Preparation ofMouse Anti VJJJ:CProducing Hybridoma Immune mouse spleen cells (10’) were fused with P3 x 63 Ag8 myeloma cells (3 x l0) in the presence of 1 ml 50% PEG 1000. Fusion was stopped by dilution after I mm. Cells were seeded in 36 wells of a Limbro tissue culture disk (Limbro Division, Flow Laboratories Inc, Hamden, Conn.) and grown on selective medium (HAT medium). After 1 3 days, supernatants were tested for the presence of antiVI1I:C activity. Clonation of antiVIlI:Cproducing hybridomas was performed by growing the cells in 0.25% soft agar (starting three times with 1000 cells well and once with 50 cellswell); after sufficient growth of the cells, individual clones were isolated using a micropipet and an inverse phase contrast microscope. These clones were expanded separately in 30% conditioned RPMI 1640 medium and assayed for the production of antiVIII:C. Cells from antiVIII:Cproducing hybridomas were stored in liquid nitrogen.
From bloodjournal.hematologylibrary.org by guest on January 21, 2014 For personal use only 1981 58: 1000-1006 A monoclonal antibody to VIII:C produced by a mouse hybridoma HP Muller, NH van Tilburg, J Derks, E Klein-Breteler and RM Bertina Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036 Copyright 2011 by The American Society of Hematology; all rights reserved From bloodjournal.hematologylibrary.org by guest on January 21, 2014 For personal use only A Monoclonal Antibody to VIII:C By H P Muller, Spleen cells of a BALB/c procoagulant mouse activity (Vlll:C) tography) were fused Ag8) After the fusion to VIll:C times Cells anti-Vlll:C tane to one well the 5000-10.000 by produced (1 B3) were U/mI the myeloma T PRESENT line) VIIIR:WF of the of IgG accepted or VIIIR:COF, factor VIII (VIII:C) VIII or LMW evidence from in vitro antibodies study of the is hampered cochemical it binds of both plasma Most VIII physi- proteins and on the specificity of the different anti-factor-VIII antibodies used Most of the homologous and heterologous antisera used will be oligoor polyclonal antibodies directed against more than one functional site or antigenic determinant An example of the limitation of, for instance, the use of anti-VIII:C antibodies FVIll: bifunctional ing VIIIR:WF and VIIICAG complex and VIIIR:AG, antigen against purified activity HMW material that of an fraction CaCI2) is retarded From of the FVIII: the the presence Human high and containing to VIIl:C von weight (in elutes the the Milstein’4 Leiden Research Hospital University void molecular of 0.25 M Unit, Leiden, The Netherlands Address and April reprint Thrombosis University 28 1981; requests Research Hospital, Netherlands © I 981 by Grune to R M Unit, & Stratton, July monoclonal study Inc 10 Ph.D AA Leiden, studies clonal isolated report of the a normal coupled plastic purified does not B3 antibody its plasma to VllI:C bind FVIII VIIIR:AG can heterogeneity technique be used of hemo- to against the antibody mouse produce such against hybridoma 1MW consisting of 15M, affinity and in eluting was VIII:C were for in a 3-step column in 30 times serum for was in the heated M Tris-HCI presence 30 mm of serum at 56#{176}C) containing (pH or A Sepha- mouse Fractions immunization Biogel FVIII diluted (BSA) 0.01 used an procedure on the affinity albumin against when by METHODS anti-HMW the this mono- produced on either serum dialyzed hr from mice; bovine one gel-chromatography collected BALB/c 0.1% CaCl2 AND In of was isolated FVIII) chromatography VIll:C, VIIIR:AG VIII:C cryoprecipitation, M CaCl2, in the field of et al.’5 They producing characterization MATERIALS (or 7.1), 0.015 M in neutralization studies Human factor IXa, factor X, and factor VIII were isolated as described.5” Immune