lmmunoassays of human prothrombin species which correlate with functional coagulant activities RITA A. BLANCHARD, BARBARA C. FURIE. STEVEN F. KRUGER. GERALD \\'ANECK, T,IARIA J. JORGENSEN, and BRL}CE FURIE Boston. ,}Iass. Specific immunoassays have been developed for forms of human prothrombin thal vary in their degree of carboxylation. Human abnormal (des-lcarboxy) prothrombin was isolated in 18% yield trom the plasma of a patient treated with warfarin. The puritied protein migrated as a single band in electrophoresis and contained an average of three ycarboxyglutamic acid residues per molecule. A specific antibody subpopulation was isolated from rabbit anti-abnormal prothrombin antiserum by using aflinity chromatography. These antibodies, which bound to abnormal prothrombin but which cross-reacted minimally with prothrombin, were used to establish an immunoassay specific for abnormal prolhrombin. ln parallel, a specific antibody subpopulation, antiprothrombin:Ca(ll), was isolated from rabbit anti-prothrombin antiserum by conformation perturbation affinity chromatography. This antibody, which bound prothromban but minimally cross-reacted with abnormal prothrombin, was used to establish a specifrc immunoassay for native prothrombin. An anti-prethrombin 1 subpopulation bound abnormal prothrombin and prothrombin equivalently and was used lor an immunoassay that measured total prothrombin. These assays permit the quantitation of abnormal prolhrombin and prothrombln in plasma and serum. The level of native prothrombin antigen coi'relates precisely with the functional proihrombin activity. These assays provide an example of the use of specific antibodies against functionally important antigenic surfaces to monitor properties of coagulation proteins with the precision and reliability of immunoassay. (J LAB CLtN MED 101:242, 1983.) Abbreviationr: antibodres agarnst prothrombin-cajcium comple>l (antiprothrombin; Ca(II)). diisopropvlfluorc-rphosphate (llFP). diethvlaminoethvl (DEAL,). ethvlenediamine tetraacerate (EDTA), borine serum albumin (BSA): the k:rms pntltrornbrn and natit,e protltrontbtn (NPTi used inter.hangeablv rr1 I h",r..rt'of proteins involvr:d in blood coaqulation has tradition:rllr'lnlolred their :rlrilin'to actcleratc thc ciotting of'substrate-deficrcnt plasrnir. Thcsc ntethods are tedious and technicallv errtcting. and the rr:sults are iinprccise and difficult ro st:rndaldize.,{thorrqh intt-ltunoassa\'-. hale bcen n'iclch'used to quantitate protein altrig('rls in plasma, antibodv to the cntir(-'pnrtcir-r dr.res not usuallr lcad to inlbrntaticn conccrning the firnctionaJ propcrties ot'the prcrtein. P..rther. the'assav iiclds tht'totril antiqcrr corcuntlltion. Iironr th,-'Ilerlalolog.;'f)nc:ohgr Dirisrtrl. Tsfts-Ncu England \lcdrc.rrl Cr'rrter. end -firt,Depaltnrt''t of \lcdrrtne. fults L'nirrrsitv St.hrnl ol }lcCicint'. Bostr)n. \ltrs. 'fhis urirl t|ai 511pi1;p1i-{ br glaltts llL-21513. llL-18$3.1, and ilL-2ti:0tl lionr rhl,\.riion.rl Insalrutrs i)f lll.r.hh rnd grant Si)-869 {ront rhe Arrrcrjcal }lcart .\ssoc.i:rUrin Stibnrtlt.J for Publiratrcrn AI,ni l. l!,lli2: arccpted Aug lrJ l9r.i:1. Rrl,nltt r('qucsts: Rita A tsianchard \i.I).. Ihornciikr. No lil0. B{,sr()n Clt\"}JcrPrr.rl 8iS lianison \r'e.. Bostrrn. \lass 0lI 18. 00ll,llJ.l/.c3i0202.1:+ I lS0 l -14|tl e tgSt'fht'C' \. Ilosl[.Co \ olurne l0l N umber 2 lmrnunoassalls o.f lntman prothrombin species 243 Recentl-v we have expiored the use of immunologic techniques that re\.eal functional information about the protein antigen. This method is based on the use of conformationspecific antibodies that are directed against discrete regions of the protein surface. Prothrombin is a l'itamin K-dependent protein synthesized in the liyer. During biosl'nthesis, 10 specific glutamic acid residues near the NHr-terminal are carboxvlated bv a'"itamin K-dependent carboxvlase.r' : In the absence of ritamin K or in the presence of rjtamin K antasonists, forms of prothrombin deficient in y-carboxyglutamic acid and functional acti\itv circulate in the blood.3 These forms, knorvn collectivei,v as abnormal prothrolnbin, ','an'in the degree of carboxyiation. We have prer.iousl,v shown that abnormal prothrombin does not circulate in normal individuals. j In the current communicarion rve describe the development and analysis of three specific immunoassavs that quantitate abnormal prothron"rbin, prothrombin. and total prothrombin. The prothrombin antigen, in contrast to the total prothrombin antigen, correlated directl-v llith the prothrombin .ougrlant activity. Methods Human abnormal prothrombin was purified from citrated plasma of patients receir.ing long-term sodium warfarin therapy by using a strateg)'previousl-v described. bui rvith critrcaj differences.:. Plasma (1825 ml) rvas made 25 mM in sodium citrate. 