_
`
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`f7" '55“ #:434- ‘
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`‘ Q! .
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`BIOEPIS EX. 1022
`
`Page 1
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`BIOEPIS EX. 1022
`Page 1
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`

`

`Nawre V ol. 256 August 7 / Y7 5
`
`Vol. 256 No. 5517
`
`August 7, 1.975
`
`0 Macmillan Journals Ltd 1975
`Published weekly
`ISSN 0028-0836
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`Cover picture
`Oeser! locusts settle for the nj~ht.
`See pages 484. 48$.
`Photo: Jean Ma nuel, FAO Rome.
`
`Volume 256
`
`Whitlam, Connor and Cameron
`INTERNATIONAL l'"'EWS
`NEWS AND VlEWS
`
`August 7
`
`1975
`
`REVIEW ARTICLE
`T he origin of nuclei and of eukaryotic cells T. Cm'filier-Smirh
`
`ARTICLES
`Palaeolithic remains at the Hadur in the Afar region-G. Con•i!rus
`Integration of viral genomes- V. J\11. Zltdtmov
`
`LETrERS 1'0 NATUJlE
`Definition of 'charge on an atom' and nuture of the inductive effect(cid:173)
`S. M . Dealt rmd W. G'. Richards
`A low velocity zone underlying a fast-spreading rise crest-J. Orcutt,
`B. Ke1mett, L. Dorma11 1md W. Prothero
`Mercury contamination in a 54-m core from Jake Huleh-U. M . Cowgill
`A resonant point absorber of ocean-wave power- K. Budar and J. Fa!J1es
`Climatic reversal in northern North Atlantic- R. R. Dickson, H . H. Lamb,
`S.·A . Malmbgrg and J. M. Colebrook
`Americium 242rn in nuclear test debris-V. T. Bowen and H. D. Livingston
`Tree remuins in southern Pennine peats- J. H . Tallis
`Regularities in duration of regional desert locust plagues- Z. Waloff and S. M. Gr~en
`Development of a desert locust plague-L. V. Bennell
`Seed-borne microorganisms stimulate seedcorn maggot egg laying(cid:173)
`C. J. Eckenrode, G. E. Harmanmtd D. R. Webb
`Defensive stoning by baboons-W. J. Hamilto11 Ill, R. E. Ouskirk and W H. Buskirk
`Eccentricity-specific dissociation of vi~ual functions in patients with lesions of the
`centro! visual pathways-E. Poppe!, D. vo11 CralrtOII o11d !{. Backmrmd
`Evidence for visual function mediated by anomalous projection in goldfish(cid:173)
`D. Yager a111i S. C. Sharma
`Thymus rudiment of the nthyrnic n ude mouse-M. Holub, P. Rossman11,
`H . Tfaskalova and H. Vidmarava
`Stria ted muscle fibres difTerentiate in monolayer cultures of adult thymus reticulum(cid:173)
`H. Wekerle. 8. Pttterson, U.·P. Ketelsen and M. Feldman
`Continuous cultures of fused cells secreting antibody of predefined specificity(cid:173)
`G. Koltler rmd C. Milstein
`Naturally occurring cytoto.~ic tumour reactive ami bodies directed against type C viral
`envelope antigents-S. £. Martin a11d W. J . Martin
`Antigen formation in metal contact sensitivity-f. M. Jones and H. E. Amos
`Induced thermal resistance in Hel a cells-E. W. Gerner and M. J. S ch11eider
`Regional turnover and synthesis of catecholarnines in rat hypothalamus(cid:173)
`D. fl. G. Ver.lteeg, J. wm der Gngren and J . 1\1. van Ree
`Rate of nucleologenesis as a measure of g~:ne activity-C. de Ia Torre,
`M. £. Fel'ltlllidn-Gomez and G. G'imenez-f,<fartill
`-5S RNA secondary structure- G. E. Fox and C. R. Woese
`Diffcrenrial effect of plasma fractions from normal and tumour-bearing rats on
`nuclear RNA restriction-D. £. Schumm and T. E. Webh
`Early role during r.hemical evolution for cytochrome P4SO in mcygen detoxilication
`R. H. lflickrvmosinglre ami C. A. Vil/ee
`Human embryonic hacmoglobins including a comparison by homology of the
`· human ~and n chains- H. Kamu:ora tmd H. Leltmamt
`Lycorine as 3n inhibitor of ascorbic acid biosynthesis-a. Arrigoni,
`R. A . Lisa und G'. Calabrese
`lntracellular killing of Listeria monocytogenes by activated macrophagcs (Mackancss
`system) is due to antibiotic- ?. Cole a11d J. Brostoff.