Mouse BALB/c injection incomplete Haemostasis of Medicine 2333 pooled in activity VIIICAG antibodies we toneal 22 1981 Bertina, Department Rijnsburgerweg 0006-497l/8l/5805-0023$Ol.00/0 1000 accepted cofactor ly, the first application of this technique FVIII has been reported by Meyer prepared a number of mouse hybridomas, Female Submitted The found a delay Depart- and the Department Hospital it for introduced previously Thrombosis its isolated VIIICAG in an IRMA in when plasma; serum (nonimmune column University of from from crysosuperIt was monoclonal antibodies from mouse hybridomas, obtained after fusion of cultured mouse myeloma cells with the spleen cells of an immunized mouse Recent- or of FVIII in the presence VIIICAG in the rose-4B.5 factor low results inhibition VIII:C FVIII plasma philia A;7’2 different results can be obtained dependent on the assay technique and antibody actually used.’3 Such problems would occur less frequently when certified monoclonal antibodies with a well defined specificity were available In 1975 K#{246}hler and 0.25 antibodies fraction ) FVIII: that VIII:C VIllR:Ag: Willebrand M CaCl2 LMW contain- precipitating molecular material Leiden Genetics, with of 0.25 on an agarose Haemostasis of Medicine, a subunit VIIIR:WF: column of FVIII glycoprotein, related measured FVIII agarose weight ment antigen as FVlll-related raised volume with VIIICAG: (in of oligomeric FVIII Sepharose; normal or VlIl:C S to in an to successfully is found of the on the factor IgG removes pooled and the in 1MW normal The dependent of information VIII:C and not antibody fibrogen (pro- for this factor VIII concept studies using homologous and against factor VIII A further molecular structure of factor by lack properties tubes used for identias procoagulant FVIII.*S6 and coupled functionally unreas an assembly of as VIIIR:AG,* on the functional aspect of the protein fication;2 the other protein is known experimental originates heterologous successful that of The of subclass binding when produced lgG1 that Hybridoma R M Bertina neutralizes activation pris- and cryoprecipitate the titers chains and it is generally FVIII, in in anti-VIlI:C factor VIII consists of two lated proteins:’ one protein, present multimeric structures, is known HMW cells antibody B3 four produces hybridoma heavy cell inhibitor by a Mouse E Klein-Breteler, natant x 63 subcloned Analysis demonstrated (P3 an that results VIII chroma- cells wells J Derks, factor affinity myeloma of mice Bethesda with by hybridoma Injection BALB/c immunoglobulin A 12/32 antibody duced mouse isolate pretreated immunized (isolated with from in order N H van Tilburg Produced Leiden The Freund Spleen mice of 0.5-2.5 adjuvant), incomplete of 0.8 U VIII:C spleen cell After removal at 200 wk) on adjuvant), on day of cellular g (5 mm), were U VlII:C and suspension gated Cells (12-14 days 79 Three debris, and 24, followed days was prepared immunized on day 56 and by an later, white Blood Vol cells mice were in HEPES 58, with 65 Freund (mixed intravenous in 10 ml HEPES resuspended by intraperi- I (mixed were with injection sacrified 1640 A medium counted, centrifu- 1640 medium No (November), 1981 From bloodjournal.hematologylibrary.org by guest on January 21, 2014 For personal use only MURINE MONOCLONAL ofMouse Preparation 1001 ANTI-VIII:C Anti- VJJJ:C-Producing Hybridoma were coated with goat these wells were added containing Immune mouse myeloma cells Fusion was wells of (3 a x in the l0) Limbro tissue After agar (starting was were after isolated microscope tioned anti-VIII:C stored supernatants clones were from x 63 Ag8 PEG were 1000 seeded were tested cells cells/ well the cells, for the and an expanded once