THROMBOSIS RESEARCH 63; 629-640,1991 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.


REGNAULT, Claude RIVAT, Michele PFISTER and Jean-Francois STOLTZ

INSERM U 284, Plateau de Brabois, CO 10, 545 11 Vandoeuvre-l&-Nancy CCdex, France (Received 19.4.1991; accepted in revised form 256.1991 by Editor J. Soria) ABSTRACT

Human protein C, isolated b conventional multistep methods, was used for immunization o fy mice. Monoclonal antibodies were prepared and screening of antibodies to human protein C was achieved usin an immunoblotting technique. Five monoclonal anti-protein as affinity ligands. cf: antibodies were compared Different parameters were studied (adsorption ca acity, specifici of adso tion, possibili of desorption under mil R conditions) an?l two anti % odies were se7 ected. One antibody allows reparation of urified protein C in a single-step rocedure Prom a fraction h?hly p containin g high levels of coagu Pation factors whereas the 0 plasma other can be used for preparation of protein C deficient plasma.


in the presence of calcium ions (4-6). Activated PC functions as an antic0 lant by selectively inactivating factor Va and VIIIa (7-9) and it also promotes ?lY 1 rinolysis (10). The association between venous thromboembolic corn lications and PC deficiencies is well documented (11). Congenital deficiency o P PC is inherited as an autosomal dominant disease. The homozy ous form is characterized by purpura folminans neonaturum at birth (12).gIf untreated with replacement therapy, this disease is often lethal. Heterozygous PC deficiency is manifested by a variety of mild to severe recurrent dee venous thrombosis and pulmona embolism or can be totally as mptomatic (J)3). Multiple conditions of acquire 3 PC deficiency and associated tKrombosis have been also described (14). Rey words : Monoclonal

antibody, protein C, immunoadsorption 629



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for Therapeutic PC replacement could be an appro riate treatment patients with PC deficiency if pure functional human pPC was available in uses fresh frozen plasma or sufficient quantity. Current re lacement therap factor IX concentrates (15 16). t$’ mce the short ha i f life of PC (6 hours), frequent infusions are needed. ThGombosis and continuous stimulation of the immune system by repeated infusions of allogenic proteins (17-19) are the rincipal adverse effects of current re lacement therap . More recently, rep Pacement therapy using a concentrate oF human protein E and S was shown to be more effective in protecting patients with severe PC deficiency from thromboembolic events (20). Since their similar physicochemical properties, separation of vitamin Kdependent proteins and purification to homo eneity is difficult. PC has been purified by conventional multistep barium salt metho f s which include aphy (1, 21adso tion, ion-exchange chromatography and affinity chromate 25). xtilization of such methods in our laboratory has resugirted in trace contamination in PC preparations. Moreover, multistep methods took a long time and ave a low yield. In contrast, immunoaffinity purification using monoclona B antibodies a peared as a sim le, ra id method which can be readily adapted to pro B uce high1J pm&d PC? in large amounts. Such immunoaffini methods have been eveloped successfully for isolation of traceamount coa ivation proteins (26-29). If recently techniques using monoclonal antibodies Y ave been reported essential1 to obtain information on the biological function and discrimination of P 6 and activated PC and to develo s assa s (30-33), only few reports deals with immunoaffinity purification metho dp (34-37). M oreover, all have used conformation-s ecific monoclonal antibodies to protein C-calcium ion complex and most of t Rem required Generation of monoclonal amet&&~es to human PC using conventional antigen multistep as was immunoadsoi tion chromatography with these antibodies as affinity ligands was develo es allowing reparation of hi hly purified PC in a smgle-step procedure. %urthemore, p lp asma deficient in F)C can be also obtained.

