CA1303532C - Determination of fibrin using fibrin-specific antibodies - Google Patents
Determination of fibrin using fibrin-specific antibodiesInfo
- Publication number
- CA1303532C CA1303532C CA000471712A CA471712A CA1303532C CA 1303532 C CA1303532 C CA 1303532C CA 000471712 A CA000471712 A CA 000471712A CA 471712 A CA471712 A CA 471712A CA 1303532 C CA1303532 C CA 1303532C
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- fibrin
- antibodies
- fibrinogen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/745—Assays involving non-enzymic blood coagulation factors
- G01N2333/75—Fibrin; Fibrinogen
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Abstract
Abstract of the disclosure:
Qualitative and quantitative determination of fibrin is possible using antibodies. The appropriate antisera are obtained when the immunogen used takes the form of peptides which contain a part sequence of the fibrin molecule, in particular a peptide containing the aminoacid sequence of the terminal groups which are exposed by splitting off the fibrinopeptides. Antisera with monoclonal antibodies are preferred.
Qualitative and quantitative determination of fibrin is possible using antibodies. The appropriate antisera are obtained when the immunogen used takes the form of peptides which contain a part sequence of the fibrin molecule, in particular a peptide containing the aminoacid sequence of the terminal groups which are exposed by splitting off the fibrinopeptides. Antisera with monoclonal antibodies are preferred.
Description
~3~ ;3~: ~
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The ;nvention relates to a procedure for the se~ect;ve determination of fibrin in plasma or in t;ssue by use of highly spec;fic antibodies, to the antibod;es required for this purpose, to a process for their pre-5 parat;on, and ~o the immunogens used for this purpose~
It ;s known that soluble fibrin is found in the circulating blood of patients wi~h dissem;nated ;ntra-vascular coagulation (consumptive coagulopathy). This f;brin has been produced from the soluble plasma prote;n 10 fibrinogen by the action of proteolytic enzymesn In capillaries and small blood vessels, soluble fibrin can associate to form fibrin strands which can be detected by light and electron microscopy ~Review in. Muller-3erghaus, G., Seminars in Thrombosis and Hemostasis 3:
15 209 - 246, 1977). Moreover, in inflammatory disorders, a material der;ved from f;br;nogen can be detected in tissue, within or outside vessels. Thus, the object ~as to develop a reagent and a procedure to be able to differentiate between fibrin and fibrinogen and to deter-20 mine fibrin specifically and quantitatively.
It is known that proteolytic enzymes~ such as,for example, thrombin, both within and outside vessels, split off peptides from the fibrino~en molecule~ so that fibrin monomer is produced. Several fibrin monomers can 25 associate to form a fibr;n polymer, a fibrin clot, which then, if this takes place within a vessel, leads to deleterious consequences for the body. Accordingly~ it is desirable to detect f;brin at a very early stage of a pathologica~ process.
To date~ there are only qual;tative methods for the detection of fibrin, since fibrin differs from fibrinogen only by the fibrinopeptides having been split off~ The qualitative or semiquantitative methods are based on, for example~ precipitation after add;tion of 35 ethanol (Godal, H.C., Abildgaard, UO, Scand. J. Haematol.
., 3~?3532 _ 3 ~. ~
3: 342 - 350, 1966), protam;ne sulfate (Nie~;arowsk;, S., Gure~ich, V~, J. Labr Clin. Med~ 77: 665-676, 1971) or agglut;na~ion of coated erythrocytes or latex particles ~Largo, R., Heller, V., Straub, P.W., Blood 47: 991-1002 5 1976). These cited methods do not permit differentiation between f;br;n and fibr;nogen. Other methods attempt to separate fibrin from f;brinogen by chromatography (Alkjaers;g, N., Fletcher, A~, ~urste;n, R., Am. J~
Obstet~ Gynecol~ 122. 199 - 209, 1975; Heene, D.L., Matthias, F.R., Thromb. Res. 2: 137 - 154~ 1973~. These methods are l;kewise not specific~ s;nce it is not pos-sib~e to sepArate fibrin completely from fibrinogen.
Fibr;nogen is necessary to keep fibrin in solution (Krell, W., Mahn, I., Muller-~erghaus, G.~ Thromb. Res~
14: 299 - 310, 1979~.
A possible method of differentiating fibrin from fibrinogen in a purified system is the determinat;on of the N-terminal glyc;ne ~Kierulf, P., ~odal, H.C , Scand~
J. Haematol. 9: 370 - 372, 197Z~. However, this method ;s complicated and requires large amounts Oc starting material, for ~hich reason it has not found ~ide use as a test. Thus, to date it ;s not possible to differenti-ate fibrin from fibrinogen in a small sample of plasma or tissue.
