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Preparation of guinea pig antiserum to quantitate serum fragment D-dimer derived from crosslinked fibrin
THROMBOSIS RESEARCH 2i; 111-115, 1982 OO49-3838/82/130111-05$03.00/O Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.
BRIEF
C 0 MM
US
I C .J,T
IO
2;
PREPbiUTIO?j OF GGINEh PIG ANTISERLW TO QUANTITATE SERL?I FUGXFXT D-DIKR DERIVED FROtI CROSSLIXED FIERIS
A.
Laboratory
Matsushima,
7i. Takahama
and T. Inada
of Biological Chemistry, Tokyo Institute of Technology, Ookayama, Meguroku, Tokyo 152
(Received 1.3.1982; in revised form 12.4.1982. Accepted by Editor N. Aoki)
Digestion of crosslinked fibrin by plasmin yields dimeric fragment D, Fragment D-dimer, while that of fibrinogen or non-crosslinked fibrin by plasmin yields monomeric fragment D, Fragment D, which arises from the COOH-terminal Discrimination between Fragment D and Fragment region of fibrinogen molecule. D-dimer in human plasma of a patient with defibrination syndrome such as disseminated intravascular coagulation syndrome has been recommended for exact diagnosis and medical treatment (1). Since Plow and Edgington demonstrated differences in antigenicity between fibrinogen and fibrin degradation products (2), isolation of antibodies against Fragment D-dimer has been tried by several investigators (3-5). Budzynski -et al. (4) prepared an antiserum against The immunoreactivity of Fragment Fragment D-dimer exposed to acid-urea. D-dimer with the antiserum was only 20 times greater than that of Fragment D exposed to acid-urea. Lahiri -et al. (5) obtained an antiserum against crosslinked Y-peptide which consists of 27 amino acid residues from the COOH-terminus of Y-chain. They demonstrated that the antiserum reacts preferentially with the crosslinked Y-peptide. Despite extensive studies, no one has yet obtained antiserum specific enough to determine Fragment D-dimer in plasma (or serum) in clinical tests. The present paper deals with the preparation of antiserum having high enough specificity and sensitivity for radioimmunoassay to determine Fragment D-dimer in human serum. It was obtained from guinea pigs immunized with Fragment D-dimer together with a large amount of fibrinogen (or Fragment D), followed by absorption of antibodies reactive with Fragment D region. Administration of a large amount of fibrinogen (or Fragment D) will lead to the suppression of antibody-formation against fibrinogen (or Fragment D) (6,7).
Key
words:
Fibrinogen, Fragment D, Fragment Radioimmunoassay 111
D-dimer,
Fibrin, Antiserum,
Fibrinogen (9;:: clottable), throobin (15.9 UE units/:21 an.! plaanin (0.5 casein unitslug) from human were kind gifts of Green Cross Cc. Factor XIII, guinea pig normal serum and anti-guinea pig IgG serum from rabbit il~~ophilized) were products of Behringwerke X.g, Xles Laboratories, ins. and Yiles-Peda Ltd., respectively. Bovine serum albumin (fraction L', fatt;: acid free) and complete Freund's adjuvant were purchased from Sigma Cnzmical Co. and Iatron Laboratcries, respectively. ?Ja 1251 sas obtained fror~ Japan Radioisotope Association. Other reagents used were of analytical grade. Preparation of Fragment D-diner and Fragment D's: Xon-crosslinked Fragment D with high molecular weight, Fragment D (fate), and Fragment D with low molecular weight, Fragment D3, were prepared by plasmin digestion of fibrinogen in 50 mX Tris-HCl buffer (pH 7.4) containing 0.15 ?I SaCl in the presence and the absence of 10 mX CaC12 (8,9), respectively. Crude preparations of Fragment D's were further purified by DEAE cellulose chromatography (10) followed by gel chromatography with Sephadex G-150. Fragment D-dimer was obtained by plasmin digestion of crosslinked fibrin polymer according to t'ne method of Purves -et al. (11) followed by chromatography with DEAE cellulose and with Sephades G-150. Fragment D (cate), Fragment D3 and crosslinked Fragment D-dimer thus prepared were in a homogeneous state and their molecular weights were determined to be 95,000, 85,000 and 159,000, respectively, by polyacrvlamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. Preparation of antiserum against Fragment D-dimer: Adult guinea pigs (600 OJ 650 g) were immunized with 