1 mtr{ in benzamidine HCl. and i mll in DFp. Banum chloride ( 1N{ ) rvas added dropwise to a fina.l concentration of B0 mM. After stirring for I hr at 23'C, the precipitate that formed was removed b.v centrifugation at 900 x g. The plasma sipematant, containing prothrombin forms that do not bind to bariurn sa.lts. r,r,as diJvzed exhaustivell at 4" C aealnst l0 vol of 10 mM potassium phosphatc lpH 7.5r. I mtrl benzamidine.0.02,l ,odium a barium phosphate precipitate- that formed during dialvsis was removed bv centrifugatron. "zide. The barium-absorbed plasma (1980 ml) was applied to of DEAE-Sephacel (3.3 iy 10.7 cm) "oiu-., equrlibrated r.vith 10 mN{ potassium phosphate (pH "7.s), 1 mil{ benzamidine,0.o2% sodium azide. After the column had been washed rvlth the equilibrarion buffer, the prothrombin forms were eluted with 0.5I{ potassium phosphate, 1 mM benzamidine.0.02% sodium azide (pH 7 5). Fractions were monitored for prothrombin species lvith the total prothrombin radioimmuno"r."y (see belos.), Fractions conuuninq abrrormal prothrombin (268 nn) were dialyzed at 4'C for l8 hr against 20 vol of 0.lM boric acid, I M NaCl' 0. 19t polysorbate 20 (T*,een 20), I mN{ benzamidine,0.02% sodium azide, pH 8 5' and divided into four equal aliquots. Each aliquot was applied to an anti-prothrombin-sephaLrose column (1.5 bv 5 cm) equilibrated in the same buffer. The column ,r'as r""shed rvith 0.1M bcric acid. lN{ NaCl,0.1 Tn'een 20 containing 1ml\I benzamidine, and 0.02% sodium azide to rrunimize nor-rspecifi c protein-column matrix interacticn.6 The bound abnorma-l prothrombin was elutcd from the column with .ll1 guanidine HCl. I mM benzamidine and dialvzed againsr.l0 mtrI Tris HCl,0.l5NI Nacl. 1 mN{ ienzamidine. pH 8.0. Abnormal prothrombin (35 ml) was concentrated in dialysis tubing bv using dry sucrose and then dry Sephadex G200, The protein was dialvzed against 40 mM Tris IjCl, 0.151\{ NaCl. 1 ml\{ benzamidine. 0 02f1' sodium azide, pll 8.0, and dirided into nro equal alquots. Each aliquot oiabnorma.l prothrombin (2 ml) u'as applied to a sephncry! 5-200 column (1.0 b\:100 cm) equilibrated in 40 mM Tns HCl, 0l5M NaCl, I ml\f benzanridine. 0.02^r sociium azide, pll 8.0. The protein rvas eluted from the column at a ratc of 6 ml/hr at 23" C. Thc fractions corresponding to abnorrnnl prothronrbin rvere pooled and stort'd at - i5" C_ llurnan des-y-carboxr'-prothnrrnbin u'as prcparcd by decarboxvlation oiprot6r6rnbin grrh the method oJ TuhI ct.rJ'lligh-nrolt'rtrlar-ut'ight agqregates that formcd dunng dccarb6xylation rr.ere rcmolcd bl gt'l frlturtron orr St pltrt n I S-10C. rhe isol;11cLI des-7-carboxv-protlirornbin i'ieided a single batrtl on sodiLlm dodcc'vl sulLrte-pclvacniamidc gcl electrophoresis arrj ir-,ntairred. or.,'rr.".u11,,. I tii2 trtol of 7-clrboxr qltttatnic acid rt'srduc's pcr rnole oi prothrombin.t Ilurnan frlqntent I u.3s pre p:ued bv linrited digestiott olhttman protltrornbin rrith bovinc thrsrnbin. Prothronrtriri u.as isoluted br b.rrium citrate lbsorptioll. il[.{I,-ccllulrtse throtnatograplrr'. and heparin-agarosr. rhrornarc,,ar"ph1' ,; pr.,frld prott'ins t|ere erur.nrnt'd trv tlt,decvl sullate gcl elt ctrcplt11r,,srr,r ancl polr,acn.larnl,le rlisc gei elt'clrophoresis. 1! , Ilutrlirrl frrotht'o:lrbin rlnd hilltt;rn abnr,nnll prothronrbin rrr.rt'lalrrlt'd $rthr:.1 br.i ]ar.- 211 Blancltard et al. I Lab. CLn \led i cbrurr\' I 98:l toperoxidase svstem (Ne1\'England \uclear Corp.. Bosron. llass.).r3 Goat anu-rabbrr immunoglobulin (CappelLaboratories. Inc..Cochranville.Pa.t.tsSAisigmaChemica-lCo..St Louis.\lo.r.andrabbit immunoglobulin (Cappelt rvete obtarned commercialil . Sepharose .l-B, Sephacn.l 5-200. and DEAE-Sephacel rvere obtained fiom Ph:u'macia Fine Chernicals. Piscatan'al. N. J. Guanidine- HCl. Tueen 20. benzamidine. and DFP ivere supplied bv Schrvarzl\'Iann. Orangeburg. N. Y.. Fisher Scicntific Co., Pittsburgh. Pa.. i\ldnch Chemlcal Co.. Inc.. Nlilrvaukee. \\'isc.. anci CalbiochemBehrinq Corp,. San Diego. Calif.. respecri\:elv. Fresh fiozen huntan plasrra uas obtained fiom the Northeast Regional Red Cross. Fresh human plasma \1'as also obtained frotn donors receirinq krng-term sodium nariarin therapr', Biood sirmples. anticoagulated in citrate (9 parts blood to I part 3.8% citrate'). rvere collected from normal subjects and patients medicated n'ith sodium *'arfarin. Consent rvas obtained from a-ll partlciparrs under guidelines estabiished br the Human Investigation Rerievr Committee at Ne\,r' Eneland trledical Center Hospital Sepharose'l-B rvas activated w.ith c-vanogen bromide (Eastman Kodak. Rochesrer. N, \'.:200 mgiml of Sepharose).'r Prothrombin. decarboxviated prothrombin. hurnan prorhrombin fragment 1, or anti-prothrornbin antibodies in 0.2II potassium phosphate. 0. 