Th;s object is achieved according to the inven-tion by determining fibrin using highly specific anti-bodies. This determination can also be carried out no~
only qualitatively but even quant;tatively in the pre-sence of comparatively high concentrations of fibrinogen.
; 30 It has been ,ound~ surprisingly, that the anti-bodies according to the invention are formed when the antigens used are peptides ~hich mimic the aminoacid sequence of certain mole~ular segments of the fibrin .~olecule~ This ~as all the more surprising since earlier 35 attempts at ;mmunization against the complete f;brin molecu~e had not led to fibrin-specific antibodies.
Ant;sera ~hich contain the ant;bodies accord;ng ~o the invention are obta;ned by ;ntroducin3 ~eptides as antiqen into the body of experimental arimals. These pep-~3~3~
~,`.
tides represent part of the aminoacid sequence of fibrin.
The pxeferred peptides have an aminoacid sequence of 3 to 12 aminoacids, which is exposed after splitting off fibrinopeptide A rom the N-terminal end of the fibrinogen molecule. The hexapeptide of the formula Gly-Pro-Arg-Val-Val-Glu has proved to be advan~ageous. Thi~ is bound in a customary manner, advantageou~ly interpolating a "spacer"
group, to carrier peptides suitable for this purpose.
The oligopeptides which mimic the aminoacid sequence of fibrin, in particular of the terminal groups which are liberated by splitting off the fibrinopeptides, are synthesized in a known manner. Suitable processes are described in, for example, E. ~unsch ln Houben-Wsyl, ~Methoden der organischen Chemie" (Methods of Organic Chemistry), Volume XV/1 and 2; The Peptides, Vol. 1, ~'Major Methods of Peptide Bond Formation", Academic Press (1979), or "Perspectives in Peptide Chemistry", A. Eberle, R.
Geiger and T. Wieland (editor~), S. Karger, Basel (1981).
This entails aminoacids or peptide fragments, whose functional groups which are not intended to participate in the reaction are protected in a suitable manner, being induced to react. The fxee carboxyl groups of aminoacid derivati~es or peptide fragments are activated in a suitable manner and reacted with the amino group of aminoacid derivatives or partially protected peptide fragments. The activation can be carried out with DCCI, DCCI/HOBt, DCCI/hydroxysuccinimide, POCl3, chloroformic esters or the azide method. The DCCI/HOBt method as described in Chem. Ber. 103, 788 (1970) is preferably used.
The synthesi~ of the partially protected oli~opeptides Boc-Gly-Pro-Arg-Val-Val-Glu-~-Capr-OH and Fmoc-Gly-Pro-Arg-; Val-Val-Glu-HMDA-H can be carried out stepwise from the C-texminal end or by coupling of fragments. In addition, stepwise synthesis on a soluble or insoluble polymeric carrier, such as, for example, polyoxyethylene or crosslinked polystyrene, is suitable. Suitable pro-", , 13~.P~'~3 tect;ve groups for the alpha-amino group of the amino-acids or peptide fragments are Boc, Fmoc, Z, ~,~ dimethyL-3,5-dimethoxybenzyloxycarbonyl~ 2-(4-biphenylyl)-2-propyl-oxycarbonyl and trityl. Boc~ Fmoc and Z are preferably 5 used.
Esterification with alkanols or araliphatic alco-hols is used to protect the alpha-carboxyl group of aminoacids and peptide fragments. The methyl, ben~yl and tert.-butyl esters are preferredO
The side-chain groups of the partially protected oligope~tides are a carboxyl group and a guanid;no group.
These can be substituted by protect;ve groups customary in peptide chemis~ry in accordance ~ith their reacti~ity~
! Esterificat;on ~ith aliphatic or aral;phat;c alcohols, 1S such as, for example, methanol, tertO butanol or benzyl alcohol, is preferably used to protect the carboxyl group ;n Glu. To protect the guanidino group in ar~inine, it can be protonated or substituted by NG-tosyl, NG-nitro, NG-adamantyloxycarbonyl and NG-benzyloxycarbonyl.
Protonation ;s preferred~
For an unamb;guous course of reaction in the coupling to the carrier protein, the N-terminal of the ol;gopeptides used must be protected so that it is pos-sible to split off the protective group on the peptide-protein çonjugate without damaging the protein. Su;tabLefor this purpose are the Fmoc protective group, ~hicn can be spl;t off under alkaline conditions, and the 2-(4-biphenylyl)~2-propyloxycarbonyl, c~ dimethyl-3,5-di ; methoxybenzyloxycarbonyl, Boc and trityl groups, ~hich can be spLit off under relatively mild acid conditions.