100 JJ~ of Fragment D-dimer in complete Freund's adjuvant via rear footpads, together with a large amount of fibrinogen (10 nd 20 mg) via a vein. In some experiments, Fragment D (cate) was administered instead of fibrinogen. Two weeks later, a subcutaneous booster injection of Fragment D-dimer (100 up) was administered in the axilla. Four weeks after the first injection, serum was collected and subjected to the following experiments. Immunological studies: An immunodiffusion analysis of Fragment D-dimer and Fragment D against antisera was performed by the method of Kabat and Mayer (12). Immunoelectrophoresis was also performed in 0.85% agar (13) with 33 V/cm for Precipitin bands or arcs were stained with Amido Black 10B. 60 min. Fragment D-dimer (10 ug) was labeled with 1251 (1 mCi) by a modification of Inhibition of binding between the dimethylsulfoxide-Chloramine-T method (14). labeled Fragment D-dimer and the antiserum by unlabeled Fragment D-dimer (or The antigen-antibody complexes others) was tested by radioimmunoassay (3-5). To 0.1 were separated from free antigens by a second antibody technique (4). ml of a sample solution were added 0.1 ml of 1251-labeled Fragment D-diner (10 ng/ml, 10,000 cpm) in saline containing 1% guinea pig normal serum, 0.1 ml of antiserum in phosphate buffered saline (PBS) (pH 7.0) and 0.5 ml of 0.5% bovine The mixture (0.8 ml) was incubated at 4°C for 48 hr, serum albumin in PBS. and 0.1 ml of antiserum against guinea pig immunoglobulin G (diluted 10 times After the with saline) was added and incubated at 4°C for another 24 hr. centrifugation of the mixture at 3,000 rpm for 30 min at 4"C, the radioactivity of precipitate was counted with a Beckman automatic gamma counter 4,000. RESULTS AXD DISCUSSION Fig. lb shows immunodiffusion patterns of Fragment D(cate) and Fragment D-dimer against antiserum obtained from guinea pigs immunized with Fragment
it3
3-dir.er ~100 ;g) subcutaneously and a large a~unt :f fibrinogen i-0 mg) IntravaneXslv I Precipitin lines between aaciserum and these fragments ware slightly fused but a spur distinct from the fused line was observed between This indicates that the antiserum contains antiserum and Fragment D-dimer. not only antibodies which react vith both Fragment D and Fragment D-diner but As a control, also ones which react specifically vith Fra,-ent D-dimer. another antiserum was obtained from guinea pigs i-munized with Fragment D-diner (100 'jg) alone. Immunodiffusion patterns of Fragment D (sate) and Fragment A fused line D-dimer against the antiserum thus obtained are snow: in Fig. la. fcrmed by two fragments was very strong and only a minor spur appeared between h quantitative precioitin study revealed that antiserum and Fragment D-dimer. the content of anti-Fragment D antibodies in the antiserum used in Fig. lb is These reccnsiderably lower than in the other antiserum (used in Fig. la). suits suggest that the formation of anti-Fragment 5 antibodies is suppressed by a massive administration of fibrinogen to guinea pigs as we expected (h,7). Anti-Fragment D antibodies in the antiserum (used in Fig. lb) were absorbed by the addition of Fragment D (cate), and the antigen-antibody complexes were The antiserum hereafter referred to is the one thus removed as precipitate. prepared. Immunodiffusion patterns of Fragment D (cate) and Fragment D-dimer So precipitin line was observed against the antiserum are shown in Fig. lc. between Fragment D and the antiserum but a sharp precipitin line appeared Antiserum obtained from guinea between Fragment D-dimer and the antiserum. pigs immunized with Fragment D-dimer (100 ~g) and a large amount of Fragment D (5 'L 10 ng) instead of fibrinogen showed similar specificity after the absorption. These results indicate that the antiserum binds specifically regions unique for Fragment D-dimer and not regions common to Fra_ment D and Fragment D-diner. This conclusion was further confirmed by immunoelectrophoresis of the antiserum with normal plasma anticoagulated with citrate together with Fragment D-dimer, Fragment D (cate) and fibrinogen. A single and strong . . precipitin arc was recognized only between Fragment D-dimer and the antiserum (data not shown).