contrast in 30% for the anti-VIII:C-producing 50 clones phase separately assayed soft with individual inverse condi- overnight with the were with for the presence pooled assay; buffer; 200 residual of anti-VIll:C normal plasma l supernatant of the (2 hr, of 0.1 with hemophilia 5-mm addition diluted than were considered VIIl:C as 0.1 was sample The of a patient (VIIIR:COF) at 37#{176}C.After more the by the severe 10 sec antigen than without for of the Samples was started medium antibodies), presence anti-VlII:C BALB/c mice or pristane were to induce were injected was punctured whenever ascites About antibodies a solid day tumor later, were Freund was 14 (sometimes more developed, tumor were cells cells daily; till by ofAnti-VIII:C treated than tate was removed by centrifugation tant was dialyzed against NaCI M (3 ml PBS gel/mI and IgG 2.5) activity or after subsequent Anti-VIII:C against 0.01 4#{176}C in the Subclass hybridomas antibody M presence was sandwich (pH of 0.02% of analyzed ELISA was found (400-1000 citrate specificity eluted elution preparations sodium plasma sodium 7.7), isolated two technique: very either at with were methods wells (1) of to dry protein/g for measured by means was under conditions of described antibody (40 g/ml) used in an IgG was coated W Noyens carried out in (1) (about mg to the prescriptions and was antiserum were immobilized Sepharose-4B Sweden), were rabbit was antibodies according Uppsala, VIIIR:AG a specific Immunodiffusion CNBr-activated weight) of using FVIII.5 10 of the manufac- (2) by coating in 0.1 M of plastic NaHCO3 (pH immunoradiometric assay FVIIICAG anti-VIII:C of KCNS at by Dr activity to plastic Organisation, phase tubes as described and reference assay monoclonal a ‘25l-labeled cmp/mg protein) Institute TNO, are was (IRMA) hybridoma human details curve l0 was Health Leiden) using the Experimental x (Gaubius A two site immunoradiometric identical constructed for VIIICAG anti-VIII:C anti-VII1:C in the to described from those serial was in the second first phase earlier.”3 dilutions of A pooled plasma normal (pH (specific provided Blood was collected in 1/10 volume of 0.11 M sodium citrate Pooled normal plasma was prepared from the platelet-free plasmas (30 mm, 20,000 g, 4#{176}C)of 32 healthy volunteers (14 women, 18 men; average mouse serum was modified double glass for a microtiter plate A Xa anti-VIIl:C developed stored by in monoclonal coupling Research 0.15 azide produced or present 52337 unit with of activity Purified ‘251-fibrinogen dialyzed and as a patient or antigen by Ouchterlony.#{176} kindly of the high the of activity Factor One to measured x 50 ml; start activity was from plasma earlier HMW tubes M glycine M NaCI, polyacryl- according plasma as described These volumes at the 251 a p1-I range cofactor VIII:C 1.2% agar 8.6) column (2 U/mg) 0.15 immunoglobulin by with gradient of with immunoelectrophoresis purified 7.3), al.’8 using towards as described with in et amount activity tubes Superna- (pH washed with analyzed prefabricated Sweden) ristocetin as substrate normal once); precipi- A Sepharose KCNS activity anti-VIII:C concentrations was in a linear Anti-VIII:C No gradient A (Pharmacia, Ascites any rpm) phosphate a protein column eluted after (5 mm, I 2,000 through The was KCNS) which M sodium 0.01 passed ascites) before M 0-2 (PBS), From 30 mm, on platelets et al.,” as the turer cultured Antibodies at 56#{176}C for and Localization Broma, for Weiss Veltkamp is defined Human was AB, formalin-fixed by hemophilia (IRMA) Ascites performed assayed using ascites vitro Purification lyophilized previously.” by adjuvant hybridoma 106 observed U.K.) by autoradiography was described against complete days Mice