The Journal of Immunology
`
`2453
`
`Reprinted with permission from Nature 256 (5517): 495– 497 (1975). Copyright © 1975 Macmillan Publishers Ltd.
`
`Nature Vol. 256 August 7 1975
`
`495
`(MOPC 21) is an JgGJ (K) which has been
`The protein sccreled
`Equal numbers of cells from each parental
`fully sequenced""·
`of fused cells
`cultures
`Continuous
`line were fused using inactivated
`Sendai virus• and samples
`contining 2 10' cells were grown in selective medium in
`specificity
`of predefined
`antibody
`secreting
`separate dishes. Four OUI or ICn dishes shO\\Cd growth in
`hybrid
`independent
`medium and these "ere taken as
`selective
`by means of
`of predefined spcciftc antibodies
`THE manufacture
`from single fusion events. The karyotype
`lines, probably derived
`interest. There
`permanent tissue culture cell lines is of general
`of the hybrid cells after 5 months in culture was just under the
`number of permanenl cultures of
`are at present a considerable
`sum of the 1wo parental lines (Table 1). Figure I shows the
`been used to
`procedures have
`and screening
`myeloma cells'·'
`(IEF) pattern of the secreted products
`of
`isoelectric focusing'°
`in some of them. This, hov.ever, is
`reveal antibody activity
`different lines. The hybrid cells (samples c-li in Fig. I) give a
`source of monoclonal antibodies of predefined
`not a satisfactory
`much more complex pattern than either parent (a and b) or a
`of a number of
`We describe here the derivation
`specificity.
`mixture of the parental lines (m). The important feature of the
`red blood
`tissue culture cell lines which secrete anti-sheep
`new pa11ern is the presence of exlra bands (Fig. I, arrows).
`cell (SRBC) antibodies. The cell lines are made by fusion of a
`These new bands, however, do not seem to be the result of
`mouse myeloma and mouse spleen cells from an immunised
`this is indicated by the lEF
`in primary structure;
`differences
`of the
`donor. To understand the expression and interactions
`separate the heavy
`pattern of the products after reduction to
`lines. fusion experiments between
`l g chains from the parental
`and light chains (Fig. IB). The IEF pattern of chains of the
`two known mouse myeloma lines were carried
`out.
`hybrid clones (Fig. IB, g) is equivalent
`to the sum of the IEF
`chain result� from the integrated
`Each immunoglobulin
`pattern (a and b) of chains of the parental clones with no
`of one of several V and C geues coding respectively
`expression
`evidence of extra products. We conclude 1hat, as previously
`only
`Each cell expresses
`and constant sections.
`for its variable
`shown with interspecies hybrids ... , new lg molecules arc
`revie"ed in
`exclusion;
`one of the two possible alleles (allelic
`ref. 3). When two antibody-producing
`produced as a result of mixed association betv.ecn heavy and
`cells are fused, the
`light chains from the two parems. This process is intracellular
`and although
`products of both parental lines are expressed'·',
`as a mixed cell population does not give rise to such hybrid
`the light and heavy chains of both parental lines are randomly
`(compare 111 and g, Fig. I A). The individual cells
`joined, no evidence of scrambling of V and C sections is
`molecules
`must
`This result shows
`be able to express both isotypes.
`therefore
`These results, obtained in an heterologous system
`observed'.
`of one isotypc and idiotype
`the expression
`that in hybrid cells
`cells of rat and mouse origin, have now been con­
`involvin,g
`firmed by fusing two myeloma cells of the same mouse strain,
`does no1 exclude the expression of another: both heavy chain
`
`Chains B
`
`H(Pl)f I H(P3)
`
`L(P3)
`
`L(Pl)
`
`. ' ' .
`--
`
`Fig. l Autoradiograph
`of labelled compo­
`nents secreted by the parental
`and hybrid cell
`lines analysed by IEF before (A) and after
`were incubated
`reduction {8). Cells
`in the
`and the supernatant
`presence of "C-lysine"
`applied on polyacrylamide slabs.