METHODS Anti-protein C monoclonal antibodies : Purification

of human nrotein C bv conventional


PC was isolated from lasma cryo recipitate supernatant by a procedure similar to that described by B ahlback (3I&). roteinase inhibitors were All ex eriments were run at 4°C. The following added to t! e lasma : benzamidine h drochloride R 0 mM), soybean trypsin inhibitor pe PI S Sigma (50 mg/l). TXkn 1 M BaC12 was added dropwise (80 ml/l of pr asma). After stirring of the mixture for 1 h, the barium citrate precipitate was collected by centrifugation at 4000 g for 20 min. The barium citrate recipitate was suspended m 0.15 M NaCl, 5 mM benzamidine hydroch Poride, H 7.4 (200 ml/l of plasma) and centrifuged once again. The washing proce B ure was repeated once. The barium citrate preci itate was dissolved in 0.2 M EDTA, 10 mM benzamidine hydrochloride, p IF 7.4 (150 ml/l of lasma). The sus ension was stirred for 6 h and then dialyzed overni ht H 6. A i!!er against %0 mM Tris-H3 $ 04, 10 mM benzamidine hydrochloride, dialysis, a small recipltate was removed by centrifugation at 10 &O for 30 min. The dial ze % barium citrate solution was applied to a column P.6 x 50 cm) of DEAE- B epharose Fast Flow (Pharmacia Fine Chemicals) equili 6 rated in


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20 mM Tris-H PO , 2 mM benzamidine hydrochloride, pH 6. Chromatography was run at 3 2 m f+h in order to obtain o timum resolution. Proteins were eluted with a linear gradient of NaCl (0.1 - B .7 M) for 24 h. PC in the column effluent was monitored immunochemically (for details, see below) and PC containing fractions were ooled. The PC pool was dialyzed against 50 mM Tris-HCl, 0.1 M NaCl, 1 m R EDTA, pH 7.5 and applied to a column (1.6 x 33 cm) of Blue-Sepharose CL-6B (Pharmacia Fine Chemicals) in the same buffer. Chromato a h was run at 35 ml/h. Proteins were eluted with a linear adient oFJ a (!!1 (0.1 - 1 M) for 16 h. PC was monitored immunochemically. fractions were pooled and concentrated b ultrafiltration on an F C containin Amicon YMl 8 membrane. Purified PC was stored at -30”F . Production

of monoclonal


Murine monoclonal antibodies to human PC were produced by injectin to Balb/c mice 11.5 U of PC (partially purified as above mentioned) in camp 4ete Freund’s ad’uvant. Another injection, containing 11.5 U of PC in incom lete Freund’s aa’ juvant was given on day 23, followed by three injections of P0 U without adjuvant on days 37, 97 and 118 respectively. Fusion with mouse myeloma cell line SP 0 was performed on day 121 according to the method described by Hammer&g et al (39). In a first time the groxg cultures were assayed usin an enzyme linked assay (for details, see below) m which partially purified BC was adsorbed to polystyrene microtiter wells. In a second time_, among hybridoma cells secreting antibodies, screening of monoclonal antibodies to human PC was carried out usin an immunoblotting technique (for details, see below). % onoclonal anti-protein C antibodies were purified from ascitic fluids usin protein A-Sepharose CL-4B (Pharmacia Fine Chemicals). The gel was equi Yibrated in 1.5 M lycine, 3 M NaCl, pH 8.9. Ascitic fluids were assed through the column $1.0 x 6.3 cm) at a flow-rate of 10 ml/h. %ound immunoglobulins were eluted with 0.1 M sodium citrate, pH 6. The gel was regenerated with 0.1 M sodium citrate, H 3 and then equilibrated in 1.5 M glycine, 3 M NaCl, pH 8.9. Purified anh% odies were dial zed against a 0.1 M sodium bicarbonate, 0.5 M NaCl, pH 8.5 buffer and store B in this buffer at 4°C after sterilization. Preparation

of immunoadsorbents


Anti-protein C antibodies were covalently coupled to Sepharose CL4B (Pharmacia Fine Chemicals) activated with cyano en bromide according to the modified method of Cuatrecasas et (40). A voglume of a 10 % solution of CNBr in distilled water was added to an identical volume of packed Sepharose and the H was maintained at 11.0 using 10 M NaOH. After extensive washin at 4”C, Pirst using distilled water, then, a 0.1 M sodium bicarbonate, 0.5 I& NaCl, pH 8.5 buffer, the antibody solution (at a concentration of about 3 mg/ml) in the same buffer was added to the activated Sepharose and the mixture was left overnight at 4°C under gentle stirring. The unbound proteins were eliminated by washing with sodium bicarbonate buffer. The amount of antibodies linked on the el was deduced from the unbound roteins. The excess rea ent groups were B locked b incubatin with 1 M ethano plamine, pH 8 for 2 h. Al?ter washing with 3M NaS Z N, 0.1 M fiaCH%OO, pH 6 to eliminate possible rotein- rotem interactions, the immunoadsor ents were stored before use at 4”g m ’ 0.0 !!i M Tris-HCl, 0.15 M NaCl, pH 7.4 containing 0.02 % NaN3.