intraperitoneally from either Slough, A Sepharose Methods amidolytic its Antibodies with were rabbit precipi- subclass-specific protein electrophoresis Producter Two-dimensional treated eluates of Miles, the treated tl normal specifically IgG the I X2-l00 was I 50 were from performed were ml of pooled of anti- units ofMonoclonal Ascites 2.9); focusing performed Production in Mouse of subclass; eluted gel was Analytical described times, (pH gels (LKB IgG bound lgG Dowex preparation bound Sepharose were acid proteins added for 25 mm indication potency was method Clotting (culture a positive tl for of a phospholipid/kaolin (37#{176}C),coagulation A SDS-polyacrylamide amide radiolabeled anti-mouse complexes acetic with immunoglobulins protein Antibody the using iodinated was of 3.5-9.5 200 assayed (plasma by radioim- Michaelis with mixture ml tested inhibitor in incubated the ml undiluted M CaCI2 in Bethesda times plasma and of a control antibodies expressed 0.1 and a modified was which ) incubation that by been preincubated had of 0.1 ml 0.033 higher plasma A- centrifuged diluted ml VIII:C-deficient which a further were activity was 37#{176}C),after to a mixture suspension, cells to this purpose, VIII:C; severe hybrid obtained treatment A Sepharose (rabbit labeled Activity of cultured subclass and 5-amino antibodies supernatant After labeled Isoelectric Supernatants IgG IgG, material specific zl culture dialysis, protein antisera diluted VIII:C 50 before tated of nitrogen ofAnti- of the subclass technique.’7 and serum, production hybridomas from chloramine-T by Assay By analysis with anti-mouse sheep anti-rabbit to U.K.); supernatant Radioimmunoprecipitation Protein in 0.25% and rabbit Slough, j.d culture Flow medium of anti-VIlI:C-producthe (2) munoprecipitation (Miles, 50 antibody, peroxidase-labeled acid IgG subsequently hybridoma antibody, benzoic in 36 Division, on selective of and medium Cells in liquid 1000 growth 50% Cells by growing a micropipet 1640 ml P3 (Limbro Clonation with with grown activity times These RPMI disk and days, sufficient using of mm I culture performed three cells/well); fused presence Conn.) of anti-VI1I:C hybridomas were after Hamden, medium) ing (10’) by dilution Inc, presence cells stopped Laboratories (HAT spleen anti-mouse volunteers age 26.7 prepared hr from at yr) and the 37#{176}Cand stored sera at - (supernatant overnight at 70#{176}C.Pooled of blood 4#{176}C)of normal stored 30 healthy in From bloodjournal.hematologylibrary.org by guest on January 21, 2014 For personal use only MULLER 1002 Table RESULTS Production Producing After and Selection Anti- VJII:C fusion cytes VIII:C, of of mouse a mouse the fusion of Mouse ( I B3) myeloma I B3 hybridoma ing the cells in 0.25% soft wells produced anti-VIII:C in vitro (Pooled cells with Residual (mm) spleno- was cloned 0.08 0.41 0.08 60 0.35 0.10 120 0.33 0.10 pooled normal antibody ( B3) time intervals, different plasma was U/mI) (500 samples were one All of presence and of an IgG2 chains The monoclonal immunoglobulin was and polyacrylamide electrophoresis of antibodies inhibitor anti-VlII:C anti- in cell culture superassay was used; the specificity of the assay was checked by replacing the VIII:C-deficient plasma by FIX-deficient plasma: a result was considered to be positive only when no anti-factor-IX activity could be demonstrated AntiVIII:C titers in supernatants were maximally about one volume of buffer; assayed for gel Anti- Activity V1I1:C ofthe from IgG, ascites (after by reduction) when analyzed by 7.09 (see Fig 1) iso- Hybridoma Antibody (1B3) Table equilibrium I shows that the time necessary binding of purified monoclonal 50 both elution) was found to be as analyzed by SDS- and