Suitable "spacer" groups are the bridge ~o~ming reagenls customarily used in peptide chemistry, such as are also used, for example, for the i~mobilization of enzymes~ The appropriate reagents contain t~o identical or different - 35 reactive groups at the ends of an alkylene group having, for example, 2 to 8 carbon atoms~ suitabLe reactive groups being those ~hich can undergo covalent bondins with the functional ~roups of the aminoacids. Suitable ~3~?3~-~.2 examples are dialdehydes such as glutaraldehyde, di;so-cyanates such as 1,6-hexamethylene diisocyanate~ dia~ines s~ch as 1,6-hexamethylenediamine, or ~aminocarboxylic ac;ds such as -am;nocaproic acid. These b;funct;onal reagents are, ~here appropriate after activationD linked in a known manner ~ith, on the one hand, the oligopeptide and, on the other hand, the carr;er.
In the case of Boc-Gly-Pro-Arg-Val-Val-Glu- -Capr~
OH~ the react;on of the partially protected oligopept;des ~;th the carrier protein ~akes place via activat;on of ~he carboxyl group us;ng a water-soluble carbod;imide, such as, for example, N~ethyl-N'-(3-d;methylaminopropyl)-carbodiimide, or ~ith DCCI/hydroxysuccinimideO ~oc-Gly-Pro-Arg-Val-Val-G~u-HMDA-H is bonded v;a the free amino group using a bifunctional reagent such as, for example, MBS. The Boc protective group on the peptide-protein conjugate can be spl;t off with 1.2 N HCl/HAc only ;n the case of BSA~ With many carrier proteins, such as, for example, BSA or KLH, it ;s possible to split off the Fmoc protect;ve group using ~.2 N NaOH~
Any des;red prote;n ;s su;table as a carrier~
It is advantageous to select those carr;er prote;ns which cause no cross-reaction. Suitable examples are albumins, such as bovine serum albumin, or hemocyanins, such as keyhole limpet hemocyanins.
Using the antigens thus obtained, exper;mental ani~als, such as m;ce, rats, rabb;ts or goats, are immun;zed in a known manner, and thus antisera conta;ning polyclonal antibodies are obtained~
In a preferred embodiment, monoclonal antibodies are produced in an appropr;ate Manner by the method of G. Kohler and C~ Milstein, Nature 256, 495 - 497 ~19~5).
The known mouse myeloma cell lines can be used for th;s purpose. It has proved to be particularly favorable to use a cell line ~hich itself produces no im~unoglobulin.
~ he polyclonal and monoclonal antibodies accord-ing to the invention do not react ~ith fibrinogen~ but ~o react ~1-th des-A-fibrin ~fibrin typ~ I), des-AB-~L3~~ 3~
-- 7f;br;n (fibrin type II), the peptide conju~ate and the synthetic pPptide. This binding takes place ~ith such ~reat sensit;v;ty and specificity that nanogram amounts of monoclonal ant;bodies are able specifically to detect 5 nanogram amounts of antigens (des-A-fibrin and/or des-AB-fibrin) even in the presence of fibrinogen in the physio-logical concentration (3 mg/ml)O
In the above text and in the examples ~hich ~ollo~, the abbreviations shown below have been used:
1Q Arg L-argin;ne soc t-butyloxycarbonyl ~ ~-Capr ~-aminocaproic acid (or radical derived - therefrom) DCCI dicyclohexylcarbodiimide 15 DCU dicyclvhexylurea des-AA-fibrin type I
des-AABB-fibrin type II
DMF dimethylformamide ELISA enzyme linked immunosorben~ assay 20 Fmoc fluorenylmethoxycarbonyl Glu L-glutam;c acid Gly glyc;ne HAc acetic acid HMDA 1,6-hexamethylenediamine ~or radical derived therefrom) HOBt 1-hydroxy-1H-benzotriazole Ig immunoglobulin ;.p. intraperitoneal i.v~ intravenous 30 KLH keyhole limpet hemocyanins MBS m-maleinimidobenzoyl-N-hydrsxysuccin-imide ester OBzl benzyl ester OtBu t-butyl ester 35 PBS phosphate buffered saline Pro L-proline BSA bov;ne serum albumin s.c. subcutaneous ~L 3 ~ 3 S 3 ~
TFh trifluoroacetic acid Yal L-valine Z benzyloxycarbonyl Preparat;on of th ~
S The peptides of Examples 1 and 2 are charac-terized by elemental analysis, aminoacid analys;s and th;n-layer chromatography. The chemical purity is established by thin-layer chromatography in a variety of solvent m;xtures. The racem;zat;on is checked by gas chroma~ography usin~ a glass capillary column coated with RCHIRASI~-Val by the ~ethod of J. ~hromat. Sci. 15, 174 ~1977)~ and is belo~ 2 per cent for each aminoacid.