of protein

i’ni. 63, No 08

C :

PC was purified from either plasma or a chromatographic sub-fraction prepared dunng the fractionation of plasma and containing high levels of coagulation factors. This sub-fraction had underwent viral inactivation usin a solvent-detergent solution (41). An excess of plasma or an excess of the BCcontaining fraction was applied to the immunoadsorbent at a low flow-rate to ensure maximal adso tion of PC. The immunoadsorbent was washed with 0.05 M Tris-HCl, 0.15 M x aC1, pH 7.4 containin 0.02 % NaN3 until absorbance reached zero. Bound rotein C was desorbe d: by 0.1 M triethanolamine, 1 M NaCl, pH 10.5 or by 3pM NaSCN, 0.1 M NaCH COO, H 6. Immunoadsorbent vvvNregenerated with 0.05 M Tris-HCl, 0.15 M &aCl, p!I 7.4 containing 0.02 % rotein C was dialyzed against 0.15 M NaCl, 0.01 M . The purified sodit?m phosphate, p Hp7.2. Analytical



Antibodv measurements Solid phase enzyme linked immunosorbent assay : Microtiter plates (Costar) were coated with 0.25 U/ml of partially purified PC in 0.05 M sodium H 9.6 (100 ul/well) at 4°C overni ht. After washing 3 times carbonate buffer, with 130 mM Na 8 1, 5 mM Na2HP04, 1 mM KH2P 8 4, pH 7.2 containing 0.05 % tween 20 (PBS-tween), h bridoma culture su ernatants in PBS-tween (100 ~1) were added to the coate B wells and incubate B for 2 h at room temperature or at 4°C overnight. The wells were then washed 3 times with PBS-tween. 100 ul of peroxidase conjugated anti-mouse &G-goat immunoglobulin were added to each well in the same washing buffer and incubated at room temperature for 1 h. After washing 5 times with PBS-tween and then once a ain with 0.5 M sodium acetate-citrate, pH 6, 100 ~1 of a freshly re ared so Bution constituted of 0.1 mg/ml of TMB and 0.01 Yo H202 in 0.5 MpTt so ium acetate-citrate, H 6 ~=z!F laced in each well. The enzyme reaction was stopped b *addition of 3 5 ~1 rp H2SO4. The coloration was measured at 450 nm m a ? ttertek Multiskan plus micro ELISA reader (Flow Laboratories). of pure preparations of antibodies was measured Concentration spectrophotometrically at 280 nm using A rofR = 14. Puri of antibodies was ascertamed by pol acrylamide el electrop l!oresis. Electropx oresis was carried out using Phastge f gradient 4- !?5 (Pharmacia Fine Chemicals) in presence of SDS. Protein C assays PC antigenic level was determined by rocket immunoelectro horesis. 1.5 ml of 1 % a arose (Litex HSB agarose, Tebu) containing 25 ul o P anti-human protein C ra Bbit serum (Diagnostica Sta o) were poured onto Gelbond sheet. 1 ~1 samples were applied m wells. # lectrophoresis was performed on a apparatus (Pharmacia Fine Chemicals) according to Phasts stem manu Iy acturer’s instructions. A local pool of normal plasmas (arbitrarily assigned to 100 Yo or 1 U/ml) was used as standard. PC activity level was determined by a chrome enic assay (Berichrom Protein C, Behring) using a Chromotimer apparatus (Be!! ring). Assavs for other coagulation factors were

Factor II., V, VII, VIII, IX, X activi 1eve 1s and fibrinogen concentration determined by clotting assays. Z ntithrombin III activity level was