13XI NaCt. pH 7..1. n.ere coupled to the acdvated sepharosc in rarios o1 3.0 mg of polvpeptrde to 1.0 mI of activared Sepharose, Nerr' Zealand lvhite rabbits u'ere immunized rvith either human prothrombin or human abnorrnal prothrombin as prcviouslv descrjbed." Anti-prothrombin:Ca(ilt antibodies rvere prepared bv numerous modiJlcatrons ot the ntethod preriousll'described,'' Rabbit anti-human prothronrbin antiserum (-1 ml)uas applied to a humiur prothron.rbin-Scpharose column (2 br'7 cm)equilibrarcd $'ith.10 mXI Tris FICI.0. I5II \aCI. I mN{ CaCl.. pH B. 1. After the nonbindine prorein had been remor ed br. rvashing *.ith equilibrarion buffer. a smali arnouttt of antibodl $'as eluted $,ith -10 mi\l Tris I icl, 0. 15II NaCi. 3 mll EDTA. pH 8. i. and considcred to be anti-prothrombin:Ca(-ll ) antibodies. Antibodics to human abnormal prothrolxbin n'ere raised in rabbits. Anti-abnormal prothrombin antiserum (-1 ml) rras passed orer a prothrorxbin-Sepharose coiumn r2 bi'7 cmrequilibratecl in -10 ml\l Tris HCl. 0. l-xl NaCl pII 8. 1. .{nttserurn thar lailed to bind rras applir-d to a ciecarboxvlated prothrombin-Sepharose co.lumn (1.5 br 7.0 cm) equilibrared in 0. l\l boric acid. 1trI NaCl.0. 192 Tneen 20. pH 8.5. Bound antibodv. speci{ic fbr abnorntal prothrombin. las eluted *ith III guanidine fiCl. pooled. and dia.lvzed against J0 rn}{ Tris }'lcl.0. 1\{ NaCl. plI 8. 1. Antibodies that hor-rnd both protirronrbrn and abnormal prorhrombln equiralcnth rvere isglated liom rabbit anti-human abnonnal prothrombin antls..rum..{s a bv-producr o{'isol.ition of antrb6dies sPecifiLc fbr anti-abnorrnal prothrombin. tht abnormlrl prothrornbin antibodr fiac,tion thar bound to prothrombin n'as entploved. A{icr dialvsis. thrs antibodv prepa-ration nas applicd ro a humiut iiagment 1-Sepharose colutrn i1.2 bv 10 cm) cquilbrated in 0. 1\1 bodc acid. 1\l \aCl. Lr. lli Trr.eel 20, pH ti.5. to rctnttre.rntrbodi that bilrds thc anrinoterminai rt,qion ol lrrothronrbin. The antibodics that did not hind arc directc-d against ilntiqcnic dclcrrlin:rnts shal.r,d bv hurr;rn protltr(,ntbirl:rnc1 atrnorrral J)r'othrombin. Prothrontbit't actj\itl \r'i1s assarcd br mcasurinq tlre accrlcration of tl-rt ciottiltq ot lirctor ll-and ftictor VII clcficrent pliisnra f Siqmai bv test sirmples. 100 t{.1 :rbrluot o1 rabhrt br':rur ccphrilin suspen'\ sion (Thromirolar: Ortho Diaqnostic'S1'srcms Inc.. R:rritan, \. J.ti0.l nrllnrl of {J.1lf Tris tlCl. ptJ j1 T.6lcontaintngRLrssell sviDt'rrcnorrr(0.1nrl of a p{ilrnl sohriioltin I ml ol 0. lllf lsll('l.pFl 7.6) tas addcd to:r l00,sl santple ol the I:rctor-dcficierrt plasnra and i00 pi ol rht tesr soiurion 1nd inctlbrtled;it.17" C. Cir,ttinq uils rnrtrated bv the lddition of 101-i p.l of 25 rn\l ('aCl. Pr.othrombin acti\it\'\f ils detcrmtn('(l br'1;lottinq tlrt'lrigarithrn o{ thc ckrttin,; tjmc as a iunctirrn ol tlrc lo.{anthrtr ol tht'lrlothrolltbln t()I)c('lttliitiott [)urtficd hrrnran protirrorrrhut ol knosn cirnientllli()n \1its uscr] to pr( l)arr(, tlrc stancl;rrrj curre. 'l-ttlrl prothrr;rrrhinarlilitrnasnrt,rrsrpr'rl irr.rntodifiratilr.ill'tlir. I-cltrsrrrr-piat1\\(,t).11:lsslrv.r,. Ilad ioimmun()assal' I)tt{Ir l nir;ulrr; .{s!AY. Thc l}rt('r.rctiou oi pulrfit'd rrntil)r)d\ tith r: I i.rbt.letl or lu.othrr,rirlrrn IlS.'\r'r0 llrqill1l 1s()i!\totti.tle tlrt'lirr,ileonct,ntr.lti{)lll.; rrrilS.\.\'iu.rrn{.int,riinii0l lrntiirrriltr0li-t l()1) llgr \\(r{' lL<lLlt'd tt, lit'itl.utti}rrxir ({rrir('nir.ltlrtns rilt{u}r, ttoirr l() rr\1 tir l0 ;\l Brrllt.r'tJO trtll Trts II( I 0. 1ir\l NlrCl. lrll S. l. I tr\l hlnr.rnrrrlrrir. .rnri ': l-irrlrt itci irrortin rinirgt'rr lfinrri Lotlcllllrilti{rl I { l0 "'\1, trr,}i'il(i(1(,{i tii ql\e,r \.)luntc ()i _5i pi. \\rtlt.Lrrti,lrrotlrlr,rrrl,nt:(.t Ilt to i \ ol!rtrrl l0l \Llrrber 2 lmmtLnoassuys ct.f ltuntun ltrrttltrontbin spccies 24a Tablc l. 'f utal pr0tein yOL S (ntl tcp Plasma fror.n parients 1 NPI Itrg) lnlq) , 825 80.300 APT (mg) (rng) \ tek! AP't (%) 12.8 105 8 i00 0.010 5.9 5.9 -t 0 021 0.008 5.4 4.6 4.1 4.i 42 to 3.817 r8 6.285 58.4 7-otaL PT Puri.ftcation receiving sodiurn rlarl:rln a .1. Barium citrate supernatant DEAE-Sephacel An ti-prothrombin- 1.950 .18,750 2.512 268 35 6.8 0.75 t) 13.41 Sepharose Sephacn'l S-200 \PT - nonlal prothrombin: APT 2.3 - 2.3 abnomral prothronlbin; pl = plsthrombin antibodies. solutions $ere adjusted to I mI{ Ca(li). With anri-abnonna-l prorhrombln al)d anti1 antibodies. soiutions uere adjLrsted to I mNI EDTA. Each incubation rube rvas mrxed vigorouslv and rncubatcd or,ernrqht at .1" C. Afier this time. 25 pl of goat anti-rabbit inrmunoglobuhn (12 n-rgtrnl)uas added. Tu'entr mrcroliters of rabbit irlmunoqlobulin i2 mg/ml) was added 30 mln later'. 'f he solution u a-s incubated {br -l hr at 25' and assaved in toto for r:ri. The precipitate that ibrmed prethlombin rvas removed br- centriiugation in a Beckman model .1B microfuge iBeckman Instruments. Inc.. I'-ullerton. Calif.;. rvashed t$ice $ith 200 pl of -10 m\I Tris flcl. 0.1511 NaCl. pti 8,1. I mx{ benzamidine. and assa]'ed ior '!'I in a Beckman Gamma 8000 scintillation spectrometer. The data are pr€sented as th(' percentage of antiqen bound as a function of antibodv concentration plorted on a sernilogadthmic sca.le. Corrprrrrrox AssAy. The dr-splaccmenr oi 'r'l-labeled prothrombin or abnormal prothrombin fuom antl-plothrontbtn:Caill, antibodies. anti-abnormal prothronrbin antibodies. or anri-prethrombin 1antibodies bl prothrombin or abnormal prothrombin nas studied with a comperltion radioirnnrunoassav. .