contains two components electric focusing: p1 6.98 and U/ml; however, titers were difficult to estimate due to the peculiar properties of this antibody (see below) When the hybridoma cells were cultured in the ascites of BALB/c 10,000 U/mI were found in the serum of the fusion and containing demonstrated antibody (isolated gradient preparation bodies For the detection natants, a modified diluted with volume (IgG,, IgG25, IgG2, IgG3) By means of a modified double antibody sandwich ELISA technique and the analysis of the radiolabeled supernatant protein precipitated with subclass-specific antisera, the protein A Sepharose a homogeneous IgG of one VIII:C positive wells One of the anti-VIII:Cclones, which on statistical grounds only one type of hybridoma, has been used for production incubated or with by grow- agar (1000 cells/well) activity; cells from the in vivo Anti-VIII:C + 0.38 One volume by VIII: C (U/mI) Anti-VIII:C 15 monoclonal residual - Plasma) Ascites) 30 with isolated human seeded in 36 wells; 14 after Normal (From these wells were expanded and again distributed over different wells on soft agar This procedure was repeated times; at the fourth cycle the initial number of cells was reduced to 50/well; under these circumstances it was possible to isolate individual clones from one of producing contains Anti-VIII:C Incubation Time immunized products were The of Vlll:C Hybridoma Hybridomas days later, the supernatants of wells were found to contain significant amounts of anti-VIII:C activity This study details the properties of anti-VIII:C produced by a hybridoma isolated from one of these wells Inactivation ET AL to reach antibody r mice, anti-VIII:C titers of 5000obtained; this titer equals the titer of the immunized mouse on the day HYBRIDOMA ANTI Vlll.C U = > IgG Subclass Hybridoma VJI1:C Produced by -j B3 Culture supernatants anti-VIII:C presence ofAnti- of activity mouse p1 of were lgG hybridomas analyzed of the 698 producing with respect to the different subclasses H :D LU I 10 ANTIBODY 100 1100 DILUTION Fig human Fig globulin Isoelectric focusing pattern of isolated B3 immuno- D00O Dose-response curves for mouse anti-VIll:C serum anti-VIII:C serum and monoclonal antibody One volume of pooled normal plasma was incubated with volume antiserum (dilution) for hr at 37’C Residual VIll:C was measured and expressed in U/mI; (#{149}-#{149}) human anti-VlIl:C; (00) mouse anti-Vlll:C; (x-x) monoclonal antibody (1 B3) From bloodjournal.hematologylibrary.org by guest on January 21, 2014 For personal use only MURINE MONOCLONAL Table 1003 ANTI-VIII:C Neutralization Cryosupernatant of VIII:C Cryoprecipitate Hybridoma Activity in Plasma and Isolated 1MW Preparation Cryosupernatant LMW VIII:C Binding of tThlFibrinogen to Immobilized Hybridoma Anti-VIII:C ( : 3) Tubes (U/mI) Anti-VIII:C - plasma Cryoprecipitate by Anti-Vlll:C Residual Pooled normal Table FVIII Anti-VIll:C + 0.30 0.05 0.04 0.45 0.15 Coated Additions With Human anti-VIll:C Buffer Human anti-VIII:C VWD anti-VIII:C Buffer Hybridoma anti-VIII:C VWD FVIII 1:2 3.70 1.80 purified 1:4 2.15 0.70 (42 1:8 1.00 0.35 patient 1:16 0.38 0.16 fibrinogen) tubes coated hybridoma x i03 von After 2.9 purified disease of contents, anti-Vlll:C incubated or absence Willebrand’s removal 2.1 human ( 1B3) were in the presence severe 2.4 plasma with anti-VlIl:C cpm) with either 3.2 plasma Hybridoma Plastic ‘251-Fibrinogen to Tube (%) Bound with of 0.01 (as tubes with ml plasma a source were or ‘25l-fibrinogen of counted of a unlabeled for bound radioactivity One with volume either of VIII:C-containing volume of purified volume of BSA in buffer After residual VIII:C samples were monoclonal tenfold incubated antibody dilution, samples 