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determined by chromogenic electroimmunoassay. Polvacrvlamide


assay and protein S antigenic level was assayed by

gel electroohoresis

and immunoblotting

Electrophoresis was carried out using Phastgels gradient 8-25 in presence of SDS on a Phastsystem apparatus according to manufacturer’s instructions. The amounts of proteins loaded on the gels were about 100 ng and the gels were stained with silver nitrate. Immunoblotting : the transfer to a nitrocellulose membrane was performed for 30 minutes at 70°C. After blockin for 30 minutes with 3 Yo gelatin in 20 mM Tris, 500 mM NaCl, the mem %rane was incubated for 60 minutes with either a l/50 dilution of anti-human protein C rabbit serum hybridoma culture supernatants. The (Dia ostica Stago) or undiluted memI??rane was washed with distilled water and then incubated with a l/500 dilution of anti-rabbit or peroxidase conjugated anti-mouse IgG immunoglobulin (TAGO). After washing with distilled water, the membrane was incubated in eroxidase substrate constituted of 5 m /ml il-chloro-lnaphtol, 0.015 % $202 in 20 mM Tris, 500 mM NaCl. T!l e reaction was stopped with distille water.

RESULTS Purification of protein C by conventional multistep methods : PC was purified from c oprecipitate supernatant by a multistep procedure as described in “Methods”. ?I he purification procedure was re eated several times and consistently yielded a partially purified PC (Figure K Includin a (NH4)2S04 fractionation of the barium citrate eluate, or an addiiional DEIESepharose chromatography in resence of CaCl before affinity chromatogra hy rovided no improvement in tie purity of PC. 3’imilarly, using dextran sul Pateg epharose or heparin-Se harose rather than or in combination with BlueSepharose chromatogra K y resulted in contaminated PC (Figure 1). Furthermore the PC yiel a s were general1 less than 10 %. When samples of partially urifie dv PC were analyzed b gel electrophoresis and immunob Yotting, only the tightly space J JZZZZZ!: apparent molecular wei ht of 62 000 and 59 000 is recognized by a specific anti-human rotein C ra%bit serum (Figure 1). Althoug R PC preparation contained contaminants, it was used for immunizing mice. Preparation of monoclonal antibodies to human protein C : Hybridoma cultures were shown to produce antibodies to one of the components of PC preparation as determined by solid-phase enzyme linked immunosorbent assay in which conventially isolated PC was adsorbed to wells. In order to discriminate antibodies a ainst PC and antibodies against PC contaminants, the binding of each anti %ody to one of the corn onents of PC preparation, which have been electrophoresed on SDS-PAEE and then transferred to a nitrocellulose membrane, was tested b immunoblotting. As shown in Figure 2 immunoblotting allowed screening o P antibodies to protein C doublet and antibodies to PC contaminants. Five hybridoma cells producing antibodies to human PC were obtained.



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1. Puri of PC isolated b Conventional Methods. (A) SDS-PAGE analysis on 8-? 5 % adient gez of different PC sam les. The first chromate aphic step of Q C isolation was erformed on DEA I?-Sepharose. The further c f?romato a hit steps were CarrieB out usin Blue-Sepharose (lane 6), dextran sulfate- YR e arose (lane l), he arm-Se a arose (lane 5), DEAESepharose and BPue-Se harose (lane 27, DEAE-Sepharose and heparinAE-Sepharose, heparin-Sepharose and Blue-Se harose Sepharose (lane 3) or D Ep (lane 4). (B) Immunoblottin with a specific anti-human protein Cp rabbit serum. PC was isolated by D I?AE-Sepharose and Blue-Sepharose. Lane 1 : SDSPAGE ; lane 2 : immunoblotting. Figure

2. Screening of Monoclonal antirotein C Antibodies by Immunoblotting. ! a&all purified PC was electro horesed on a 8-25 X/o gradient gel (lane 1). * he proteins were transferred to a nitrocellulose membrane and strips cut from the membrane were incubated with a s ecific anti-human rotein C rabbit serum e ane 2) or various g bridoma culture supernatants (lanes 3 to 8.7 Figure



+ 12345676




Comnarison of the antibodies as afinitv lieands The five anti- rotein C monoclonal antibodies obtained, C12C5, 9E2, 12H8, 8HlO and 9 I! 7, have been studied as affinity ligands. Purified antibodies were immobilized on activated Sepharose with similar yields of cou ling (91 to 97 %). To determine the PC adsorption capacity of the monoclon ap antibodies immobilized on the gel, an excess of plasma or PC-containing fraction was a plied to each column at a low flow-rate and desorption was achieved usin c fi aotropic a ent. It was ascertained for four of the antibodies, C12C5, 12H B , 8HlO and 9 #i 7 that the amount of PC desorbed corresponded to the difference in the plasma PC content before and after passa e throu h the column. The adsorption capacity (defined as the amount of P Cgdesorbe !!i from the gel) was observed to va widely among these antibodies. For 9E2 antibody, the amount of PC desorbe 2 was markedly low as compared with the difference in plasma PC content, thus, the adsorption capacity cannot be quantify b the amount of PC desorbed from the gel. The adsorption capacity of the 9 H2 antibody was