\s abor.e. the double-antibodv technique ivas emploved. The reacrron mixtures included lr'l-labeled prothrombrn i1 x l0- r'r\I) or abnormal prothrontbin r'1 x l0-"'\1); anti-prothrombin:Car.ll) i3.2 >i l0 r"\ll. anti.-abnomralprothrombin i7.2 x l0''!ll). oranti-prethrornbin l i2 x 10 1ri\I): l'i alburnin: .10 mJI Tns ilcl 0. 1511 NaCl, pH 8. 1. I mX{ benzamidine: unlabeled prothrornbin or lbnontr.rl prothrombin or citrated plasma diluted into I mg/nrl albumin. as indicated. Stilutions*-ereacl.lustcdto' lmllCaCl, lbranti prothrombin:Cai II)ortrbcdirsandrolnrl\{EDTA fbr arti.-abnonrrril prothronrbin end antr plcthronbin 1 artibodies. The total volurne u'as 2i:5 pl. Data are prrescntrrd as the percentaqe of racliolabeled antigen bound to antibod_v as a function ol the concentration ol the unlabelrd cr-.rnperitor on a scmiloqarlthntic scale. Results Purification of human abnormal prothrombin. Abnormal prothron)bin 11as pu- rificd from plrsrna obtained frorn prticnts treated ovcr the long tcnri n ith sodiurn n irrf'ann. Tlris plasml containcd botlt irirnorrnai prothrombin and l)r()thronlbin, as evidenced lrr. r'tngulation rs,savs irircl the -sItiific inrntunoassa\s (Tablc I). Prothrrmbin and rvcllcarbt:,svlatcd Ionris ol airnolnrrrl Ilrotlrromliin \\'cre rl.rlo\-cd lirni tirc pllsrna bv banLrnr citnttt itbsorlltion. Alrnotnlrl prritlrrorntrin r;irt;iincd fionr the se'.ond plotcin pcak li'orn thc DF.,'\E-Stph.r(cl colrrr)lr1 rr.rs .rpplird to an anti-protlrrornbin-Scnh.irosc column I'lre lr<rLrnd yrcak. rluttil r',itir l\l qit.rniclirrt'IICI. c'urrt.rinrril airnc,lnlrl l)roiluourbin tlri.igln. .\lrnotrnrtl I)rotllll)nlllinu.rsiirltlrcrpur-ifirdl)\ q('l filtliltionolts,.lill.lrtrlS-200tol'tln)()\r, 'l-his Itic.lt-tttL,k't'ttlrrrtr e i':,ht ( olli.inriuitilts tu'r'p,rraiir.e sche ni,-' olir'rcd a 6000-lirld prtrr{ic.itirttt ol ril.nr':ttttrrl i}r'!rtirr.nli}ll) .rntigln tlorrr plasrtra n itit rur l8'i rilld, Char:rcterization of abnorrnal prothrombin. PLrriirr'd .rlrnt,r'nrrrl plotlrronrlurr 216 I Blancherd et ul. Lab Cirn \1ed Ft,br uan I 9S3 Or Fig. 1. Anal.vsis of human abnormal prothrombin br gel electrophoresis. A, Sodium dodecvi sulfatepolvacnlamide gel elcctrophoresis. The eel. containing.10 pg of protein. \vas staincd uith Coomassie blue. B, Polvacnlamlde disc gel electrophoresis. The gel. containing 10 pg of prorern. u,as srarned s'ith amido black. lielded a single band rvhen subjected to disc gel electrophoresis or gel electrophoresis in dodecl'l sulfate (Fig. 1).,\bnormal prothrombin appeared more than g5qi pure br.these criteria. Clotting ircti\itl of human abnormal prothrombin as measurcd b.v the prothrornbin assal was less than l% of that expecied from a simiiar quantit_r of purified human prothrombln. but it had an activit,v comparable to that of prothrombin rvhen assavecl b1'theE. ('arinatus assar'. Abnormal prothrombin contained, on a|erage, 3.0 mol of 7-carboxvglutamic acid pcr mole oi abnormal prothrombin. In comparison. hurnan prothrornbjn contains l0 mol ol 7-carboxl'glutamic acid per rnole of protcin. prothrolnbin rvas radiolabcled n'ith r:;'J bl the lactoperoxiciase mcrhod lnd '\bnornral nrigratcd a-s ii singlt,band on gel elcctrophoresis rn dodec.1l sulfate. Purification and specificity of anti-abnormal prothrombin antibodies. Rlbbit arrtt-abnorrnal pl'othronrllin iriltisera lormcd precipirating cornl;lcrcs nith both 1;rothrontbitt nttd abnortnal prothronrbrn. Af tcr lrnrorirl of arrtibodics thar cr.oss-react n jtlr I)rothloltlbilt. bl using :t ptothrotnlrin-Sepharosc, crrluutn. and purification o{'Ihe rcltilining arttibodies on a dccaltrorvlrrlcd ltrothrotrrbin-scpitaicse colirntn. onlv 5fi of tht,ttrtal .lnti-.ll)n()nliil |rotitron'rbrn lrntibodics nere spr'cifir lirr rurti-rrlrrtortnal plttlrrornbut. Tht' ittttractron ol purificd ;urtilrriclics spccific lirr anti-rtlinorrnal prcthrontlrirt n'nlt l)I'othrolrli)itr and itltttorntlrl protht'onrbin n'as rr elrr.ited br rlrrlioilnrlLtnoussil\. ln tht, .lir.e'rt brrrtiinrl irss.r\'. iltlti-irbnornrril lrlrtlrlornllrn antiboclir,. rr t'rt. urc,rrbittcd n ith I:'l-l.ilrr.lt'd Volrrrne 101 Itnmunoassa!:JS of human prothrombin Nu nrber 2 [arurraoo'r_t-'l t species 247 u) R N N 10-ro 10-s 10-8 10-€ {ANT/gODY] (M) 2 interaction of' radiolabeled abrrormal prr)thrornbin and radiolabeled prothrombin \rith anriabnotmal prothron)bin. anti-prothrombin:Calll), and anti-prethromtiin l Direct binding assal'describing thc ilttelaction of '!"1-labeled abnormal prothrombln (6"nribodi"r.A, x 10 rt,trI) g.ith inclicated concelrtrarions of enti-abnormal prothrombin (r). anti-prothrombin:caill) (o). and anti_prethrombln