`A, pH range
`6.0 (bottom) to 8.0 (top) in 4 M urea. 8, pH
`range S.0 (bonom) to 9.0 (top) in 6 M urea;
`the supernatant was
`incubated for 20 min nt
`37 •c in the presence of 8 M urea, 1.5 M
`and 0.1 M potassium phos­
`mercap1oe1nanol
`phate pH 8.0 before being applied to the right
`from parental cell lines
`slab. Supcrna1an1s
`in: a, Pl Bui; b, P3-X67Ag8; and m, mixture
`of equal number of Pl Bui and P3-X67Ag8
`cells. Supernatants from two independently
`derhed hybrid lines are shown: c-f, four
`sub<:lones from Hy-3; g and h, 1wo subclones
`out"' using
`from Hy-B. Fusion was carried
`10' cells of each parental line and 4,000
`Sendai
`units inactivated
`haemagglu1ina1ion
`virus (Searle).
`Cells were divided into ten
`equal samples and gro\\ n separa1ely in
`s<:lttthe medium (HAT medium, ref. 6).
`Medium was changed every 3 d. Successful
`hybrid lines were obtained in four of the cul-
`Hy-B
`IEF pallerns.
`1ures, and all gave similar
`and Hy-3 were funher cloned in soft agar".
`L, Light; H, heavy.
`
`a b c d e mf m g b
`
`a /i b g t1
`
`and under­
`and provide the background for the derivation
`and y2a) and both V" and both VL regions
`·sotypes (yl
`hybrid lines in which one of
`standing of antibody-secreting
`markers for
`There are no allotypic
`are expressed.
`(idiotypes)
`the C" region 10 provide direct proof fortheexpression of both
`spleen cell.
`the parental cells is an antibody-producing
`parental C,. regions. But this is indjca1ed by the phenotypic
`Two myeloma cell lines of BALB/c origin "ere used. Pl Bui
`link between the V and C regions.
`is resistant
`does not gro" in
`to 5-bromo-2'-deoxyuridine',
`a myeloma protein,
`6) and secretes
`hybridi­
`medium (HAT, ref.
`different
`Figure l A shows that clones derived from
`selective
`Adj PCS, which is an lgG2A (K), (ref. I). Synihcsis is not
`of one line are indistin­
`and from subclones
`su1ion experiments
`The second
`balanced and free light chains arc also secreted.
`This has also been observed in other experiments
`guishable.
`is resistam to
`cell line, P3-X63Ag8, prepared from P3 cells•,
`(data 001 sho"n). Variants were, however, found in a survey of
`20 µg ml-1 8-azaguanine and <lOl.'S not grow in HAT medium.
`100 subclones. The difference is often associated
`with changes
`
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`2454
`
`496
`
`PILLARS OF IMMUNOLOGY
`
`Nature Vol. 256 August 7 1975
`Fig. 2 Isolation of an anti-SR BC antibody­
`secreting cell clone. Activity was revealed by
`SRBC. Direct plaques
`a halo of haemolysed
`given by: a, 6,000 hybrid cells Sp-1; b, clones
`grown in soft agar from an inoculum of 2,000
`Sp-I cells; c, recloning of one of the positive
`clones Sp-1/7; d, higher mag nification of a
`positive clone. Myeloma cells (107 P3-X67A
`gS) were fused to 108 spleen cells from an
`im munised BALB/c mouse. Mice
`were im­
`munised by intraperitoneal injection of 0.2 ml
`packed SRBC diluted I :JO, boosted after l
`
`month and the spleens collected 4 d later.
`After fusion,
`cells (Sp-I) were grown for 8 d
`in HAT medium, changed at 1-3 d intervals.