`
`447
`448
`455
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`463
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`468
`47l
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`473
`
`41S
`476
`478
`
`479
`482
`482
`484
`486
`
`487
`488
`
`489
`
`490
`
`491
`
`493
`
`495
`
`498
`499
`500
`
`SOl
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`S03
`505
`
`S08
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`509
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`511
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`513
`
`SIS
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`BIOEPIS EX. 1022
`Page 2
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`

`

`495
`
`The protein sc.:rctcd ( M 0 PC 21) •~ ·n I gG 1 ( 1\) ·.vhich has been
`f~lly scQuenc.:cd' · 1
`. ~qu?l nu~1bers o r cel:s from each parental
`line were fused usmg ma.;:uvatcd Sena.,; virus• and samples
`contining 2 J( 10' cells \\ere gr0 11 n in seketive medium in
`sep:1ratc dbhes. Four out of ren disbes shOI\•d Krowth in
`selective medium and these were taken as indepenue,,: "-tb~lu
`lines, probably derived from single fusion events. The karyotype
`of the hybrid cells after 5 months in culture \\US just under the
`sum of the two parental lines (Table !).',Figure 1 shows the
`isoelectric focusi ng 10 {IEf) pattern of the secreted products of
`difl'erent lines./The hybrid cells (samples c-h in Fig. I) give a
`much more complex pattern than either parent (a and b) or a
`mixture of the parental lines (m). The important feature of the
`new p:lttern is the presence of extra bands (fig. I, arrows).
`T hese new bands, however, do not seem to be the result of
`diO'erences in primary structure; this is indicated by the IEF
`pattern of the products after reduction to separate the heavy
`and light chains (Fig. I B). The IEF pattern of chains of the
`hybrid clones {Fig. 1 B, g ) is equivalent to the sum of the lEF
`pattern (a and b) of chains o f the parental clones with no
`evidence of extra products. We conclude that, as previously
`shown with interspecies hybrids'·', new lg molecules are
`produced as a result of mixed nssociation between heavy and
`light chains from the two parents. This process is intracellular
`as a mixed cell population does not give rise to such hybrid
`molecules (compare m and g , Fig. I A). The individual cells must
`therefore be able .to ·express both isotypes. T his result shows
`that in hybrid cells the expression of one isotype and idiotype
`does not exclude the expr('SSion of another : both heavy chain
`
` \Cttw<' Vol. !j6 ...1 u~usr 7 1\175
`L--·
`1 i Continuous cultures of fused cells
`\ 5ecrding antibody of predefined specificity
`l fll• r .anufa~:ture of predefined specitic antibodies by means of
`:L ~rr.; Jnent tissue culture cell lin~s is of general interest. There
`:r. at present a considerable number of perman.:nt cultures of
`
`1:
`
`I ~cloiT'a cells'· 2 and screeni:lg procedures h;>ve been used to
`
`lr~>eal antibody activitY. in some of them. This, however, is
`:111 a satisfactory source of monoclonal anti bodies of predefined
`,"ecificity. We describe here the derivation of a number of
`• ,;;sue culture cell lines which secrete anti-sheep red blood
`,~11 (SRBC) antibodies. The cell lines are made by fusion of a
`1nouse myeloma and mouse spleen cells from an immunised
`dl)nor. To understand the expression and interactions of the
`Jg chains from the parental lines, fusion experiments between
`1110 known mouse myeloma lines were carried out.
`Each immunoglobulin chain result~ from the integratl!d
`!·.pression of one of several V and C ge.aes coding respectively
`iJr its variable and constant sections. Each cell expresses only
`on(. of the two possible alleles (allelic exclusion; reviewed in
`ref. 3). When two antibody-producing cells are fused, the
`1 products· of both parental lines are expressed•·•, and although
`rh~ light and heavy chains of both parental lines are randomly
`JOined, no evidence of scrambling of V and C sections is
`Jb.ened'. These results, obtained in an heterologous system
`IDIO)ving cells of rat and mouse origin, have now been con·
`tinned by fusing two. myeloma cells of the same mouse strain,
`
`A
`•
`
`I
`
`Chains
`
`• • ._..-~._ -
`
`H(P3)
`
`. . .