I antihodies (r). B. Silme expcnment but utilizingr:.1-labeled prothrombrn (6 l0-'l'1,{) ^ 1 anti_ alld altti-abnrrrrnal prorhrornbin (,'-). anti-prothronlbin:Caillt (- ). and:rnti_Jrrethronibin Fiq. bod_v ( :-,), ;tbnonttal prothrotrtbitr or I:il-lirircled prothronrbin. Binding of abnornrril protirronrbin anti[iocll to radiollbclcd itt)tlgcn \rils obscr\'.id onll nitlr abnorntitl prot]tronlbin iFiu 2. ..1 ). No siqrrificlnt brrding of antibodr ro plotl'ironrbin uirs obscrrcd iFic. l. fl). Tirc l.riltrrc ol tl-re irnti-abtrolrnal l)r{)tllrollbln antil)odj(,s to rear-.h the nraxirnurl lercl ol r:t'l-lrrbc'icd rrbrtonnll prothlorlbin binding \\..Is in c,ontl.ilst t() the resulrs obscr,.trl rvitl-r tht'anti-])l't'tllrontl)iIt I arttibodics This is nrost likch dr-rt,to tlrt'hetrro.lcncitl oi abltor, ttl.tl pt'otltrtrtttbin. 'Ihe antibodies. r'aisr'd against irblrornr.rl pr(rtlu()r)tbins that do lrrr billd to batjtttn salts. rtc're ptrlilied on thc brrsls o{ theil abilrtv rti brnd to sc-rnisr.rrt}retrc t]ecar, 2.18 Bhnthard et al I Lab .-]rn. \led februan. Igtl o zl @ f @, ;.olnorter,ro*1 rur o )z o o * :COMPETITOR. (M) Fig' 3 A, specificitv of antr-irbnonnal prothrornbrn antibodies, Displacement ol *,'l-labeled abnormal prothrombin fiom antibodl'bv abnomrul prothrombin tr) or prorhrcimbin (: ). B, Displacenrent of '!"1-labelcd rrbnormd prothronlbin from antibodv bl punfied abnorrl,rl prr)throrlrbil inro BSA {o)' purified abnornr;rl prt.rthrot'btn diluted into boiine plasrna ir). and abnrrnraldilptt,d pl-othrombin in plasnta {iom a patier)t rr(,atcd \1lth rr.iiflann (l ). borvlrrtcd prothr-onrl;irrs. Thc,atrtiboclies alc tlrrrs hetcroqenr,ot-ls in thrir spcc,ificitr.. and eath indirjdrrtil urrtiboclt.rrill interact tlttli varr.inq lrfhnit,,.to i:lrr.iorrnd prorhrcnrbins of. dillt'r'in': st'ttt s ,f t ,it l'r'tr l,tti'n. lt rr,'rtld tlrrrs r1,1,, .11 rlr.tt tlrt. ll l),rn rrr'hrrrtlirrq ( (rl)stilrt, Kr. clcsct'ibing int(,ru(tion ol rt ht,tt.r-ugr.nC,ouS iilltil)ridr popul:rtrorr uirh a hctcroqcrr,-.oirs ilrltig''11 llopullrtitrt-t. is Iort et tlralr rhat of' the .rirti Irrcrhrorlrbili I rrrrtilrr16ie s iur4 tlie Itotltoqt'ttt'0tts dt'ttlrrutrtrrrls ()lt ii)t,c.u.bo\r'tr.r,ntirrrrl JX)rtl(,\ns t,l .rl-.loint,rl lrt.otli*',bir_r. A Posstlrilih rt'ttt'titts tli.tI s'rtttt',,l tit. .rlrr,rlrrr.rl protir,rrnibirs Lc.itr pooLlr.or rrot at ril] *.itlr the alrtibtxlit's slrtcifii lirr rrrrri .rirtlr)nti.ll pl.()tltr{llti)it) us rsoi.tlcd. \ olumt I0l \ rrnrbrt 2 50 I Imntunoassatls o.f' human pr(ttltrantbin s/)rcics 2-19 t 40 o i zl I ; i 630 s @, to-e to- lo I o-8 [corrrerrrron] tu) PLASMA DILUTION-1 1000 100 o zl o (D I @"1__ 10-rl 10-e lcor,leerrroRl 10-8 (M) ig. L t\, Specificrtv of anti- prothrol'nbin : Car I I ) xntibodies. Displacernent o{ !i'l-l:rbcle d prothrombin iionr anttbodl bv rbnormal prothrombrn {a) or prothrombrn (:). B. Displecernent oir5l-labelcd prothronrhin lirrrn anttbodv br thc protlrrornbin in normal plasmr (.1 ) md purifred prothrombin F supplementld tti IlS,\ i,-. ). C'unclitions li;r the assir\'()l rtbttornra-l protl]ron)bin in p!;rsnru \\r'rc cxl)l(rre(l. \\'ith n i'()nrirltitiorl rl(lir)inrl)un{)assii\'. thr displace'ment oi ':-'l-lll'rk'cl i)Lrl}()rmal pl'othrontllin llonr enti-ab!r{)rnr.rl irrothrc)lnbin .lntihodies bv a}rnulnta-l pir)thronrlrin in 1t'i rrlLrr.rrrrin. rtitlt n<;rnrrrl bovini' Irlrr:r'nu. rii,licrnrd pf i)tllr()nll)in in Pl.rsrnii {tcnr .r p.ttient tnrt['(] rlith chronic uarlirrin t]rer.r1,r. (]r'ilir'tin'onrbin nas critluatcd. .\s slrortn irt Fitl, :i..1 tltt'se rtnlibodics cross-l-r.rctrd nuriirl.lili nith pltrtltrt,trrbirr undel thr' ( ()lr(ii{i(\n\ r'rrr1-'lort'rl IIr r'(-\lltrirst. .rbnolrlal I)r(}llllonbili c,rrrscd L()nlple tl dis- .rbrrrirnrrrl proihrr,rnlrin suppli'ntctttcd 250 Blanchard et aI. J. Lab. CIn l\'led Februan. r983 s o a N 10-5 ICOMPET/TORI(M) lo-4 Fig' 5' spec.ificity of anti-prethrombin 1 antibodies. Displacement of rr;I-labeled abnormal prothrom. bin from antibody to abnorma.l prorhrombin (o) or prothrombin (c). placement of '2il-labeled abnormal prothrombin fron.r the antibodr.. Furthermore, the iniribition slopes of abnormal prothrombin in 1% arbumir-"b;;;;;prothrombin in borine plasma' and plasma fiom a padent receiving chronic H'arfarin therapl. rvere identical (Fig. 3' B) These experiments indicate trvo points. one is that although abnormal prothrombin and prothrombin are structuralh' similar, the anti-abnonnal prothrombin antibodies have specificiti' for abnormal prothrornbin. Second, since the inhibition slope fbr purified ab- normal pr.thrombin in l9i albumin rvas identical ro that oi'abnomral prothrornbin in plasma from a patient receir"ing long-term u'arfarin therapr'. purified abnorrnal prothrombin in albuniin ma' be used as a standard to quantitate abnornral pr'thrornbin 'fhe hrr.rit ol sensitir-itr,of the throntbin. Purification and assa,v ,ras about 3 in plasma. x l0-,,Xi (2 ngird) ibr