E~ample 1 ( The partially protected pept;de Boc-Gly-Pro-Arg-Val~Val-Glu-~-Capr-OH is synthesized in accordance ~ith synthet;c scheme I, including -aMinocaproic acid as a spacer.
Example 2 The part;ally protected peptide Fmoc-Gly-Pro-Arg-Yal-Val-Glu-HMDA-H ;s synthesized in accordance with synthetic scheme II, ;nclud;ng hexamethylenediamine''as a spacer.
Example 3 20 mg of Boc-Gly-Pro-Arg-Val-Val-Glu-~-Ca~r-OX
tExample 1~ are dissolved ;n 0.4 ml of ethanol, and 4 mg of DCCI and Z.5 mg of hydroxysuccinimide are added.
After stirring at room temperature for 2 hours, the pep-tide hydroxysucc;n;mide ester is slowly added drop~ise to a solution of 760 mg of BSA ~extra pure) in 7 ~l of buffer (0.25 M HEPES, 0.2 M CaCl2, pH 7.5). After 20 hours at room temperature, the DCU is filtered off, and the filtrate is dialyzed against dist;lled ~ater and freeze-dried~ '-300 mg of the peptide~BSA conjugate thus obtained are d;ssolved in 1 ml of 1.Z N HCl/HAc, and the solution;s stirred at room temperature for 20 minutes. The sol-vent is removed by distillat;on ;n vacuo~ and the res;due is d;ssolved ;n ~ater and ~reeze-dried~
~31~3~
"~ ~. 5, E ~
20 mg of KLH are d;ssolved in 1 ml of 0.05 M
sodium phosphate buffer (pH 6). Then 3 mg of Mss~
dissolved ;n 0.2 ml of ~MF, are added, and the mixture is stirred at room temperature for 30 minutes~ Lo~ mole-cular ~eight constituents are removed by gel f;ltration on RSEPHADEX 6 25. The protein fractions are combined and concentrated to 4 ml by ultrafiltration. After addi tion of 10 ml of sodium borate buffer ~pH 9), 5 n9 of Fmoc-Gly-Pro-Arg-Val~Val-Glu HMDA-H (Example 2~ dis-solved in 0~5 ml of DMF, are added dropwise, and the solution ;s stirred at room temperature for 3 hours~ To spl;t off ~he protect;ve group, 15 ml of 0.2 N NaOH are added, and the m;xture is stirred at room temperature for 30 minutes. After neutral;~ation, the solut;on ;s di-alyzed against d;stilled water and freeze-dried.
Preparation_of heterologous antibodies Example 5 A peptide unidirectionally coupled to a carrier 20~ protein (~SA,KLH) is d;ssolved in P9S and mixed with equal parts of Freund's adjuvant. 200 y~ port;ons of th;s m;xture are injected iOp~ and s.c. ;nto BaLb/c mice 6 - 8 ~eeks old. These injections are repeated twice at ;ntervals of 3 - 4 weeks. Antisera of the majority of the animals thus treated show, in an ELISA assay, a posi-tive reaction to fibr;n monomers and the unconjugated peptide, but no ;mmune reaction ~ith fibrinogen~
Des-AA- and des-AABB-fibrin monomers are prepared by treatment of a fibrinogen solution ~;th batroxobin (2.5 U/mg fibr;nogen) or thrombin (5 Ulmg fibr;nogen).
The resulting clots ~re isolated and dissolved in 0~05 M
tr;s buffer (pH 7.4) ~ith the add;t;on of 3-molar urea~
Repolymerization is brought about by removing the urea by d;alys;s. The clot is again dissolved in the tris/urea buffer described above.
The presence in the ~ouse serum of antibodies against certain antigens (fibrinogen~ des-A-f;brin ~fibrin type I), des AB-fibrin ~fibrin type II) and un~
3 ~ 3 ~
., .;
conjugated pept;de) is determined by ELISA~ The pr;n-c;ple is based on a serum dilution being incubated ~ith ant;gens immobilized on a carr;er. After a wash;ng step followed by add;tion of an ant;-~ouse Ig solution labelled w;th an enzyme such as phosphatase or perox;dase, on addit;on of a substrate a change i~ the optical density is measured only ;n the reaction vessels in ~hich the serum antibodies have reacted with the corresponding ; antigens.