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evaluated from the amount of plasma totally de leted of PC after pass e through the immunoadsorbent and was found to P,e largely higher than at? t e adsorption capacity of the other antibodies. The adsorption capacity for each antibody was found to be similar whatever the startmg material used. The results are summarized in Table I. Because of the toxicity of sodium thiocyanate and the intended thera eutic use of purified PC, possibility of total deso tion of the adsorbed P e was investigated with a non toxic basic elution bu 2 er. Each immunoadsorbent was eluted successively 0.1 M triethanolamine, 1 M NaCl, pH 10.5 and 3M NaSCN, 0.1 M NaC COO, pH 6. Results are ex ressed as the percent e of PC desorbed by each uffer in relation to the tota P amount of PC desorbeT . As shown in Table I, quasi-complete desorption of PC was achieved with the basic buffer for only two lmmunoadsorbents. TABLE I Comparative Study of the Monoclonal Anti-Protein C Antibodies as Affinity Ligands


ant ibody density on gel mg/ml

adsorption capacity U/ml

sequential desorption __----__________--__----~ % of PC desorbed with basic chaotropic condition PH


























The purity of PC, desorbed by these two buffers, was checked out for the four immunoadsorbents, C12C5, 12H8, 9H7, and 8HlO columns. When analyzed by SDS- olyacrylamide gel electrophoresis Fi re 3), each sample of human purified 1pC migrated as a ~h~~H~~~d~~~~~~~~~~o~~~~~~ n9f reducin agents. The eluates from 12 detectab9 e contaminants. Interestingly,’ the PC obtained from the C12C5 column consistently contained two contaminants (one with a large molecular weight and another with a 55 Ooo molecular weight). Attempts were tried unsuccessfully to remove these contaminants by using washin buffers containing either CaCl or EDTA prior to desorption. After partial re ! uction of the 12H8 eluate with &I mM dithiothreitol, a heavy chain which migrated as a ti htly spaced doublet (Mr = 41 000 and 39 000) and a light chain (Mr = 21 o&;o) were observed (Fi re 3). The binding of eatY antibod to human unreduced and reduced PC, which has been electrophoresed on SD 6 -PAGE and then transferred to nitrocellulose membranes, was tested by immunoblottin . Eve t,“_“;;~$i~~~~Fi~re “f’. Four antiboties, ?lzE!!!t ?$?%l bt%! c am o PC. However, it could not be determined which of the two chains of C the antibody 9E2 was bound to.



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Figure 3. SDS-PAGE anal sis of immunopurified PC. (A) Eluate Prom the 9H7 column. (B Eluate from the 8HlO column. (C) E1’ uate from the C12C5 column. (D) Eluate from the 12H8 column. 1 and 2 indicate that desorption was achieved with basic H or NaSCN respectively. 3, reduced P 8 .

Preuaration of hiphlv nurilied PC concentrates PC was isolated from the PC-containing fraction by immunoaflmi chromatograph on a 12H8 column. The purified PC was concentrated to 10% U/ml b ultra Pdtration. Levels of others coagulation factors, factors II, V, VII IX and 3; and level of protein S were less than 1 O/o.Similar results are obtaine d for immunological and functional assays of PC. The frozen or freeze-dried purified PC was shown to be stable over at least a one ear eriod. Specific activity of the concentrate was found to be of about 300 U Ivmg (tR e total protein content was determined by the Lowry method). The amount of antibodies released from the immunoadsorbent (assessed by an ELISA assa ) was found to be less than 5 ng of antibody per unit of PC (data to be publishe B). Prenaration of PC deficient ulasma