abnorrrral pro_ of anti-prothrombin :Ca(II) antibodies.,\nti- .specificity prothrombin: ca( ll'r antibodies were purified from and-prothrombin antlsera. \\hole rabbit anti-human prothr.mbin jn antiserum *.as applied to a prothrombin-sepharose c,r-rlumn equrJibrated in I rn\I caclr. The antibrdics rhat eluted with 3 nixl El)TA *.ere considered anti-prothro'rbin:calll)_:rntibodies and compriscd about i.t to 7ti ot thc tot:r.l antiprothr'mbin antibodies. Tlre sPccificitv o{' anii-prothrombin:car II) anribodies ibr protltr.lttbitr *as sho*n rtillt;t drrcrt hincling lssa'and u cornpt'tititirr r.rclioirrrnrrrno:.rss:rr.. ln the direc t brnding assav. the i'rcracrions ol anti-pr<ithrombin: crarg) ,,;;;;;;;;, ;,;;, ill thronlbjn ard abnortlt:ll |rothronrbin uere eraluattd. Thtse anribo<iies b.uncl to protlirotllhitl (f ig' 2 B)but slror',c'd niininral r'ross-rcirctnjtl uitli 2 1r. atrnornrallr.oth.onrbrn rlriq. Thc displirc.e.nrt,trt ot u r:il-lirbelt,cl PrrithLtrnrlt,ilr lirrrn 31111_O,.r,rhror.ntrin:CaL II) irntil'odI br 1rro1[111111[1111 or lbttottttel pr,rt]rr<.rnrbin uas ililtstiq;itetl L,'cle' t5c c.rditi.'s t'lttpltivcd lltirthrotltllin ctrrtld contplt'telv displace plothroprbrrr 6ur, rurtrbrd' 'r'l-l.ibelcd (Fig. '1..'1 ). ll.r'c'r'r. rr. disPllc.r'clrt \\,.1s.,b."r,"d r'itrr abrrornrr,r 0",rrr,.,,,,rr,,;; "-,:;;;:; Volurnr'l0l \Lrmber lntmunoassaus 2 human prothronlbin species 251 o,f Table ll . Anallsis o{ plasrna from norrnal subiect and pationts treated with warlafin Prolhronbin Naf iae actiT'ittl prothronfuin t, pglml) ( tmli pg Abnormal prothrombm ( pglml) Total prothrontbin (. pq lnil) Natit'e PT + abnormal PT "l"t"l Pf (7o) Patients treated lvith uarfarin 16 l9 25 75 22 28 22 27 JJ 8l 26 23 92 58 57 57 62 57 69 66 o i5 J 18 21 19 12 16 19 17 14 34 11 13 )A 12 13 l6 l6 24 ZJ 20 24 l6 -i 1l 25 28 26 30 62 62 59 64 37 62 76 73 5 6 't? 7 10 11 11 12 12 19 7 6 8 106 11 124 48 4 14 20 q'7 10 43 28 B 45 I B 59 68 58 60 6l 61 77 82 53 59 73 58 63 61 69 1 .17 85 47 73 143 2 71 3 56 t04 0 0 96 B4 r0B 98 0 0 67 121 B6 94 0 0 0 0 0 0 0 0 76 o.) 7B 127 74 126 129 Normal subjects 1286 380 496 566 695 770 96 82 B1 82 70 99 74 82 s2 B1 93 10 B7 106 11 86 95 12 77 91 B I 65 73 72 B6 r23 B6 123 77 It8 PT = prothrombin. conccntrations in excess ol about 3 x l0 '\'1. The degree of reactii'itv of anti-prrlthrombin : Car II ) antibodies tlith ahnornri,rJ plothrorabin n as about 0.0-19i tlrat of prothrombin. Thc cflcct rtf plasrna proteins orr the irlteraction o{ anti-pt'ot}rrornbin:Ca( ll:anti- bodies and prc,tiu'ornbin u'as (oirparcd, As shown in Fig. I B. the slol.lc of the displacettleltt clll'\'e o! prothronrbirt in 1'7 albLurrin t'as cquir'.rlent to that o1'prothronrbin in normal i,rllsnta. On this basis. ytrotltrorrtbin in l'i lltrunrin rvas rcgLrlarlr c'rtrl.rloicd as il quantita- tilc stuldlrrd. Characrtcrization of anti*prethrombin I antibodies. ..\nu-hunran pr('thronrbin ;inttbiidics nt'tt'1;rrrifird, nhith intt'r'lctr'd t'clrrilaicntlr uith Lroth prothnrrubin;rrd abttortttal prothrotttbitt. Itt tltt'dirt'ct bindinq ltss:rr tlris .ultibodl subpopulation bound tri lroth rrrdiolaix'lcd liLiru:rn.rbnounrrl prothi'()ntllin iurd pn,tlrlotnbin tt'iq. l...{ and B). Jir:isscss I 252 i BLanclturd et al Lab CLn \l FcbrLian 1 ed 963 a a a - a c \ :60 L F a 240 d) o PT Activity = O.74 . O.92 (PT Antiqen) 0.30 = F r=O99 (r^^ LZV lo 30 40 50 PROTHROMBIN 60 70 80 90 roo ANTIGEN (F.9/mt) ilo 6 Companson of prothrombln antigen ,uld prothrornbin r PT actiutt.. Prothrombin antigen \vas measured \11th anti-prothrolnbin:Car'lliantibodies. The plotllrornbin acri\.it\'\1as quantilareci in coagulation assavs us.ing prothrombin-deficieni plasma. Studies inclutled nomal subjects 1a ) and patients trcared $ith srjdium rvadann ir ). Frq. whether the interactions ol anti*prethr'olnbin 1urth prothr.ombin and abnormal prothrombin \r'erc equivalent. a contpetirion assar' \\'as peili)rmeci in ,,r,hicl.r r!.1-labeled abnormal prothrombin was dtsplaced frorr the antibodl bv eirher prothropbin or abn{.r1rlal prothrorllbill iFig. 5;. Thesc tuo antigens generatcd idcntical displaccr.r.rent cun.cs. indic.itirlg that:lnti-lluman prcthronrbin I antibodics bind cclLriralenth to hoth Iunran pro- ald ltumln abnormal lrrothronrbin. lteasurement of prothrombin species in plasma. The three irnribodies \\rre used to de|elop threc radioimrnunoassavs to cluantirare the plasma levels of total prothrornllrn thronrbin. prothrombin. artd abnormirl prothrombin in a group of paticnts recelving sodium lrarf)rin and in norntal sub,iccts (Tiible ll). Thcse rrssals yr,:lcled a prccision ot'abgut * 15'i. Abnortnal 1;rr.rthrort-rbin in the normlil subjccts \1 Js undct(.cl.rLr]e. Toral prpthro6rbin lelcis agrced re:rson:rblv n'cll njth thc lelels ol prothlombin. Prothrorpbin acrjvitv. as Illeasurccl trY coagrrlritit)n.tssirvs. nas in exccllent agtecnlcnt rrith the l)rothftitnbin lcvel. ars dctcrmtlrt'd hl radioirrnrunoilssilv tFig. 6). Thc correlirtion cocfTrcrelt,"vas 0.!lt,;. dcscl'ibing the lincar rt'lrrtiorrship be twr'r'n the l,rrothlonrbin antiscn antl rlre irrorhR)rltbin acli\ it\'. 