`
`L(P3)
`
`L(Pl) r
`. ' ' -- ' -
`
`Fig. I Auaoradiograph of labelled compo(cid:173)
`nents secreted by the parentalnnd hybrid cell
`lines analysed by IEF before (A) and after
`reduction (8). Cells were i n~ubatcd in the
`presence of 11C-Iysine11 and the supernatant
`applied on polyacrylamide slabs. A. pH range
`6.0 (bonom) to 8.0 (top) in 4 M urea. B, pH
`range 5.0 (bottom) to 9.0 (top) in 6 M urea;
`the supernatant was incubated for 20 min at
`37 •c in the presence of 8 M urea, 1.5 M
`mercaptoelhanol and 0.1 M potassium phos(cid:173)
`phate pH 8.0 before being applied to the right
`slab. Supernatants from parental cell lines
`in: a, PI Bu I ; b, P3-X67 AgS ; and m, millture
`of equal number of PI But and PJ-X67Ag8
`cells. Supemiltants from two independently
`derived hybrid lines are shown: .:-[, four
`subclones from Hy-3; g and h, two subclones
`from Hy-B. Fusion was carried out•·• using
`to• cells of each parental line and 4,000
`haemagglulination units inactivated Sendai
`virus (Searle). Cells were davaoe<i into ten
`equal samples and grown separat;!y i:J
`selet:tive medium (HAT medium, ref. 6).
`Medium was changed every 3 d. Successful
`hybrid lines were obtained in four of the cul(cid:173)
`tures, and all gave similar JEF panerns. Hy-B
`and Hy-3 were further cloned in soft agar".
`L, Light ; H, heilvy .
`
`..
`!_, H(Pl)/ 1
`.
`~ -ta=:;i;
`. -
`
`. <
`'
`4. ._.·,
`... . . . ~~ ... .,.. 1 ,_
`_;
`.. ,....
`.. ;.
`.
`-·
`
`•
`
`-
`
`~
`
`I
`
`I.
`
`e m f m g
`
`b
`
`.....
`a h b g
`
`{l
`
`d provide the background for the derivation and under·
`
`~
`j
`\ ,~~nding of antibody-secreting hybrid linc:s in whit:h one of
`
`1:"~: parental cells is an antibody-producing spleen ct:ll.
`f,vo myeloma cell lines of BALB/c origin were used. PI Bul
`11 resi:;tant to :i-bromo-2'-dcoxyuridine•, does not grow in
`!elective medium (HAT, ref. 6) and secretes a myeloma protein,
`.\•li PCS, which is a n lgG2/\ (K), (ref. 1). Synthesis is not
`~lan~~d and free light chains arc also secreted. The wcond
`~~·1 line, PJ-X6JAg::l, prepan:d from P3 ccJb!, i~ resis tant to
`)) ~1g mt - • S-aw guani ne and ttoc~ not grow :n HI\ T medium.
`
`1
`
`:sotypes (yl and y2a) and both V" a nd bo th VL regions
`·(idiotypes) are expressed. There are no allotypic markers for
`the C K region to provide direct proof for the expression of both
`par.::ntal C K regions. Out this is indicated by the phenotypic
`lia.k between the Vand C regions.
`Fi11ure lA shows that clones derived from different hybridi(cid:173)
`satio~ experiments and from subclones of one line are indistin·
`guishable. This has also been observ.:d in other experiments
`(data not shown). Variants \\ere. however, found in a survey of
`JOO subcloncs. The dill'.:rence is o ften associated with changes
`
`BIOEPIS EX. 1022
`Page 3
`
`

`

`NoturC' Vol. 256 All;i lllt 7 l'ii.'
`
`Fig. 2 Jsolatiun of an ami-SRBC antibc~v.
`secreting cell cl(lne. Activity wns re' ea:cct 6,
`a halo of hacmolyscd SRBC. Dir.:ct plaq~~
`given by: a. 6,0ll0 hybrid cells Sp-l ; 1>, cl<n..,
`grown in soft agar from an inoculum or 2,111..-.:
`Sp-1 cells: c. recloning of one of the positi~t
`clones Sp-1/7; d, higher magnification or a
`positive clone. Myeloma cells (10' P3-X67A
`g8) were fused to 10' spleen cells from 'In
`immunised BALB/c mou;e. Mice were im(cid:173)
`munised by intraperitoneal injection of0.2ml
`packed SRBC diluted 1:10, boosted afler 1
`month ami the spleens collected 4 d lat~r.