0
D-D
FIG. 1 Immunodiffusion studies of Fragment D (fate) and Fragment D-dimer against three different kinds of antisera. Antisera in wells were obtained from the following guinea pigs. I, immunized with 100 ug of Fragment D-dimer; II, immunized with 100 ug of Fragment D-dimer and with 20 mg of fibrinogen; III, same as II, except that Fragment D antibodies were removed from the antiserum by absorption. Concentrations of Fragment D and Fragment D-dimer were 0.5 mg/ml. D-D and D stand for Fragment D-dimer and Fragment D (cate), respectively.
The ne?ct series of experiments is concerned with i=nunorea:cti-it;:of :'he antiserum with Fragment D-diner as well as fibrinogen and its degradation products. Results of radioimmunoassays are shown in Fig. 2. Inhibition of binding between 12'1-labeled Fragment D-dimer and the antiserum by unlabeled Fragment D-diner is demonstrated by curve A. The antiserum diluted L,OOO times was used in the experiment. The binding is inhibited by increasing the amount of unlabeled Fragment D-dimer and 50% inhibition takes place with 100 ng/nl of unlabeled Fragment D-dimer. Inhibition curves were also obtained -,_ithFragment D (cate), fibrinogen and Fragment D3 which are shown by curves B, C and D, respectively. The concentration of antigen that caused 50% inhibition was LO Kg/ml for Fragment D (cate) and 20 l;g/ml for fibrinogen, and these values are 100-200 fold higher than that of Fragment D-dimer. Fragment D3 did not inhibit even at 20 i;g/ml. Fragment D3 lacks the COOH-terminal region of ,f-chain in which the crosslink reaction takes place, while Fragment D (cate) has the full amino acid sequence of the COOH-terminal region of y-chain. The minimum concentration of Fragment D-dimer in plasma detected by this radioimmunoassay is only 10 ng/ml as is shown by Curve A in Fig. 2, while the concentration of Fragment D-dimer in plasma was reported to be 230 ng/ml for normal plasma and 14,000 ngfml for a patient with disseminated intravascular coagulation syndrome Since the concentration of Fragment D in plasma was determined to be (15). comparable to that of Fragment D-dimer in normal subjects as well as patients with dissiminated intravascular coagulation syndrome (15), the antiserum could specifically detect Fragment D-dimer. The potential problem of the inhibition with fibrinogen, whose concentration in plasma is extensively high, can be eliminated by using serum instead of plasma for analysis. The antiserum obtained in the present study offers high enough specificity and sensitivity in radioimmunoassays to permit determination of Fragment D-dimer in serum of normal subjects as well as some patients with defibrination syndrome
’
loo-
I
I
I
I
I
750
8 A
5
B
.= 50-
c
'5 ._ z : 25/
.