1-ht' llltit'rrts rf c(,i'\ ll)g liirrg-tr,r'rn n.irl;f irr tIr1ir1;r. r I.rblt, ll) r'. itlr pr.1rir111t]tip tiprcs tt,' tht rliirt'Lrti( r:lll'tc r l),ttie rtts i to 8 r lr.rd ulrnt,r'rrr.ri itli,tlr1llrIil .lcte l-s tltlit rrlr.r' lrrglrr.'r tlnn Ptothlrintlrin lert'ls. Plrtitnt tl ltlrd sultoPtinrril irxrkrrriririr,rr ol tlrt. ltroilrr.ornl.rin tirnc. I);ttients 10 to 1l lt,rd lrtr'tluorrrbrn tinics iltilt \\r'rL, l)r't\\r'i.lt .lr: to :l frrrrcs th.rt ol rhc Ir,rrrtlrrl to|rtrol. -Ilrcit l.rl.rsrnir lt'rr'ls o1' 1.r1'1r1|111y11111iri siretit,r ut,rt c.h.inri tct-i:rt,ri br rcn llrril lt'lrls ol,tirttotttt.il Ir1r1i11,,,rt1rtn,irtcl lorr lrrcls ril ir','tiuoirrilrr 'l-irt,r',,rr.r: rrr.t,[irnt (()1l'tliltl('11 lli trti''.'tt tlti' lltutlttirtttlrtl lt'rt'ls. .ts ntrr:ru'r'i] lrr tllih,;iiltrlrrrrr!,15:,1\ u\llg Volumr I 0l \ Imntunoasstttls untbcr 2 o,f' Intman prothr()nil)itl.spec.ies 253 III' colrtparisot.t of quantitation of prolhrornbin. abnormal prothronlbin. ard total prothrornbin in seruin ancl plasrna T'ttblc Prothronbin ( pg lntl) ,lbnorntal prt 'fotal Plusma" Serrlz Plasnta' 141 0 0 0 0 Serrr rn prothrontbin (wlnl) throntbtn ( pg I tnl I Plasrna" Seru nt i) 0 1.16 42 ao 0 1BB l5 0 t1/ 23 Nornral subjects I 1.15 2 ll0 3 rBr t:: 4 130 1.16 Patients treated n ith n.arJarin I 2 3 10 12 12 29 67 3.1 z1 2J to )) to .)O nl l9 4 5 6 7 8 t0 I 'Piasrna correcred 'alues'ere 108 37 i8 27 51 t0 11 29 37 10 35 26 36 uJ 31 38 101 50 83 62 61 32 20 lB 24 60 20 l\) tc 15 5.+ 21 26 tiir the dirutron of the n'hole blood bv arrricoaguiant. anti-prothrotrrbirl: Caill) antibodies. and the prothrornbin coasulanr actiyit\,. as clcter- rnrned bl coaqulaiion In contr:ist assav iFig. 6). the normal subjects. tl.re total prothrombin levels ir.r the piiticrts receir.inq lot.rg-ternl $'arLarin therapv consistenih erceedecl the surn oi the prothror-nbin plus the abnormal pl'othl'olni)irl. This rcsult suqg{:srs that these patients har-e prothrorabin spcc,ies in their plasnla rthich eithel do not rcaci ol reacr poorlv u'ith the antiboclics spccific. lbr tr'r anti-ilbnormaj prothrorrlbin llost impolturtll'. this emphasized rlie quantiratiye lations of thc abtlorllal prothrombin assar. This rncasurement represcnts a li*l- loy.er lirnit of the ahnortn;il nrrrtlrtolnltin eonL(ntrrt on. Tirr: prothrorr:bin assar'11llasures a hor1t6geneous aniiqcn ltnd is thus qLlarltitiitl\:e. fhC qtlarltitrttiott of abnorttial prothlonrbin ar-rd proihrornbin bv speciiic irnlrrr-roirssal. \ras colllp:lrt'd in scrtttrl and plasrnlr littn nolnrai,subiects and p,rucnis tr,j.rt(,d rin rJablc Illt 'I'he prinsnra irnd st'rutrt fi'om one patielrt 1ii5 r'u1.- receiling rvrrrJlril disl;lac.d ':'l-labcled prothromhin lrotrr anti-prothronrbjn:cailI) eq.riralentlr: anci drsplaccdr::,flabeled itbnornlirl prothrolnl)in fiorn errti-abnorLlal prt.rthnrnbin t.qLriral,.,1tlv rdara sh'*n;. The ti;trrl llrothx)lrl)in nrunit.rccl i.^. a'ti-prettronrbin i iu,rtib.dics \ras not sigIrificarrtiv lr-r$ct itl scrtlllt thrln tn iri.rsnr;r. Tlrcsc rcsults indicate t5.rt prothrorll-,in ard Itlrtlorn]lil pr0tltloltlbin c'rtn hc nti:usurr'ci rn cithur scrurn or plrrsrna il ilrc spcc,ilic ir'rarno- as-cAvs lil'e r'rnPklved. Discussion SIr'tific itlllltl:tlollssll\s fitr lilrtiornrlil ploilironrbiu. il.othnrrrr5irr. irptl teral Ir.ri6*'rlrin lrlnc lrr.r.,1i 611-51 1.i1;r,rl. [,nllkc tlrc cloninl] ris\.t\.s ll\ld tc c;11"r.,11,,,," tlrc !ir'.ti.rrlrl il(tiVilir.\ t,l tfrtse sl)(,(tr..s tltt.s(,lt\s,l\s lu,t.|q,1{iy11111,d lritlr sprrifir.iilttill()il! srrirpi,lrrrl;r- ti0t1i 1i1111 a L.rrli,,;illntril().lss.l\ Tlt,,.tr.Ji,r.t,ti:t,r lt.trr,lill thc.ic,]r.l])t.lqi,s,{.qrrltntit,ttitt' llt illtlllttltrliss.tt L rrlikt irntrrrinollssli\s g:lli nliLrlc ,tt.r!isttt.a. gilrt,h (iLp 6nlr aip.illtit.lt(, l)lot('ill :llltl'{i'n't '" thi't('lissrl\'! rr'r('.t\ult'stiirctLrL.rl antlqens ti.r.rr tont,l.iit,tlirt.trir iirrrtirort.i.l I)fr)t)r'nt(.: rtl tltt rrit}t rnrili,r Lrit.. I-ltt,sl rtrt,titt,rjs rir.ti I,t, (rrti\ir]r,1td .t\ .l rlr,\\ :lp_ 254 Blanchard et al. .l Lab Ciin. Med. I-ebruan.. lgiJ3 proach to the application of immunoassay to the evaluation of the functional properties of the biood coasulation proteins in compiex hiological flulds such as blood. plasrna. and serum. The correlation of the prothrombin acti\ritv to the prothrombin antigen is exceilent. This is true both in normai plasma and plasma obtained ftom patients treated with rvarfarin. It n'ould thus appear that the prothrombin antigen, in contrast ro the total prothrombin antigen, is convenientlv measured in serum or plasma and contains information identica_l to the prothrombin coagulant activit,v. This lvould be expected to be rrue in plasma from normai subjects, from patients rvho are ritamin K-deficienr. from patients treated with rvarfarin, and from patients rvith liver disease.