`After fusion, cells (Sp-1) 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 week1
`with hypoxanthine and thymidine. Forty day~
`after fusion the presence of anti-SRBC act.
`ivity was revealed as shown in a. The rotio o(
`plaque forming cells/total number of hybrid
`cells was 1/30. This hybrid cell population
`was cloned in soft agar (50% cloning ef.
`ficiency). A modified plaque assay was used to
`reveal positive clones shown inb-das follows.
`When cell clones had reached a suitable site,
`they were overlaid in sterile conditiom witll
`2 ml 0.6% agarose in phosphate-buffeR(}
`saline containing 25 111 packed SRBC and
`0.2 ml fresh guinea pig serum (absorbed with
`SRBC) as source of complement. b. Taken
`after overnight incubation at 37 •c. The ratio
`of positive/total nwnber of clones was 1/33. A
`suitable positive clone was pick.ed out and
`grown in suspension. Th.is clone was called
`Sp-1/7, and was recloned as shown inc: over
`90:1. oft he clones gave positive lysis. A second •
`experiment in which 106 P3- X67Ag8cellswcre
`fus~d with 10• spleen cells was the source or
`a clone giving rise to indirect plaques (clone
`Sp-2/3-3). Indirect plaques were produced by
`the addition of I :20 sheep anti·MOPC 21
`antibody to the agarose overlay,
`
`assays13 have been used to detect specific clones and represenla·
`tive clones o f both types have been characterised and studied.
`T he derived lines (Sp hybrids) are hybrid cell lines for the
`following reasons. T hey grow in selective medium. Their
`karyotype after 4 months in culture (Table l) is a little
`.nailer
`!han the sum of the two parental lines but more than twice the
`chromosome number of normal BALB/c cells, indicating that
`the lines are not the result of fusion between spleen cells. Io
`addition the lines contain a metaccntric chromosome also
`present in the parental P3-X67Ag8. Finally, the secreted
`immunoglobulins contain MOPC 2 L protein in addition to new,
`unknown components. T he latter presumably represent the
`chains derived from the specific anti-SR BC antibody. Figure JA
`stwws the JEF pattern of the material secreted by rwo such
`Sp hybrid clones. The IEF bands derived from the parental Pl
`line are visible in the pallern of the hybrid cells, althougb
`obscured by the presence of a number of new bands. TJ:c
`pattern is very complex, but the complexity of hybrids of thiS
`type is likely to result from the random recombination or
`chains (see above, Fig. 1). Indeed, IEF patterns of the reduced
`material secreted by t he spleen- PJ hybrid clones gave a simpler
`pattern of lg chains. The heavy and light chains o f the P3
`pa rental line became prominent, a nd new bands were apparent.
`Th<! hybrld Sp-1 gave direct plaques and this suggested t~al
`it produces an lg M antibody. This is confirmed in Fig. 4 whiCh
`shows the inhibition of SRBC lysis by a specific anti-IgM
`
`Table I Number of chromosomes in parental and hybrid cdl lines
`
`55
`65
`
`
`
`r
`9
`~~
`
`Cell line
`P3-X67Ag8
`PI Bul
`Mou~espleen cells
`Hy-B(PI - PJ)
`Sp-1/7-2
`Sp-Z/l-3
`
`Numbet' of chromo;,on1es per cell
`66,65,65,65,65
`Ref. 4
`
`112,110, 104,1U4, i01
`9J.YU.1!9.t:9.~7
`97,n,1!5.96,\14,X~
`
`_ , ·-
`
`!
`I
`
`:
`
`M~lll l
`1~ I
`- · •. l
`
`49h
`
`Ji\ In the ratios of the different chains a nd occasionally with the
`
`l lotaf disappearance of one or other of the chains. Such eve~ts
`
`are best visualised on IEF analysis of the separated chams
`(fo1'example, Fig. lh, in which the heavy chain of P3 is no
`lon er obser ved). The important point that no new chains a re
`c!et ·-::ted by IEF complements a previous study' of a rat-mouse
`5 hybrid line in which scrambling of V and C regions ~rom the
`a
`light chain' of rat and mouse was n:>t observed. 'n this study,
`... .,th light chains have identical c. regions and therefore
`d
`1 scrambled VL-CL molecules would be undetected. On the other
`p hand, the heavy chains a re of different subclasses a nd we
`p exp:ct scrambh:d V11-CH to be detectable by IEF. They were
`li not observed in the clones studied and if they occur rnust do
`so at n lowet frequew:y, We conclude that in syngeneic cell
`~' hybrid> (as well a.; in interspecies cell hybrids) V-C integration is
`not th ~.csu1 , C.>IOplasrnic events. Integration as a result of
`;";'~ A ::·11nslocation or rearrangement during transcription is
`also suggested by the presence of integrated mRNA molecules11
`tmd by the existence of defective heavy chains in which a
`deletion of V and C sections seems to take place in already
`•:l'mmitted ceJisH.