0 loo
. IO'
IO2 Antigen
D'r IO'
la'
IO5
(ng/ml)
FIG. 2 Inhibition of binding between lz51-labeled Fragment D-dimer and the antiserum by unlabeled proteins such as Fragment D-dimer (curve A), Fragment D (cate) (curve B), fibrinogen (curve C) and Fragment D3 In the absence of unlabeled antigen, binding of (curve D). lz51-labeled Fragment D-dimer with the absorbed antiserum was 31% when the antiserum was diluted 4,000 times.
.tiTISERUM XGAI&ST
FIBRIS D-DI?%E&
A.2
REFERES:ZS 1.
?UBXR, i'. J. and XDZYSSKI ) .i\. * z. Degradation prsdiicts of fibrinosen a:;2 crosslinked f ibrin-projected clinical applications. ThrombJs. 3ia:hes. haeaorrh. -' 32 i9-56, 1975.
.!.
PLO:;, E. F. and EDGI?;GTOS, T. 5. Discrimi:l'lti::.Y j z:e3a1ltigen:.: differentes bet*deen fibrinogen and fibrin derivatives. Proz. Tat. Acad. Sci. YSb 70, llh?-X73, 1973.
3.
T_EE-mT,
i.
BUDZVSSSI, i. Z., XARDER. V. J.. PARKER, >I. E., Sh:.;L‘IES, P., and OLE.U., S. :I. Antigenic markers on fragment DD. a unique plasmi< deril:ative aif human crosslinked fibrin. Blood 5S , 795~SOI. 1979.
3.
.
.
1,‘. , GORDOS, T. B. and CH.\RD, I. The de:eztion of sites c:? the D-dimer peptide isolated from plasmi:: digested -., tiorin. Thrombosis Research l& 77-8L, 1979.
LAhIRI, B., KOEHX, J. A., CANFIELD, R. E., BLRKES, 5. and LEXIS, J. Development of an immunoassay for the COOH-terminal region of the gamma chains of human fibrin. Thrombosis Research 2 23 103-112, 1981.
Immunological 6. HESSEY, C. S. and ISHIZAKA, K. antigenic determinants in human ,{G globulin. 335-341. 1967.
unresponsiveness to specific Droc. Sot. Exp. Bioi. lljl
7. HEXSEY, C. S. and ISHIZAU, K. An antigenic determinant of human +globulin susceptible to papain digestion. J. Immunol. -2 99 695-702, :9jT. 8. XIEUWENHUIZEX, W., VERMOND, A. and Hr\VERKATE, F. Factors influencing structure of terminal plasmin degradation products or‘ human fibrinogen fibrin. Biochim. Biophys. Acta. 667, 321-327, 1981.
the and
9. FERGUSON, E. W., FRETTO, L. J. and MCKEE, P. A. A re-examination of the cleavage of fibrinogen and fibrin by plasmin. 3. Biol. Chem. 250, 72107218, 19i5. 10. DOOLITTLE, R. F., CASSMAN, K. G. COTTRELL, B. A., FRIEZNER, S. J. and TAKXI, T. Amino acid sequence studies on the a chain of human fibrinogen Covalent structure of the a-chain portion of fragment D. Biochemistrv 16 p--.2 1710-1715, 1977. 11. PURVES f L. R ., LINDSEY, G. G. and FRANKS, J. J. Sites of D-domain interaction in fibrin-derived D diner. Biochemistry 19, 4051~$058, 12. KAB.-IT,B. A. and MAYER, X. Il. Experimental immunochemistry, Springfield, Ill: Charles C. Thomas, 1961, pp. 85-88.
1980.
2nd edn.,
13. PLOY, E. and EDGINGTON, T. S. Molecular events responsible for modulation of neoantigenic expression: the cleavage-associated neoantigen of fibrinogen. Proc. Nat. Acad. Sci. USA & 208-312, 1972. lb. STAGG, B. H., TEFIPERLEY, J. W. and ROCl-XQi, I?. Iodination biological activity of gastrin. Nature 228, 58-59, 1970.
and the
15. YOSHIOKA, K, XIYATA, H. and FlAKI, S. Fibrinogen and fibrin derivatives in serum from normal and sick children. Thrombosis Research 2&, 25-32, 1981.