`Cells were then grown in Dulbecco modified
`Eagle's medium, supplemented
`for 2 weeks
`
`
`with hypoxanthine and thymidine. Forty days
`after fusion the presence of anti-SRBC act­
`ivity was revealed as shown in a. The ratio of
`plaque forming cells/total number of hybrid
`cells was 1/30. This hybrid cell population
`was cloned in soft agar (50% cloning ef­
`A modified plaque assay was used to
`ficiency).
`
`reveal positive clones shown in b-d as follows.
`When cell clones had reached a suitable size,
`conditions with
`
`they were overlaid in sterile
`2 ml 0.6% agarose in phosphate-buffered
`saline containing
`25 µI packed SRBC and
`0.2 ml fresh guinea pig serum (absorbe d with
`SRBC) as source of complement. b, Taken
`at 37 °C. The ratio
`
`after overnight incubation
`nwnber of clones was 1/33. A
`of positive/total
`suitable positive
`clone was picked out and
`This clone was called
`grown in suspension.
`Sp-1/7, and was recloned as shown inc; over
`
`
`90% of the clones gave positive Jysis. A second
`in which 10• P3-X67Ag8cells were
`experiment
`fused with 108 spleen cells was the source of
`a clone giving rise to indirect plaques (clone
`plaques were produced by
`
`Sp-2/3-3). Indirect
`the addition of I :20 sheep anti-MOPC 21
`antibody to the agarose overlay.
`
`in the ratios of the different chains and occasionally with the
`
`assays13 have been used to detect specific clones and representa­
`
`total' disappearance of one or other of the chains. Such events
`
`
`tive clones of both types have been characterised and studied.
`
`are best visualised on IEF analysis of the separated chains
`The derived lines (Sp hybrids) are hybrid cell lines for the
`(for example, Fig. !h, in which the heavy chain of P3 is no
`
`following reasons. They grow in selective medium. Their
`karyotype after 4 months in culture (Table I) is a little smaller
`
`longer observed). The important point that no new chains are
`detected by £EF complements a previous study• of a rat-mouse
`than the sum of the two parental lines but more than twice the
`hybrid line in which scrambling of V and C regions from the
`
`chromosome number of normal BALB/c cells, indicating that
`light chains of rat and mouse was not observed. �n this study,
`the lines are not the result of fusion between spleen cells. In
`both light chains have identical c. regions and therefore
`
`addition the lines contain a metacentric chromosome also
`scrambled VL-CL molecules would be undetected.
`On the other
`
`present in the parental P3-X67Ag8. Finally, the secreted
`MOPC 21 protein in addition to new,
`
`
`hand, the heavy chains are of different subclasses and we
`immunoglobulinscontain
`expect scrambled VH-CH to be detectable
`by IEF. They were
`unknown components. The latter presumably represent the
`not observed in the clones studied and if they occur must do
`chains derived from the specific anti-SRBC antibody. Figure 3A
`
`so at a lower frequency. We conclude that in syngeneic cell
`sh.ows the lEF pattern of the material secreted by two such
`cell hybrids) V-C integration
`hybrids (as well as in interspecies
`is
`Sp hybrid clones. The lEF bands derived from the parental P3
`
`
`not the result of cytoplasmic events. Integration as a result of
`line are visible in the pattern of the hybrid cells, although
`
`
`
`DNA translocation or rearrangement during transcription is
`obscured by the presence of a number of new bands. The
`
`also suggested by the presence of integrated mRNA molecules11
`pattern is very complex, but the complexity of hybrids of this
`and by the existence of defective heavy chains in which a
`
`
`type is likely to result from the random recombination of
`deletion of V and C sections seems to take place in already
`chains (see above, Fig. !). Indeed, lEF patterns of the reduced
`committed cells12•
`
`material secreted by the spleen-P3 hybrid clones gave a simpler
`The cell line P3-X63Ag8 described above dies when exposed
`pattern of lg chains. The heavy and light chains of the P3
`to HAT medium. Spleen cells from an immunised mouse also
`parental line became prominent, and new bands were apparent.
