines
`secreted lgG (Fig. 1). Ouchter10ny analysis of culture super-
`natants using class-specific antisera (Litton Bionetics) indicated
`that hyl.2 makes an lgGZa, and that hy2.15, hy3.3 and hy5.19
`make IgGl and that all four TNP-specific hybridomas make a K
`light chain. The SDS-PAGE analysis shown in Fig. 1 indicates
`that the clones can be distinguished by the mobility of the light or
`heavy chain, implying that these cell lines are making difierent
`antibodies.
`We have examined the TNP-specific hybridomas for re-
`expression of the SRC-specific lgGZb of the progenitor cell line
`Sp2/HL-Ag14 by testing for SRC-specific plaques. No plaques
`
`© Macmillan Journals Ltd 1978
`
`Mylan v. Genentech
`|PR2016-00710
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`Merck Ex. 1117, Pg. 1
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`Mylan v. Genentech
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`Merck Ex. 1117, Pg. 1
`
`

`

`270
`
`were found and we conclude that revertants constitute fewer
`than one in 5 X 104 of the TNP—specific hybridoma cells.
`The karyotypes of the TNP-specific cell lines provide evi—
`dence that these cells are hybrids of Sp2/O-Ag14 and mouse
`cells. Normal mouse cells and Sp2/0-Ag14 contain 40 and 73
`chromosomes, respectively. The TNP—specific cell lines hy1.2,
`hy2.15, hy3,3 and hy5.19 contain approximately 100, 105, 106
`and 99 chromosomes, respectively, and we suppose that fusion
`yielded hybrids that segregated better growing cells that had lost
`7—14 chromosomes.
`Evidence has been presented that X63-Ag8 cells pref-
`erentially fuse with the dividing cells of the spleen2‘6'7, which for
`the B—cell compartment consist mainly of Ig-secreting, plaque-
`forming cells. Sp2/O-Ag14 seems to display the same pref—
`erence. In the population of spleen cells used for this fusion,
`approximately 0.4% of the cells made indirect (IgG) TNP-
`specific plaques and 0.004% made direct (IgM) TNP plaques.
`Assuming that 1% of the spleen cells are plaque forming cells8
`one would anticipate that about 40% of the hybridomas would
`make lgG with TNP specificity, as was observed. On the other
`hand, about 90% of the nonspecific plaque-forming cells of the
`spleen make IgM rather than IgGs, so that one would anticipate
`that among the nonspecific hybridomas, 90% would also make
`IgM, a figure that is consistent with our observation that all four
`nonspecific hybridomas examined made IgM.
`To obtain high—titre preparations of the TNP—specific lg, 106
`TNP-specific hybridoma cells were injected intraperitoneally
`into BALB/c mice. All caused tumours and yielded ascites fluid
`with titres generally about 100 times higher than for the culture
`supernatants.
`The efliciency of fusion with Sp2/0-Ag14 has been variable.
`In our early experiments, such as the fusion described above, we
`obtained fewer hybrids than we ordinarily obtain with X63—
`Ag8. However, in more recent experiments, fusions with Sp2/0-
`Ag14 have often been more efficient, in particular in fusions
`with lipopolysaccharide stimulated spleen cells or with large
`spleen cells (F. Melchers, Clark and GK, unpublished obser—
`vations). We consider that Sp2/0-Ag14 can yield as many
`hybrids as X63-Ag8.
`Alternative methods exist for obtaining hybridomas that do
`not express the myeloma Ig. It has been possible to isolate
`re—clones that have lost the expression of the nonspecific heavy
`or light chainsz. This problem has been reduced by fusing with
`NSI-Ag 4/ 1, a derivative of X63 that makes the K but not the yl
`chain of the myelomaz. We anticipate that Sp2/0-Ag14 will
`prove yet a better cell line for generating hybridomas making
`truly monoclonal antibodies.
`
`MARC SHULMAN*
`C. D. WILDE‘l‘
`GEORGES KOHLER*
`
`*Basel Institute for Immunology,
`Grenzacherstrasse 487,
`Postfach, 4005 Basel 5, Switzerland
`
`TMRC Laboratory of Molecular Biology,
`Hills Road, Cambridge, UK
`Received 21 July; accepted 20 October 1978.
`
`Kohler, G. 31 Milstein, C. Nature 256, 495497 (1975).
`Kohler, G. & Milstein, C. Eur, J. Immurti 6, 5 l l—Sl9 (1076)
`were
`. Current Topics in Microbiology and Immunology (eds Melchcrs, R, Potter, M, & Warner, N .}
`(Springer, Berlin, in the press).