BRIEF
C 0 MM
US
I C .J,T
IO
2;
PREPbiUTIO?j OF GGINEh PIG ANTISERLW TO QUANTITATE SERL?I FUGXFXT D-DIKR DERIVED FROtI CROSSLIXED FIERIS
A.
Laboratory
Matsushima,
7i. Takahama
and T. Inada
of Biological Chemistry, Tokyo Institute of Technology, Ookayama, Meguroku, Tokyo 152
(Received 1.3.1982; in revised form 12.4.1982. Accepted by Editor N. Aoki)
Digestion of crosslinked fibrin by plasmin yields dimeric fragment D, Fragment D-dimer, while that of fibrinogen or non-crosslinked fibrin by plasmin yields monomeric fragment D, Fragment D, which arises from the COOH-terminal Discrimination between Fragment D and Fragment region of fibrinogen molecule. D-dimer in human plasma of a patient with defibrination syndrome such as disseminated intravascular coagulation syndrome has been recommended for exact diagnosis and medical treatment (1). Since Plow and Edgington demonstrated differences in antigenicity between fibrinogen and fibrin degradation products (2), isolation of antibodies against Fragment D-dimer has been tried by several investigators (3-5). Budzynski -et al. (4) prepared an antiserum against The immunoreactivity of Fragment Fragment D-dimer exposed to acid-urea. D-dimer with the antiserum was only 20 times greater than that of Fragment D exposed to acid-urea. Lahiri -et al. (5) obtained an antiserum against crosslinked Y-peptide which consists of 27 amino acid residues from the COOH-terminus of Y-chain. They demonstrated that the antiserum reacts preferentially with the crosslinked Y-peptide. Despite extensive studies, no one has yet obtained antiserum specific enough to determine Fragment D-dimer in plasma (or serum) in clinical tests. The present paper deals with the preparation of antiserum having high enough specificity and sensitivity for radioimmunoassay to determine Fragment D-dimer in human serum. It was obtained from guinea pigs immunized with Fragment D-dimer together with a large amount of fibrinogen (or Fragment D), followed by absorption of antibodies reactive with Fragment D region. Administration of a large amount of fibrinogen (or Fragment D) will lead to the suppression of antibody-formation against fibrinogen (or Fragment D) (6,7).
Key
words:
Fibrinogen, Fragment D, Fragment Radioimmunoassay 111
D-dimer,
Fibrin, Antiserum,
Fibrinogen (9;:: clottable), throobin (15.9 UE units/:21 an.! plaanin (0.5 casein unitslug) from human were kind gifts of Green Cross Cc. Factor XIII, guinea pig normal serum and anti-guinea pig IgG serum from rabbit il~~ophilized) were products of Behringwerke X.g, Xles Laboratories, ins. and Yiles-Peda Ltd., respectively. Bovine serum albumin (fraction L', fatt;: acid free) and complete Freund's adjuvant were purchased from Sigma Cnzmical Co. and Iatron Laboratcries, respectively. ?Ja 1251 sas obtained fror~ Japan Radioisotope Association. Other reagents used were of analytical grade. Preparation of Fragment D-diner and Fragment D's: Xon-crosslinked Fragment D with high molecular weight, Fragment D (fate), and Fragment D with low molecular weight, Fragment D3, were prepared by plasmin digestion of fibrinogen in 50 mX Tris-HCl buffer (pH 7.4) containing 0.15 ?I SaCl in the presence and the absence of 10 mX CaC12 (8,9), respectively. Crude preparations of Fragment D's were further purified by DEAE cellulose chromatography (10) followed by gel chromatography with Sephadex G-150. Fragment D-dimer was obtained by plasmin digestion of crosslinked fibrin polymer according to t'ne method of Purves -et al. (11) followed by chromatography with DEAE cellulose and with Sephades G-150. Fragment D (cate), Fragment D3 and crosslinked Fragment D-dimer thus prepared were in a homogeneous state and their molecular weights were determined to be 95,000, 85,000 and 159,000, respectively, by polyacrvlamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. Preparation of antiserum against Fragment D-dimer: Adult guinea pigs (600 OJ 650 g) were immunized with 100 JJ~ of Fragment D-dimer in complete Freund's adjuvant via rear footpads, together with a large amount of fibrinogen (10 nd 20 mg) via