{ However, the antigen-actir.ity relationship may Vary considerably in patients u'ith mutant prothrombins (d1'sprothrombinemias). Since the prothrombin antigen may be measured in serum as u,ell as plasma. this assay ol' functional prothrombin obviates the need for fastidious handhng of anticoagulated blood. lvith rapid transport of fresh citrated blood in piastic rubes on ice from the bedside to the laboratory. Previous investigators of prothrombin species in plasma obtained from patients treated n'ith vt'arfarin have indicated that multiple populations of parti:rllv carboxylated forms circulate in the biood of these patients.:0 2r Our assavs of total prothrombin. using anti- prethrolnbin I antibodies. substantiate this. There are present small. but consistent pro- thrombin components that bind poorl,v to anti-abnormal prothrombin and anti-prothrombin:Carll) in the plasma of patients receiving long-term rvarfarin therap-v. These mav represent the partialiy carboxr'lated forms that can be absorbed bv barium citrate. We do not knorv rvhether these components fluctuate nith thc beginning or cessation of u'arfarin therapy or rvhether there is marked individual varjarion in the amount o{ these intermediate prothrombin species benveen patients rvith equilalenr prothrombin times and levcls of prothrombin. The assays descnbed offer potential for the diagnosis of disorders assoclated rvith abnormalities of prothrotnbin biosynthesis. As reported preriouslv, lorv leyels of abnomral prothrombin circulate in the piasma of patients r','ith liver disease.r Since this antigen is not detectabie in normal plasma. it mal,sene as a useful antigenic marker of abnormal 5ver function. In ihe absence <-rf rvarfhrin. high levels of abnormal prothrombin relative to prc-rthrombin immediatell' suggest r-itamin K deficicncl. These assays are a sensitive measure o1'clinicai and subclinical vitamin K deficiencr,, uhich off'er considerable improvement over the orothrombin time. This assay has been used to diagnose surreptitious war{arin inqestion in a rvoman vr'ith factitious purpura, In the face of'a normal diet and normal prothrornbin time. a significant abnormal prorhrombin level (6 pglml) was measured. These assaYs shorr promise as an approach to monitoring oral anticoagulant therap1'.2r [n a prclirninan' report, 89qi of patients u'ith a bieeding or tlrron.rbotic complication during $'arfarin therapv rvere identifiable rvith the assav of prothronrbin antigen. Onlv 33'i w'ould have been prcdicted to be at risk on the basis of the protl-rrornbin time. Finlllv. nc har"c rccentit shor.r'n that abnormal prothrombin is an antiqenic scrunr marker 16r pnnrary heJratic carcinonra, pres€.llt in gllct oi tire 7ll pati{.nrs testcd.r:t ,'Uthough thcse;tssals shorv considt'rable potential and diaqno-.tic utiiitv. their r,rsc is crrrrcntlv lirnited. The antibodv subpopitlation-s reqrril.e ertensile prrdfication front lar"ge qLlarlt;ties of rultiscra. 1'he1'arc not corrrnr'rcialiv available..\ labcrr.afory that c,hooses to cstabhsh tltcsc ass:rvs tnu-st tttake a considcrablc cornuritmt'nt llr rirrre and clibrl. Rece.nt ad|atlces tnltv llelp tr-r ntake tltese assavs nrcrre arailable. F-or exarnple. ne havg rcc,cltly prc'p;ired a tltttrittt. tnonoclon;il antilrodl ur hunr.rn ltrothlrintbtr-r. u'hic.lr binds plrthlrptbir-r \olurnr l0l N lmnunoassaAs unrber 2 o.f human prothrombin species 255 onh' in the presetrce of calcium and does not bind to abnormal prothrombin.2{ Such reagent nlav allolv the development of an inexpensive diagnosric test for general use. We thank lls. a Eileen O'Brien for her assistance in the preparation oi'this manuscript. REFERENCES 1. Stenflo J, Fernlund P. Eean \\'. and Roepstorff p: A ritamin K-dependent modification of glutamic acid resrdues in prorhrombin. proc Natl Acad Sci usA zl:27i0. 1974. 2 NelsestuenGL.Z-vtokoriczTH.andHouardJB:ThemodeofactionofritaminK: identificationof 7-carboxvqlutamrc acid as a component of prothrombin. J Biol chem 24g:634 7, 1974. 3. Canrot Po and Nilehn JE: Plasma prothrombin during teatmenr rvith dicumarol. Sca_nd Clin J Lab Invest 22:23. 1968. 'l Blanchard RA. 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Kluqt'r SF. and l'-rrnc'B: Pllsnrri pnitlrrcnlbin end abnonnal pror5rombirr.rntiqt'rr: (onr'iliti{jn rrirh lrict,cjjnq.rncl thrornbritic compLc,itions rn p.ititnts rreated lirh 1,,.,rirnn. iJltxrcl 5ti, Su1-.p1. ;::-r.3i..\. lflSl. Li' bnlen ll'\. ['rrric [Jt-'. [Jl:irr,,]rard Il.\.'fcrrg \lj. I-o KJ. Li.r,SD. arC Furic BC: l)es-7-iruboxv :,ilrtiomt.il, Jtrotlrtrntliin: .t I)c\\' s{,nlnt rntrrke,r ol }lririt:rn lrc,}tatot,,liulJI irirrc.iljontil Chrr ltts iin l-rl uss r. ll' Lt'$is Il\1. Ir'tlrie BC lrtd littlrr'B: llrbrtdorna-prodtrced ron{irnn,riion-s1x.cific. 1rgler!11J 3n- trlrixlic: to ltrrrrlrr lirothrtrntbin. Ill<xrl 58i Sup1;l ):120,\. l9ul.