`4. Kohlcr. G.. Hengartner. H. & Shulman. M. Eur. J. Immun. 8. 82—88 (1978).
`5. Shulman, M. & Kohler, G. Nature 274, 917919 (1978).
`6. Kohler, G. & Shulman, M. in Current Topics in thhifllflgy and Immunology 1434149
`(Springer, Berlin. 1978).
`7. Andersson, J. & Melchers F. in Current Topics in Microbioiogy and Immunology 130—140
`(Springer, Berlin. 1978).
`8. Andersson, J., Coutinho, A. dc Melchers, F. J. exp. Med. 145, 1520—1530 (1977).
`9, Galfre, CL, Howe, S., Milstein, C,, Butcher, G. & Howard, J. Nature 266, 550—552 (1977).
`10. Gronowitz, E., Coutinho. A. & Mclchers, F. Eur. J. Immun. 6. 588—590 (1976).
`11. Cotton, R., Secher, D. & Milstein, C. Eur. 1. human 3, 135—140 (1973).
`12. Shulman. M.
`in Research Methods in Immunology (eds Lelkovits,
`I. & Pernis, 13.)
`(Academic, New York, in the press).
`
`Nature Vol. 276 16 November 1978
`
`
`Activation of latent Epstein—Barr
`virus by antibody to human IgM
`
`EPSTEINvBARR VIRUS (EBV) is the causative agent of most
`infectious mononucleosis‘ and is also associated with two human
`tumours, Burkitt’s
`lymphoma (BL)2 and nasopharyngeal
`carcinomal. Lymphoid cell
`lines of B-cell origin have been
`established from patients with these diseases, and from
`lymphocytes transformed in vitro by EBVE. In lymphoid cell
`lines the lytic viral cycle is usually repressed even though the
`cells carry multiple copies of the EBV genome. These cells
`therefore provide a valuable model for the study of latent EBV
`infection. Although EBV is ubiquitous,
`its association with
`human malignant disease is to a large extent both ethnically and
`geographically restricted2 and it
`is therefore of interest
`to
`understand the host factors involved in the regulation of the
`expression
`of
`the
`latent EBV genome.
`In
`infectious
`mononucleosis, EBV infection is associated not only with a
`specific anti-EBV antibody2 and T-lymphocytc response4 but
`also with an increase in nonspecific lgM productions. EBV—
`infcctcd B lymphocytes produce polyclonal lg (ref. 6) and the
`majority of established EBV genome-positive lymphoid cell
`lines possess surface IgM (ref. 7). We report here that treatment
`of several human lymphoid cell lines with antisera to human IgM
`activates the latent EBV genome to give a marked increase in
`EBV—specific early antigen (EA). Also, in some cell lines this
`treatment induces an increase in virus capsid antigen (VCA).
`Treatment of BL-derived Raji cells with antiserum directed
`against human immunoglobulins increased the number of
`cells expressing EBV EA by up to 5,000—fold, to give 11%
`ISA—positive cells determined by either direct or
`indirect
`immunofiuorescencc (Table l), and confirmed by EA-spccific
`complement fixation". The fluorescence was specific for EA, as
`no positive cells were observed after staining with either control
`EBV-negative human serum, or with anti-VCA—positive EA-
`negativc antiserum. EBV—specified antigens can be induced in
`lymphoid cell lines by halogenated pyrimidincsg’m, inhibitors of
`protein synthesis“ or the tumour promotor 12—O-tetradecanoyl
`
`Induction of EA in Cultures of Raji cells treated with
`Fig. 1
`various dilutions of antiserum. Cells were cultivated for 72 h with a
`particular dilution of antiserum in the presence of
`IUdR
`(ZSug ml”). The number of EA—positive cells was then deter—
`mined on acetonevfixed smears by indirect immunofiuorescence.
`None of the antisera had any eflcct on cell viability at the dilutions
`shown in the figure. 0, Sheep anti»total human immunoglobulins;
`0, goat anti«human IgM; 13, goat anti»human IgA; I, goat anti»
`human IgG; A, goat anti~human lgD; 0, goat anti-human IgE; A,
`IUdR alone (25 ug ml”).
`
`50
`
`NwAOOO
`
`'5
`
`
`
`3;",EAApositivecells
`
`§:
`
`23456
`
`log antiserum dilution
`
`0028v0836/78/0276-0270301.00
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`© Macmillan Journals Ltd 1978
`
`Merck Ex. 1117, Pg. 2
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`Merck Ex. 1117, Pg. 2
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