a vein. In some experiments, Fragment D (cate) was administered instead of fibrinogen. Two weeks later, a subcutaneous booster injection of Fragment D-dimer (100 up) was administered in the axilla. Four weeks after the first injection, serum was collected and subjected to the following experiments. Immunological studies: An immunodiffusion analysis of Fragment D-dimer and Fragment D against antisera was performed by the method of Kabat and Mayer (12). Immunoelectrophoresis was also performed in 0.85% agar (13) with 33 V/cm for Precipitin bands or arcs were stained with Amido Black 10B. 60 min. Fragment D-dimer (10 ug) was labeled with 1251 (1 mCi) by a modification of Inhibition of binding between the dimethylsulfoxide-Chloramine-T method (14). labeled Fragment D-dimer and the antiserum by unlabeled Fragment D-dimer (or The antigen-antibody complexes others) was tested by radioimmunoassay (3-5). To 0.1 were separated from free antigens by a second antibody technique (4). ml of a sample solution were added 0.1 ml of 1251-labeled Fragment D-diner (10 ng/ml, 10,000 cpm) in saline containing 1% guinea pig normal serum, 0.1 ml of antiserum in phosphate buffered saline (PBS) (pH 7.0) and 0.5 ml of 0.5% bovine The mixture (0.8 ml) was incubated at 4°C for 48 hr, serum albumin in PBS. and 0.1 ml of antiserum against guinea pig immunoglobulin G (diluted 10 times After the with saline) was added and incubated at 4°C for another 24 hr. centrifugation of the mixture at 3,000 rpm for 30 min at 4"C, the radioactivity of precipitate was counted with a Beckman automatic gamma counter 4,000. RESULTS AXD DISCUSSION Fig. lb shows immunodiffusion patterns of Fragment D(cate) and Fragment D-dimer against antiserum obtained from guinea pigs immunized with Fragment
it3
3-dir.er ~100 ;g) subcutaneously and a large a~unt :f fibrinogen i-0 mg) IntravaneXslv I Precipitin lines between aaciserum and these fragments ware slightly fused but a spur distinct from the fused line was observed between This indicates that the antiserum contains antiserum and Fragment D-dimer. not only antibodies which react vith both Fragment D and Fragment D-diner but As a control, also ones which react specifically vith Fra,-ent D-dimer. another antiserum was obtained from guinea pigs i-munized with Fragment D-diner (100 'jg) alone. Immunodiffusion patterns of Fragment D (sate) and Fragment A fused line D-dimer against the antiserum thus obtained are snow: in Fig. la. fcrmed by two fragments was very strong and only a minor spur appeared between h quantitative precioitin study revealed that antiserum and Fragment D-dimer. the content of anti-Fragment D antibodies in the antiserum used in Fig. lb is These reccnsiderably lower than in the other antiserum (used in Fig. la). suits suggest that the formation of anti-Fragment 5 antibodies is suppressed by a massive administration of fibrinogen to guinea pigs as we expected (h,7). Anti-Fragment D antibodies in the antiserum (used in Fig. lb) were absorbed by the addition of Fragment D (cate), and the antigen-antibody complexes were The antiserum hereafter referred to is the one thus removed as precipitate. prepared. Immunodiffusion patterns of Fragment D (cate) and Fragment D-dimer So precipitin line was observed against the antiserum are shown in Fig. lc. between Fragment D and the antiserum but a sharp precipitin line appeared Antiserum obtained from guinea between Fragment D-dimer and the antiserum. pigs immunized with Fragment D-dimer (100 ~g) and a large amount of Fragment D (5 'L 10 ng) instead of fibrinogen showed similar specificity after the absorption. These results indicate that the antiserum binds specifically regions unique for Fragment D-dimer and not regions common to Fra_ment D and Fragment D-diner. This conclusion was further confirmed by immunoelectrophoresis of the antiserum with normal plasma anticoagulated with citrate together with Fragment D-dimer, Fragment D (cate) and fibrinogen. A single and strong . . precipitin arc was recognized only between Fragment D-dimer and the antiserum (data not shown).