Using an ELISA, the following optical dens;ties were measured using antisera of mice ~hich had been immunized against a synthetic hexapept;de:
A n t i g e n 5 u s e d Mouse Fibrinogen Des-AA-fibrin Des-AABB-fibr;n Unconjugated hexa~ePtide ., . . . _ .
1 0 1.45 1.45 0.691 2 0.046 1.35 1.û8 0.51 3 0.017 1.64 1~25 0.49 4 0.03 2.0 1.55 0.51 Th;s result shows that heterologous antibodies in sera of m;ce wh;ch have been ;mmun;zed against conjugated pep-tide can differentiate unambi~uously between fibrinogen and fibrin.
Preparation of monoclonal antibodies - 25 Example 6 M;ce immunized ~ith conjugated peptide ~BSA, KLH) in accordance ~ith the abovementioned scheme receive, 3 days before removal of the spleen, an i.v. injection of 100 ~g of conjugated peptide dissolved in PBS.
About 108 cells from the spleen of an immunized mouse are fused ~ith 5 x 107 myeloma cells (x63-Sp8-653, a line which synthesizes no immunoglobulin; obtainable from the Salk Inst;tute, Cell Distribution Center, San D;ego CA 92112, USA) in the presence of polyethylene 35 glycol (MW 3,400). Fused cells are d;str;buted over 4 plates each of which contains 24 ~ells. Each o~ these wells contains 5 x 107 spleen cells of non-;mmuni~ed syngeneic mice in a nutrient medium which contains hypox-....
anth;ne~ aminopterin and thymidine~
The supernatants of these fused cells (hybridoma) containing the antibodies are'assayed, using the ELISA
assay described above, 10 - 14 days later in respect of 5 the;r specif;c;ty toward the following ant;gens: f;brino gen, des AA-fibrin, des-AABB-~ibrin and unconjugated peptide.
In order to obta;n monocLonal antibodies which ; are directed only against fibrin monomer and against the 10 synthetic peptide, hybridoma cells whose supernatants contain no antibodies directed against fibrinogen are cloned tw;ce.
, :,.. . .. .
~ ~ 3~3S3;2 Synthetic scheme I
h U C~ ~ P C r~ ~ ~, ~ C P , W ~ ~ ~ _ __ ~
H P..
U ,~ æ ~ ~ ~ ~ ~
N ~:C U N N N N N N N
O ~ O O O 8 o m C7 1 ~ \~ _ \ _ __ ~Y7 ~) H
I ) U
U o R r . g t4 U
,C~ ,U ,~C C~ , , , , C7 PC~ p C ~ ~ ~ P ~: U
pO :C V
: ~C?
~ ~. , , , ~ ~
p~ U
~, U C~
m m m 13~J3~2 ~ 13 --Synthetic scheme I I
u u u u o u O u m m Pq m ~ I , :
lll ~1 ~ ~
u m æ ~ m m m m m l$ C,.) l l l ~ l l ~o a~ ~ ~ ~ ~ ~
o I o o o o o o o , c \ ~ ~ \ \ _ P~
H
f P I __ _ ~ ~ C~ ~
a E~
: p-- f ' ~_ ,'~
Z
P: ~ :C
~t~
P~ .
N
:
~r~ C~
U
O O O
" Z
The ;nvention relates to a procedure for the se~ect;ve determination of fibrin in plasma or in t;ssue by use of highly spec;fic antibodies, to the antibod;es required for this purpose, to a process for their pre-5 parat;on, and ~o the immunogens used for this purpose~
It ;s known that soluble fibrin is found in the circulating blood of patients wi~h dissem;nated ;ntra-vascular coagulation (consumptive coagulopathy). This f;brin has been produced from the soluble plasma prote;n 10 fibrinogen by the action of proteolytic enzymesn In capillaries and small blood vessels, soluble fibrin can associate to form fibrin strands which can be detected by light and electron microscopy ~Review in. Muller-3erghaus, G., Seminars in Thrombosis and Hemostasis 3:
15 209 - 246, 1977). Moreover, in inflammatory disorders, a material der;ved from f;br;nogen can be detected in tissue, within or outside vessels. Thus, the object ~as to develop a reagent and a procedure to be able to differentiate between fibrin and fibrinogen and to deter-20 mine fibrin specifically and quantitatively.