`The cell line P3-X63Ag8 described above dies when exposed
`to HAT medium. Spleen cells from an immunised mouse also
`die ln growth medium. When both cells are fused by Sendai
`" !rus and the resulting mixture is grown in HAT medium,
`:>urviving clones can be observed to grow and become estab(cid:173)
`hsh~d after a few weeks. We have used SRBC as immunogen,
`\\·hil;h enabled u~. after culturing the fused lines, to determine
`the ~presence of specific antibody-producing cells by a plaque
`un~!\} technique13 (Fig. 2a). The hybrid cells were cloned in
`soft agar'~ and clones producing <~ntibody were easily detected
`by an overlay or SR BC and complement ( Fig. 2b). Individual
`dt•nes were isolated and shown to retain their phenotype as
`;(lr'lost all the clones of the derived purified line arc capa ble of
`1:/~· ng SRBC ( Fig. 2c·). The _Iones were visible to the: naked
`c.'"~~ \for example, Fig. 2d). Both direct and indirect plaque
`
`'
`
`BIOEPIS EX. 1022
`Page 4
`
`

`

`t~<.:hniques u~uu lly do not reveal 19~ l gM
`I D '
`.J11tlll,,dy.
`.,
`1
`.. ,,•c~llks. lgi\1 I'>
`th.:r..:fo re u nlil-..;ly to be pr<:'i<!nl 111
`the
`11 '1rc.ltaceu ~<llllpk 11 (Fig. J [I) bur p <:hllins sh<~ttld <:Ontribute
`.~, rhc patter11 o bta ined a fter rcdu-:tio n (.;ample u, Fig. 3 .'1).
`o1
`1 · Tho:: abo,·<: re;ulls $hvw that <.:dl fu>io n techniques are u
`I' ;,~1\crful tool to produce ~pecific antibody directed a);ainst a
`1 'fctkt•;rminc:d antigen. It further shows that it is possible to
`·vl~ te hybrid lines producing different antibodies directed
`r
`·Jinst the s ame anti!;en and carrying d lffcrent eflector func-
`*
`·"' (direct and indirect plaque).
`;I
`r :1c uncloned population of PJ- spleen hybrid cells seems
`n ,uirc heterogeneous. Using suitable detection procedures it
`T. ;hould be possible to isola te tissue c ulture cell lines making
`it Jiil'aent classes of a ntibody. To fac ilitate our studies we have
`lt Jl~d a myeloma parenta l line which itself produced an lg.
`re vari.llltS in which one of the parental chains is no longer ex(cid:173)
`:d ore.s;ed seem fairly common in the case of PI- PJ hybrids
`ri (fig. lh). Therefore selection of lines in whic h only the specific
`· mdb!Jdy chains are expressed seems reasonably simple.
`g~~ltcrnatively.' ~on-producing variants ~r myeloma lines could
`:i. be us.:d for fuston.
`e We used SRBC as antigen. Three different fusion experiments
`u~ were successful in producing a large number of antibody(cid:173)
`N' proJ .tcing cells. Three weeks after the initial fusio n, 33/ 1,086
`:r.:
`·d
`
`A
`
`B
`
`1
`I
`
`.•
`
`;
`
`,
`
`t
`
`,. -·
`
`'
`
`c
`
`.......
`
`b
`
`c
`
`......
`
`a
`
`c
`
`Fill. 3 Autmadiograph of labetl.:ll co~1ponents sccr~teJ by anti(cid:173)
`SR BC' specific hybrid lines. Fractional tOn before ( 8) anti after (A)
`
`l redt1ction was by IE F. pH gradient was 5.0 (h<lttom) to 9.0 (top)
`
`in th~: pres~nce of 6 M urc:a. Oth~r <.:OntlilillnS as in Fig. I. S\tper(cid:173)
`natants fro m: u, hybrill clune Sp-1/7·2: h, hybrid clone Sp-2/J-J:
`r, myeloma line P :l-X67A~:8.