0
D-D
FIG. 1 Immunodiffusion studies of Fragment D (fate) and Fragment D-dimer against three different kinds of antisera. Antisera in wells were obtained from the following guinea pigs. I, immunized with 100 ug of Fragment D-dimer; II, immunized with 100 ug of Fragment D-dimer and with 20 mg of fibrinogen; III, same as II, except that Fragment D antibodies were removed from the antiserum by absorption. Concentrations of Fragment D and Fragment D-dimer were 0.5 mg/ml. D-D and D stand for Fragment D-dimer and Fragment D (cate), respectively.
The ne?ct series of experiments is concerned with i=nunorea:cti-it;:of :'he antiserum with Fragment D-diner as well as fibrinogen and its degradation products. Results of radioimmunoassays are shown in Fig. 2. Inhibition of binding between 12'1-labeled Fragment D-dimer and the antiserum by unlabeled Fragment D-diner is demonstrated by curve A. The antiserum diluted L,OOO times was used in the experiment. The binding is inhibited by increasing the amount of unlabeled Fragment D-dimer and 50% inhibition takes place with 100 ng/nl of unlabeled Fragment D-dimer. Inhibition curves were also obtained -,_ithFragment D (cate), fibrinogen and Fragment D3 which are shown by curves B, C and D, respectively. The concentration of antigen that caused 50% inhibition was LO Kg/ml for Fragment D (cate) and 20 l;g/ml for fibrinogen, and these values are 100-200 fold higher than that of Fragment D-dimer. Fragment D3 did not inhibit even at 20 i;g/ml. Fragment D3 lacks the COOH-terminal region of ,f-chain in which the crosslink reaction takes place, while Fragment D (cate) has the full amino acid sequence of the COOH-terminal region of y-chain. The minimum concentration of Fragment D-dimer in plasma detected by this radioimmunoassay is only 10 ng/ml as is shown by Curve A in Fig. 2, while the concentration of Fragment D-dimer in plasma was reported to be 230 ng/ml for normal plasma and 14,000 ngfml for a patient with disseminated intravascular coagulation syndrome Since the concentration of Fragment D in plasma was determined to be (15). comparable to that of Fragment D-dimer in normal subjects as well as patients with dissiminated intravascular coagulation syndrome (15), the antiserum could specifically detect Fragment D-dimer. The potential problem of the inhibition with fibrinogen, whose concentration in plasma is extensively high, can be eliminated by using serum instead of plasma for analysis. The antiserum obtained in the present study offers high enough specificity and sensitivity in radioimmunoassays to permit determination of Fragment D-dimer in serum of normal subjects as well as some patients with defibrination syndrome
’
loo-
I
I
I
I
I
750
8 A
5
B
.= 50-
c
'5 ._ z : 25/
.
0 loo
. IO'
IO2 Antigen
D'r IO'
la'
IO5
(ng/ml)
FIG. 2 Inhibition of binding between lz51-labeled Fragment D-dimer and the antiserum by unlabeled proteins such as Fragment D-dimer (curve A), Fragment D (cate) (curve B), fibrinogen (curve C) and Fragment D3 In the absence of unlabeled antigen, binding of (curve D). lz51-labeled Fragment D-dimer with the absorbed antiserum was 31% when the antiserum was diluted 4,000 times.