30 mm for (500 U/mI) were assayed or for effects results of the antibody of coagulation reported assays, so far depend it was checked on the if the to plasma VIII:C is less than mm This bly less then generally found for human is consideraanti-VIII:C antibody could bind fibrinogen The experiment Table shows that the same amount of ‘25I-fibronogen antibodies.2’ monoclonal curve for the with those is bound to tubes doma anti-VIII:C In Fig antibody 2, the has dose-response been compared coated with the monoclonal as to tubes coated with an human anti-VIII:C; serum (at the time of the fusion) It is obvious that the slope of the dose-response curve is much weaker in the case of the monoclonal antibody Furthermore, it should be noted that even an undiluted IgG prepara- absence of fibrinogen tion monoclonal for a human does antibody and not inhibit In Table 2, the for plasma action the mouse VIII:C anti-VIII:C completely of purified monoclonal anti- body on VIII:C in plasma, cryoprecipitate, cryosupernatant, and isolated LMW FVIII was compared In all cases substantial but incomplete inhibition of the factor VIII procoagulant activity was obtained In the LMW FVIII preparation, no FVIIIR:AG could be detected by a specific electroimmunoassay When purified monoclonal antibodies coupled to Sepharose were incubated with pooled normal plasma, the VIII:C in the supernatant decreased significantly without affecting the VIIIR:AG concentration (see Table 3) The isolated VIII:C the mouse, from which for the fusion trace amounts Table (used for the immunization of the spleen cells were removed experiment) of fibrinogen Absorption was not always Because all With Immobilized of Plasma free from inhibitory Plasma + 0.27 0.69 Normal - 0.50 0.73 Hemophilic + - > PLASMA 10 IL) PLASMA a: 3aD #{163}1 #{163} 1/100 1/1000 SAMPLE Fig IRMA of VIIICAG using (x-x) Pooled normal plasma; () mouse I I ! I A I #{163} 1/10 Il DILUTION anti-VIII:C (1 83) in the first phase and specific labeled human anti-VIII:C plasma of a hemophilia A patient; (O-O) pooled normal serum lgG in the second phase From bloodjournal.hematologylibrary.org by guest on January 21, 2014 For personal use only MULLER 1006 as a decrease of the VIII:C); in VIII:C this also concentration explains why never could be neutralized presence of undiluted antibody completely, preparations The finding I B3 antibody the molecule that serum VIII CAG suggests that proteolytic (activation and/or accompanied sharp decrease by a structural in affinity even in the of is resulting to the antibody This abovementioned interferes with the does not bind degradation inactivation) change binding for VIII:C (neutralization VIII:C activity in a anti- FXa In the finding hypothesis the activation future the use prove to be particularly geneity of hemophilia mechanism by which is compatible ET AL with the that the effect of the of VIII:C by thrombin I B3 or of monoclonal antibodies will useful in the study of heteroA and in the elucidation of the VIII:C exerts its coagulant activ- ity REFERENCES I Bloom disorders AL, Peake Semin Zimmerman of von Willebrand’s cies of antihemophilic Clin IA: complex With factor JA, platelets Immunologic RatnoffOD, of factor and on an factor von by analysis factor Graaf 5, and specific 62 (Suppl Ill): Poon Thromb MC: FVIII Res immunologic antihemophilic factor LW: Peake lR, radiometric assay haemophilia, von Br J Haematol Bloom UM, factor 14:235, 1979 10 Muller Veltkamp antigen ii: radiometric Willebrand’s JC, Ludlam factor disease and CA: An VIII antigen: fetal plasma Barrow 52:2708, 1973 19 Veltkamp and serum carrier VIlI-related Graham JB: Radioimmunoassay antigen (Vlll:CAg) for 21 Res Thromb The HP, van Tilburg