It is known that proteolytic enzymes~ such as,for example, thrombin, both within and outside vessels, split off peptides from the fibrino~en molecule~ so that fibrin monomer is produced. Several fibrin monomers can 25 associate to form a fibr;n polymer, a fibrin clot, which then, if this takes place within a vessel, leads to deleterious consequences for the body. Accordingly~ it is desirable to detect f;brin at a very early stage of a pathologica~ process.
To date~ there are only qual;tative methods for the detection of fibrin, since fibrin differs from fibrinogen only by the fibrinopeptides having been split off~ The qualitative or semiquantitative methods are based on, for example~ precipitation after add;tion of 35 ethanol (Godal, H.C., Abildgaard, UO, Scand. J. Haematol.
., 3~?3532 _ 3 ~. ~
3: 342 - 350, 1966), protam;ne sulfate (Nie~;arowsk;, S., Gure~ich, V~, J. Labr Clin. Med~ 77: 665-676, 1971) or agglut;na~ion of coated erythrocytes or latex particles ~Largo, R., Heller, V., Straub, P.W., Blood 47: 991-1002 5 1976). These cited methods do not permit differentiation between f;br;n and fibr;nogen. Other methods attempt to separate fibrin from f;brinogen by chromatography (Alkjaers;g, N., Fletcher, A~, ~urste;n, R., Am. J~
Obstet~ Gynecol~ 122. 199 - 209, 1975; Heene, D.L., Matthias, F.R., Thromb. Res. 2: 137 - 154~ 1973~. These methods are l;kewise not specific~ s;nce it is not pos-sib~e to sepArate fibrin completely from fibrinogen.
Fibr;nogen is necessary to keep fibrin in solution (Krell, W., Mahn, I., Muller-~erghaus, G.~ Thromb. Res~
14: 299 - 310, 1979~.
A possible method of differentiating fibrin from fibrinogen in a purified system is the determinat;on of the N-terminal glyc;ne ~Kierulf, P., ~odal, H.C , Scand~
J. Haematol. 9: 370 - 372, 197Z~. However, this method ;s complicated and requires large amounts Oc starting material, for ~hich reason it has not found ~ide use as a test. Thus, to date it ;s not possible to differenti-ate fibrin from fibrinogen in a small sample of plasma or tissue.
Th;s object is achieved according to the inven-tion by determining fibrin using highly specific anti-bodies. This determination can also be carried out no~
only qualitatively but even quant;tatively in the pre-sence of comparatively high concentrations of fibrinogen.
; 30 It has been ,ound~ surprisingly, that the anti-bodies according to the invention are formed when the antigens used are peptides ~hich mimic the aminoacid sequence of certain mole~ular segments of the fibrin .~olecule~ This ~as all the more surprising since earlier 35 attempts at ;mmunization against the complete f;brin molecu~e had not led to fibrin-specific antibodies.
Ant;sera ~hich contain the ant;bodies accord;ng ~o the invention are obta;ned by ;ntroducin3 ~eptides as antiqen into the body of experimental arimals. These pep-~3~3~
~,`.
tides represent part of the aminoacid sequence of fibrin.
The pxeferred peptides have an aminoacid sequence of 3 to 12 aminoacids, which is exposed after splitting off fibrinopeptide A rom the N-terminal end of the fibrinogen molecule. The hexapeptide of the formula Gly-Pro-Arg-Val-Val-Glu has proved to be advan~ageous. Thi~ is bound in a customary manner, advantageou~ly interpolating a "spacer"
group, to carrier peptides suitable for this purpose.
The oligopeptides which mimic the aminoacid sequence of fibrin, in particular of the terminal groups which are liberated by splitting off the fibrinopeptides, are synthesized in a known manner. Suitable processes are described in, for example, E. ~unsch ln Houben-Wsyl, ~Methoden der organischen Chemie" (Methods of Organic Chemistry), Volume XV/1 and 2; The Peptides, Vol. 1, ~'Major Methods of Peptide Bond Formation", Academic Press (1979), or "Perspectives in Peptide Chemistry", A. Eberle, R.
Geiger and T. Wieland (editor~), S. Karger, Basel (1981).
This entails aminoacids or peptide fragments, whose functional groups which are not intended to participate in the reaction are protected in a suitable manner, being induced to react. The fxee carboxyl groups of aminoacid derivati~es or peptide fragments are activated in a suitable manner and reacted with the amino group of aminoacid derivatives or partially protected peptide fragments. The activation can be carried out with DCCI, DCCI/HOBt, DCCI/hydroxysuccinimide, POCl3, chloroformic esters or the azide method. The DCCI/HOBt method as described in Chem. Ber. 103, 788 (1970) is preferably used.