`die in growth medium. When both cells are fused by Sendai
`The hybrid Sp-I gave direct plaques and this suggested that
`it produces an IgM antibody. This is confirmed in Fig. 4 which
`virus and the resulting mixture is grown in HAT medium,
`
`surviving clones can be observed to grow and become estab­
`
`shows the inhibition of SRBC lysis by a specific anti-lgM
`lished after a few weeks. We have used SRBC as immunogen,
`
`which enabled us, after culturing the fused lines, to determine
`Table 1 Numbe r of chromosomes in parental and
`
`the presence of specific antibody-producing cells by a plaque
`hybrid cell lines
`
`assay techniquei3 (Fig. 2a). The hybrid cells were cloned in
`
`Mean Number of chromosomes per cell
`
`Cell line
`soft agar14 and clones producing antibody were easily detected
`65
`66,65,65,65,65
`P3-X67Ag8
`by an overlay of SRBC and complement (Fig. 2b). Individual
`55
`Pl Bui
`Ref. 4
`40
`clones were isolated and shown to retain their phenotype as
`Mouse spleen cells
`106
`Hy-B (Pl-P3)
`almost all the clones of the derived purified line are capable of
`90
`Sp-1/7-2
`lysing SRBC (Fig. 2c). The clones were visible to the naked
`95
`Sp-2/3-3
`eye (for example, Fig. 2d). Both direct and indirect plaque
`
`112,l 10.104,104,102
`93,90,89,89,87
`97 ,98,96,96,94,88
`
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`

`The Journal of Immunology
`
`Nature Vol. 256 August 7 1975
`
`2455
`
`497
`
`antibody. IEF techniques usually do not reveal J9S lgM
`
`
`
`
`
`molecules. 1gM is therefore unlikely to be present in the
`a (Fig. 38) but µ chains should contribute
`unreduced sample
`(sample a, Fig. 3A).
`
`to the pattern obtained after reduction
`
`
`The above results show that cell fusion techniques are a
`
`powerful tool to produce specific antibody directed against a
`
`
`
`predetermined antigen. II further shows that it is possible to
`
`
`isolate hybrid lines producing different antibodies directed
`
`
`
`against the same antigen and carrying different effector func­
`
`
`tions (direct and indirect plaque).
`
`The uncloned population of P3-spleen hybrid cells seems
`
`
`quite heterogeneous. Using suitable detection procedures it
`
`
`should be possible to isolate tissue culture cell lines making
`
`
`
`
`different classes of antibody. To facilitate our studies we have
`used a myeloma parental line which itself produced an lg.
`
`
`Variants in which one of the parental chains is no longer ex­
`pressed seem fairly common in the case of PJ-P3 hybrids
`(Fig. lh). Therefore
`
`selection of lines in which only the specific
`
`
`antibody chains are expressed seems reasonably simple.
`
`
`
`Alternatively, non-producing variants �f myeloma lines could
`be used for fusion.
`We used SRBC as antigen. Three different fusion experiments
`
`
`
`
`were successful in producing a large number of antibody­
`cells. Three weeks after the initial
`producing
`fusion, 33/1,086
`Fig. 4 Inhibition of haem olysis by antibody
`by hybrid
`secreted
`
`clone Sp-J/7-2. The reactioo was in a 9-cm Petri dish with a layer
`I /80
`of5 ml 0.6% agarose in phosphate-buffered
`saline containing
`2.5 µI 20 limes concentrated
`(v/v) SRBC. Cenire well contains
`and 2.5 µI mouse serum. a,
`
`culture medium of clone Sp-1/7-2
`Sheep specific anti-mouse macroglobulin (MOPC
`104E, Dr
`Feinstein); b, sheep anti-MO PC 21 (P3) lgGl absorbed with Adj
`PC-5 (lgG2a) absorbed with MOPC 21.
`PC-5; c, sheep anti-Adj
`
`After overnight incubation at room tem perature the plate was
`developed with guinea pig serum diluted I :10 in Dulbecco's
`
`mediwn without serum.