DISCUSSION Human protein C has been isolated, until now, by multistep methods. Even when lmmunoaffinity chromatography has been used, one or several additional steps are required. In order to improve yield, to reduce time of for large scale purification and to promote its development re arative purposes with therapeutic a plications, an immunopurification met kfo in one step using mild conditions t?or desorption appears more suitable. Potentially, the ma’or thera eutic use of PC is not as a zymogen but as an active enzyme to imme a’ lately ha Pt on oing thrombosis. Pre aration of activated PC concentrates would be greatly faclq itated if highly puri Pled PC concentrates are available in large amounts with a rapid one-step method. Monoclonal antibodies appear as the most convenient affinity ligands in re ard to their homogeneous quality and their well-defined characteristics (aknity constant and epitopes recognized . All our attem ts to obtain high1 urilied PC by conventional multiste met h ods were prove cf to be unsuccessfir I . R owever, partially purified PC has i een used for immunization of mice and discrimination of monoclonal anti-PC antibodies from monoclonal antibodies to contaminants has been achieved by an immunoblotting procedure. The main advantage of immunoblotting as screening procedure is to select monoclonal

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anti-PC antibodies that did not crossreact with the most frequent contaminants found in PC preparations. For the development of an immunopurification procedure, the choice of antibody to be immobilized is of particular interest since it conditions the performances of the system. Several arameters have to be taken into account : - the yield of coupling of antibo Kies to the matrix. - the adsorption capacity of the immunoadsorbent. rocedure. The system developed must allow - the efficiency of the urifie cp PC in a single step procedure whatever the preparation of highly starting material used. %or large scale purification pu oses, use of plasma as starting material will interfer with the recovery of !?actor VIII or with the fractionation of plasma. Another source of PC, a fraction of plasma containing high levels of coagulation factors was found to be largely available without alterin plasma valorization. - tB e preservation of the biological activity of the isolated PC. - the possibility of desorption under mild and non toxic conditions and regeneration of the immunoadsorbent. One side-effect of immunopurification methods is the leakage of antibodies into the purified product. Antibody leakage from gels is related to elutin buffer, type of el, mode of el activation, type of antibody and number of wash/elution cycgles of gels (42 s . Whatever the combination chosen (monoclonal antibody, activated gel and desorption buffer), it still leads to leakage. In our rocedure, a cross-linked matrix experiments, a cyanogen bromide coupling and a hi h pH desorption buffer were use s since it allowed high coupling yields, higEil adsorption specificity and low level of antibod leakage. Five monoclonal anti-PC antibodies have been pro J uced and studied as affinity ligands. Similar yields of coupling were obtained whereas considerable differences were found m PC adsorption capacities of these antibodies. The adsorption capacity and the specificity of the adsorption are identical whatever the starting material used. Detailed analysis of the results leads to the following concluding remarks : * The antibody, 9E2, that offered the highest adsorption capaci was not suitable for use in an immunopurification procedure since the antibo 3 -antigen corn lex did not dissociated even in harsh conditions. However, the a %so tion yiel % (near 100 Yo) obtained with this antibody allows preparation o ? PC deficient plasma. * One antibody, C12C5, was not suitable for use in an immunopurification procedure since it alwa s yielded PC containing contaminants although this reparations. The antigenic PC antibody bound only P urified 1 zed'bp t!i?($l!?C.! determinant reco antibo c!? y may be present on another plasma roteins wY ich were not resent in partially purified PC pre arations. * $ wo antibodies, 9H7 an K 8HlO yielded a pure PC but o Rered a low adsoytion ca acity. for use in an The Past antibody, 12H8, fulfills all requirements immunopurification procedure. Its high adsorption capacity, its specificity, the possibility of desorptlon under mild and non toxic conditions, the preservation of the biological activity of isolated PC allow preparation of highly purified PC 0 timization of such a in la e amounts with a single-step procedure. proce p ure will romote its development and provi Re highly purified PT: concentrates for tR erapeutic trials. ACKNOWLEDGEMENTS : The authors would like to thank the Hemostasis Laboratory of the Blood Transfusion Center in Nanc for assistance in latlon factors assays. This work was part1 supported g y a grant of “region ray e orraine” (Pole technologique GBM - Biotec E nologie).



Vol. 63. No C


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re 01% on 33, g 42-648,




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Monoclonal antibodies against human plasma protein C and their uses for immunoaffinity chromatography.

Human protein C, isolated by conventional multistep methods, was used for immunization of mice. Monoclonal antibodies were prepared and screening of a...
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