The synthesi~ of the partially protected oli~opeptides Boc-Gly-Pro-Arg-Val-Val-Glu-~-Capr-OH and Fmoc-Gly-Pro-Arg-; Val-Val-Glu-HMDA-H can be carried out stepwise from the C-texminal end or by coupling of fragments. In addition, stepwise synthesis on a soluble or insoluble polymeric carrier, such as, for example, polyoxyethylene or crosslinked polystyrene, is suitable. Suitable pro-", , 13~.P~'~3 tect;ve groups for the alpha-amino group of the amino-acids or peptide fragments are Boc, Fmoc, Z, ~,~ dimethyL-3,5-dimethoxybenzyloxycarbonyl~ 2-(4-biphenylyl)-2-propyl-oxycarbonyl and trityl. Boc~ Fmoc and Z are preferably 5 used.
Esterification with alkanols or araliphatic alco-hols is used to protect the alpha-carboxyl group of aminoacids and peptide fragments. The methyl, ben~yl and tert.-butyl esters are preferredO
The side-chain groups of the partially protected oligope~tides are a carboxyl group and a guanid;no group.
These can be substituted by protect;ve groups customary in peptide chemis~ry in accordance ~ith their reacti~ity~
! Esterificat;on ~ith aliphatic or aral;phat;c alcohols, 1S such as, for example, methanol, tertO butanol or benzyl alcohol, is preferably used to protect the carboxyl group ;n Glu. To protect the guanidino group in ar~inine, it can be protonated or substituted by NG-tosyl, NG-nitro, NG-adamantyloxycarbonyl and NG-benzyloxycarbonyl.
Protonation ;s preferred~
For an unamb;guous course of reaction in the coupling to the carrier protein, the N-terminal of the ol;gopeptides used must be protected so that it is pos-sible to split off the protective group on the peptide-protein çonjugate without damaging the protein. Su;tabLefor this purpose are the Fmoc protective group, ~hicn can be spl;t off under alkaline conditions, and the 2-(4-biphenylyl)~2-propyloxycarbonyl, c~ dimethyl-3,5-di ; methoxybenzyloxycarbonyl, Boc and trityl groups, ~hich can be spLit off under relatively mild acid conditions.
Suitable "spacer" groups are the bridge ~o~ming reagenls customarily used in peptide chemistry, such as are also used, for example, for the i~mobilization of enzymes~ The appropriate reagents contain t~o identical or different - 35 reactive groups at the ends of an alkylene group having, for example, 2 to 8 carbon atoms~ suitabLe reactive groups being those ~hich can undergo covalent bondins with the functional ~roups of the aminoacids. Suitable ~3~?3~-~.2 examples are dialdehydes such as glutaraldehyde, di;so-cyanates such as 1,6-hexamethylene diisocyanate~ dia~ines s~ch as 1,6-hexamethylenediamine, or ~aminocarboxylic ac;ds such as -am;nocaproic acid. These b;funct;onal reagents are, ~here appropriate after activationD linked in a known manner ~ith, on the one hand, the oligopeptide and, on the other hand, the carr;er.
In the case of Boc-Gly-Pro-Arg-Val-Val-Glu- -Capr~
OH~ the react;on of the partially protected oligopept;des ~;th the carrier protein ~akes place via activat;on of ~he carboxyl group us;ng a water-soluble carbod;imide, such as, for example, N~ethyl-N'-(3-d;methylaminopropyl)-carbodiimide, or ~ith DCCI/hydroxysuccinimideO ~oc-Gly-Pro-Arg-Val-Val-G~u-HMDA-H is bonded v;a the free amino group using a bifunctional reagent such as, for example, MBS. The Boc protective group on the peptide-protein conjugate can be spl;t off with 1.2 N HCl/HAc only ;n the case of BSA~ With many carrier proteins, such as, for example, BSA or KLH, it ;s possible to split off the Fmoc protect;ve group using ~.2 N NaOH~
Any des;red prote;n ;s su;table as a carrier~
It is advantageous to select those carr;er prote;ns which cause no cross-reaction. Suitable examples are albumins, such as bovine serum albumin, or hemocyanins, such as keyhole limpet hemocyanins.
Using the antigens thus obtained, exper;mental ani~als, such as m;ce, rats, rabb;ts or goats, are immun;zed in a known manner, and thus antisera conta;ning polyclonal antibodies are obtained~
In a preferred embodiment, monoclonal antibodies are produced in an appropr;ate Manner by the method of G. Kohler and C~ Milstein, Nature 256, 495 - 497 ~19~5).
The known mouse myeloma cell lines can be used for th;s purpose. It has proved to be particularly favorable to use a cell line ~hich itself produces no im~unoglobulin.