`
`A
`
`.B
`
`clones (3 %) were positive
`by the direct plaque assay. The
`was 50%. In another experi­
`
`cloning efficiency in the experiment
`
`
`ment, however, the proportion of positive clones was con­
`0.2 %). Jn a third experiment
`the hybrid
`
`siderably lower (about
`From 157
`
`population was studied by limiting dilution analysis.
`as many as 15 had anti-SRBC activity.
`independent hybrids,
`
`
`
`The proportion of positive over negative clones is remark­
`
`ably high. It is possible that spleen cells which have been
`
`
`
`
`triggered during immunisation are particularly successful in
`giving rise to viable hybrids.
`l t remains to be seen whether
`
`
`similar results can be obtained using other antigenes.
`
`The cells used in this study are all of BALB/c origin and the
`
`hybrid clones can be injected into BALB/c mice to produce
`
`solid tumours and serum having anii-SRBC activity. It is
`
`
`
`possible to hybridise antibody-producing cells from different
`
`origins•·•. Such cells can be grown i11 vitro in massive cultures
`
`
`to provide specific antibody. Such cultures could be valuable
`
`for medical and industrial use.
`
`M RC Laboratory of Molecular Biology,
`Hills Road, Cambridge C82 2QH. UK
`
`G. KOHLER
`C. MtLSTEIN
`
`J
`
`,.
`
`·.
`
`c a
`
`c b
`
`Received May 14; accepted June 26, J97S.
`I Polter. M .• Phy.,iol. R
`..... 52. 631-719 (1972).
`1 Horibata, K., :tnd Harris.
`A. w .. Exp( Cell RPS., 60, 61-70 (1970).
`J Milstein. C .. and Munro, A. J .• in D'rfence and Recognition
`(edit. by Porte!', R. R.),
`199·228 tMTP Int. Rev. Sci., Bu1tcrnorth, London, 1973).
`•Colton, R. O. H., and Milstein. C., Nature. 244, 42-43 (1973).
`5 Schwabcr, J., and Cohen. E. P •• Proc. 11arn. Ac11d.Sci. U.S.A .• 71, 2203-2207(1974).
`• Lilllcfield. J. w .. Srienc•, 145, 709 (1964).
`7 Svas1i, J .. and Milsiein, C., Biocliem. J .• 128, 427-444 {1972).
`Adctu"bo, K., Cowan. N. J .. and Sc<:bcr, 0. S., Pro1urss in lmmunu·
`! Milstein. C.,
`logy, II, I (edit. by
`Bren1, L., and Holborow. J.). 157-168 (North-Holland.
`Amsterdam,
`1974).
`Fig. 3 Auioradiograph
`by anti­
`of labelled components secreted
`
`
`•Harris. H .. and Wa1kin•. J. F .. N"'""· 205, 640-646 (1965).
`10 Awdch, A. L .. Williamson. A. R., and Askonas, B. A .. Nature. 219, 66-67 0968).
`after(A)
`SR BC specific hybrid lines. Fractionation
`bcfore(B)and
`E. M .. Jar\'Js, J.M., and Proudfoo1,
`11 Milstein, C .. Brownlee. G. G .. Cartwright,
`was by IEF. pH gradient was 5.0 (bollom)
`to 9.0 (top)
`reduction
`N. J., Nature, 252. 354-359 (1974\.
`in the presence of 6 M urea. Other conditions as in Fig. I. Supcr­
`11 Frongione, B., and Milstein, C., Naftlrc, 244, 597-599 (1969).
`b, hybrid clone Sp-2/3-3;
`IJ Je1 ne, N. K .. and Nordin. A. A .• Sc/Pnc'e.
`140. 405 (1963;.
`a, hybrid clone Sp-1/7-2;
`natants from:
`14 Conon. R. G. H .. Secher, 0. S., and Milstein,
`C., Eur. J. lmmun., 3, 135-140
`c, myeloma line P3-X67Ag8.
`t 1973).
`
`c
`
`a c
`
`c
`
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