`
`497
`
`Inhibition of haemolysis by antibody secreted by h ybrid
`fig. 4
`clone Sp-1/7-2. The reaction was in a 9-cm Petri dish with a layer
`of 5 ml 0.6% agarose in phosphate-buffered saline containing 1/80
`(v/v) SRBC. Centre well contains 2.5 ~tl 20 times concentrated
`culture medium of clone Sp-1/7-2 and 2.5 111 mouse serum. a,
`Sheep specific anti-mouse macroglobulin (MOPC 104E, Dr
`Feinstein); b, sheep anti-MOPC 21 (P3) lgG I absorbed with Adj
`PC-5; c, sheep anti-Adj PC-5 (lgG2a) absorbed with MOPC 21.
`After overnight incubation at room temperature the plate was
`developed with guinea pig serum diluted I :10 in Dulbecco's
`medium without serum.
`
`clones (3 %) were postttve by the direct plaque assay. The
`cloning efficiency in the experiment was 50%. I n another ex peri·
`ment, however, the proportion of positive clones was con·
`siderably lower (about 0.2%). In a third experiment the hybrid
`population was studied by limiting dilution analysis. From 157
`independent hybrids, as many as 15 h a d anti-SR.BC activity.
`T he proportion of positive over negative clones is remark(cid:173)
`ably high. It is possible that splt!en ce lls which h ave been
`triggered during immunisation a re particularly successful in
`giving rise to viable hybrids. It remains to be seen whether
`similar resu lts can be obta ined 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 a nd serum having anti-SRBC activity. It is
`possible to hybridise antibody-producing cells from different
`origins•·•. Such cells can be grown in l'l"tro in massive cultures
`to provide specific antibody. Such cultures could be valuable
`for medical and industrial use.
`~
`1
`
`M RC Laboratory of Molecular Biology,
`Hills Road, Cambridge C812QH, UK
`
`G. K oHLER
`C. M ILSTEIN
`
`Received May 14; accepted June 26, 1975.
`• Poner,. M .. Phy>iol. Rtv., 52, 631-719 (1972).
`1 Ho ribolo, K., and Harris. A. W., Expl Ctll Ru., 60. 61-70 11970).
`J Mil,lein. C .. onu Munro, A. J., in Dt/t n« and R.cognlflon(edit. by Porter. R. R.),
`199·228 (1\tTP Int. Rev. Sci., Buncrworth, London, 1973).
`• Conon, R. G. H., and Milstein. C., Naturr, 2~4. 42-43 (197)).
`'Schwobcr, J ., and Cohen, E. P., Prar. natn. Ar11d.Sd. U.S.A .• 71,2203· 2207 (1974) •
`• Lillleftclu, J . \V., SciM«, 145,709 ( 1964).
`7 Svo.ti, J., and Ml15tcin, C .. Divrhtm. I ., 128. 427-444 ( 1972).
`A Mlllo.tcin , C .• Adctu~bo. K., Co wan, N. J., and $c:,:h~r. D . S .. Prf$.r~$S in lmmuno(cid:173)
`/t11fy, II , 1 (edit. by Bren l, L., and Ho lborow. ).), I S7-168 (Nonh·Ho llond,
`Am\lerdom. 1974).
`• Horri•, II., anu Watkins, J. F .. Nawrt, l OS, 640.646 ( 196~).
`10 A"dch. A. L .. Willi•m•on. A. R., and Asko nos, B. A .. Nawr<. 21~. 66-67 ()968),
`11 Mil\tci•t. C .. Brownlee, C . G., C3rtwright, E.. M .. J11n·is. J. M., and Proudroot,
`f', J., Nultlft, 152, 3S4-J59 (19741.
`ll l'rongionc. B .. and Mll>tcin, C., Nu/11,., H~ • . ' Q7-S99 (1969).
`t ) Je• ne, N. K., and Nordin. A. A .. Scirtt<,, 1~0.~(15 (1 963).
`14 Cuuon. R. G. H., Sech<r, 0 . S., and MilStein, C., Eur. J. fmrmm ., l, t3S·I40
`( 19131.
`'
`
`BIOEPIS EX. 1022
`Page 5
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