Immunoradiometric i-P assay Clin Chim Lavergne of factor NH, Bertina assay ofprocoagulant Acta i-M, VIII: 107:1 Meyer RM, Terwiel JP, factor VIII Coagulant antigen Biggs Antibodies Haematol 22 1, 1980 D, Larrieu M-J: lmmunousing four 19:279, mode K#{246}hlerG, JB: fused cells 1975 T: Monoclonal hybrids Circulation the activation of preparation of 1980 GS: of high Rickles for Drion The specific radioactivity platelet Varma A, in von aggregation and EF, FR, deficient antigen J: Relationship content Loeliger coagulation Rogers Willebrand’s EA: to i Clin Invest Detection disorders of I Thromb the Diath 1968 Diffusion-in-gel Allergy action 23:125, of 256:495, in Glover factor activity 0: which globulin-secreting Immunol LW, 6:30, R, Austen of cultures i I 85:647, WM, of a plasma ii, II Prog Graham A comparison 1963 Ouchterlony analysis ES, Nature Pathways hormone in hereditary Haemorrh phase (abstr) Hunter procoagulant state a solid T, Edgington RM: is necessary immunoin VIII J Measurement plasma: from cellular X Biochem Hoyer assay factor Results 14, Hi, that disease 20 ES, i 89:1 Weiss 62:1048, Invest 1979 (VIIICAG) I I Girma Giddings procoagulant 42:269, Reisner coagulant for Al, 18 Br non-haemophilic Barrow Continuous 1980 growth with PM, B, Zimmerman FC, IM: specificity Bertina human Quantitative measurement J Clin Biochem are 1976 lmmunoradiometric antigen that VIII) A 5, 1980 C: factor Greenwood labeled ‘‘I K, coagulation I 1980 evidence (factor species Blood 47:657, procoagulant related 20:85, 56:61 for factor VIII 111-106, R, Nilsson (VIIl:CAg) in hemophilic of predefined D, Obert Mertens human haemophilia 1979 Blatt antigen Milstein antibody Meyer of on homologous 23:17, WA, Blood K#{246}hlerG, 15 ii: Price coagulant antibodies Veltkamp between activity HM, Willebrand’s and based Scand J Haematol VIII cases A antigen method Reisner 14 27 L, Ljung factor of multimeric in plasma RM, L, Borge of five alloantibodies a I 978 13 16 Bertina relationship molecular VIII antibodies J of 1981 Holmberg secreting Variant NH, CC, human de oligomers Study 47:269, immunoradiometric 1980 the J, Hoyer Lazarchick 12 1974 Willebrand procoagulant of a single attributes of the on 4:1 55, TS: Tilburg Slover properties V) Labruy#{232}re WT, subtypes VIII/von van FVIII antibodies of antihaemophilic and deficien- antihemophilic of homologous Res of two studies and the BN, a series Thromb factor human Immunologic (factor against Zimmerman HP, antigen its deficiency) on combined and proaccelerin J CIin Invest 65:1318, Muller AE: VIII observations Bouma VIII, ZM, of and Factor disease Characterization composition VIII 1971 proteins Ruggeri Powell (factor anticoagulant Factor of two of factor Haematol OD, disease Mourik van Ratnoff hemophilia 50:244, Mochtar genetics 1977 classic circulating Invest Molecular 14:319, TS, differentiation acquired IR: Hematol DEG, of methods for immunological 1962 Densen antibodies have second-order KWE, which Rizza CR, destroy Borrett factor concentration R: VIII graphs I Br i 1972 Howe and 6:292, 1976 SC, Milstein nonsecreting C: Fusion myeloma between cell immunolines Eur i ...From bloodjournal.hematologylibrary.org by guest on January 21, 2014 For personal use only A Monoclonal Antibody to VIII:C By H P Muller, Spleen cells of a BALB/c procoagulant mouse activity... LW: Peake lR, radiometric assay haemophilia, von Br J Haematol Bloom UM, factor 14:235, 1979 10 Muller Veltkamp antigen ii: radiometric Willebrand’s JC, Ludlam factor disease and CA: An VIII antigen:... Superna- (pH washed with analyzed prefabricated Sweden) ristocetin as substrate normal once); precipi- A Sepharose KCNS activity anti -VIII:C concentrations was in a linear Anti -VIII:C No gradient A