.tiTISERUM XGAI&ST
FIBRIS D-DI?%E&
A.2
REFERES:ZS 1.
?UBXR, i'. J. and XDZYSSKI ) .i\. * z. Degradation prsdiicts of fibrinosen a:;2 crosslinked f ibrin-projected clinical applications. ThrombJs. 3ia:hes. haeaorrh. -' 32 i9-56, 1975.
.!.
PLO:;, E. F. and EDGI?;GTOS, T. 5. Discrimi:l'lti::.Y j z:e3a1ltigen:.: differentes bet*deen fibrinogen and fibrin derivatives. Proz. Tat. Acad. Sci. YSb 70, llh?-X73, 1973.
3.
T_EE-mT,
i.
BUDZVSSSI, i. Z., XARDER. V. J.. PARKER, >I. E., Sh:.;L‘IES, P., and OLE.U., S. :I. Antigenic markers on fragment DD. a unique plasmi< deril:ative aif human crosslinked fibrin. Blood 5S , 795~SOI. 1979.
3.
.
.
1,‘. , GORDOS, T. B. and CH.\RD, I. The de:eztion of sites c:? the D-dimer peptide isolated from plasmi:: digested -., tiorin. Thrombosis Research l& 77-8L, 1979.
LAhIRI, B., KOEHX, J. A., CANFIELD, R. E., BLRKES, 5. and LEXIS, J. Development of an immunoassay for the COOH-terminal region of the gamma chains of human fibrin. Thrombosis Research 2 23 103-112, 1981.
Immunological 6. HESSEY, C. S. and ISHIZAKA, K. antigenic determinants in human ,{G globulin. 335-341. 1967.
unresponsiveness to specific Droc. Sot. Exp. Bioi. lljl
7. HEXSEY, C. S. and ISHIZAU, K. An antigenic determinant of human +globulin susceptible to papain digestion. J. Immunol. -2 99 695-702, :9jT. 8. XIEUWENHUIZEX, W., VERMOND, A. and Hr\VERKATE, F. Factors influencing structure of terminal plasmin degradation products or‘ human fibrinogen fibrin. Biochim. Biophys. Acta. 667, 321-327, 1981.
the and
9. FERGUSON, E. W., FRETTO, L. J. and MCKEE, P. A. A re-examination of the cleavage of fibrinogen and fibrin by plasmin. 3. Biol. Chem. 250, 72107218, 19i5. 10. DOOLITTLE, R. F., CASSMAN, K. G. COTTRELL, B. A., FRIEZNER, S. J. and TAKXI, T. Amino acid sequence studies on the a chain of human fibrinogen Covalent structure of the a-chain portion of fragment D. Biochemistrv 16 p--.2 1710-1715, 1977. 11. PURVES f L. R ., LINDSEY, G. G. and FRANKS, J. J. Sites of D-domain interaction in fibrin-derived D diner. Biochemistry 19, 4051~$058, 12. KAB.-IT,B. A. and MAYER, X. Il. Experimental immunochemistry, Springfield, Ill: Charles C. Thomas, 1961, pp. 85-88.
1980.
2nd edn.,
13. PLOY, E. and EDGINGTON, T. S. Molecular events responsible for modulation of neoantigenic expression: the cleavage-associated neoantigen of fibrinogen. Proc. Nat. Acad. Sci. USA & 208-312, 1972. lb. STAGG, B. H., TEFIPERLEY, J. W. and ROCl-XQi, I?. Iodination biological activity of gastrin. Nature 228, 58-59, 1970.
and the
15. YOSHIOKA, K, XIYATA, H. and FlAKI, S. Fibrinogen and fibrin derivatives in serum from normal and sick children. Thrombosis Research 2&, 25-32, 1981.