~ he polyclonal and monoclonal antibodies accord-ing to the invention do not react ~ith fibrinogen~ but ~o react ~1-th des-A-fibrin ~fibrin typ~ I), des-AB-~L3~~ 3~
-- 7f;br;n (fibrin type II), the peptide conju~ate and the synthetic pPptide. This binding takes place ~ith such ~reat sensit;v;ty and specificity that nanogram amounts of monoclonal ant;bodies are able specifically to detect 5 nanogram amounts of antigens (des-A-fibrin and/or des-AB-fibrin) even in the presence of fibrinogen in the physio-logical concentration (3 mg/ml)O
In the above text and in the examples ~hich ~ollo~, the abbreviations shown below have been used:
1Q Arg L-argin;ne soc t-butyloxycarbonyl ~ ~-Capr ~-aminocaproic acid (or radical derived - therefrom) DCCI dicyclohexylcarbodiimide 15 DCU dicyclvhexylurea des-AA-fibrin type I
des-AABB-fibrin type II
DMF dimethylformamide ELISA enzyme linked immunosorben~ assay 20 Fmoc fluorenylmethoxycarbonyl Glu L-glutam;c acid Gly glyc;ne HAc acetic acid HMDA 1,6-hexamethylenediamine ~or radical derived therefrom) HOBt 1-hydroxy-1H-benzotriazole Ig immunoglobulin ;.p. intraperitoneal i.v~ intravenous 30 KLH keyhole limpet hemocyanins MBS m-maleinimidobenzoyl-N-hydrsxysuccin-imide ester OBzl benzyl ester OtBu t-butyl ester 35 PBS phosphate buffered saline Pro L-proline BSA bov;ne serum albumin s.c. subcutaneous ~L 3 ~ 3 S 3 ~
TFh trifluoroacetic acid Yal L-valine Z benzyloxycarbonyl Preparat;on of th ~
S The peptides of Examples 1 and 2 are charac-terized by elemental analysis, aminoacid analys;s and th;n-layer chromatography. The chemical purity is established by thin-layer chromatography in a variety of solvent m;xtures. The racem;zat;on is checked by gas chroma~ography usin~ a glass capillary column coated with RCHIRASI~-Val by the ~ethod of J. ~hromat. Sci. 15, 174 ~1977)~ and is belo~ 2 per cent for each aminoacid.
E~ample 1 ( The partially protected pept;de Boc-Gly-Pro-Arg-Val~Val-Glu-~-Capr-OH is synthesized in accordance ~ith synthet;c scheme I, including -aMinocaproic acid as a spacer.
Example 2 The part;ally protected peptide Fmoc-Gly-Pro-Arg-Yal-Val-Glu-HMDA-H ;s synthesized in accordance with synthetic scheme II, ;nclud;ng hexamethylenediamine''as a spacer.
Example 3 20 mg of Boc-Gly-Pro-Arg-Val-Val-Glu-~-Ca~r-OX
tExample 1~ are dissolved ;n 0.4 ml of ethanol, and 4 mg of DCCI and Z.5 mg of hydroxysuccinimide are added.
After stirring at room temperature for 2 hours, the pep-tide hydroxysucc;n;mide ester is slowly added drop~ise to a solution of 760 mg of BSA ~extra pure) in 7 ~l of buffer (0.25 M HEPES, 0.2 M CaCl2, pH 7.5). After 20 hours at room temperature, the DCU is filtered off, and the filtrate is dialyzed against dist;lled ~ater and freeze-dried~ '-300 mg of the peptide~BSA conjugate thus obtained are d;ssolved in 1 ml of 1.Z N HCl/HAc, and the solution;s stirred at room temperature for 20 minutes. The sol-vent is removed by distillat;on ;n vacuo~ and the res;due is d;ssolved ;n ~ater and ~reeze-dried~
~31~3~
"~ ~. 5, E ~
20 mg of KLH are d;ssolved in 1 ml of 0.05 M
sodium phosphate buffer (pH 6). Then 3 mg of Mss~
dissolved ;n 0.2 ml of ~MF, are added, and the mixture is stirred at room temperature for 30 minutes~ Lo~ mole-cular ~eight constituents are removed by gel f;ltration on RSEPHADEX 6 25. The protein fractions are combined and concentrated to 4 ml by ultrafiltration. After addi tion of 10 ml of sodium borate buffer ~pH 9), 5 n9 of Fmoc-Gly-Pro-Arg-Val~Val-Glu HMDA-H (Example 2~ dis-solved in 0~5 ml of DMF, are added dropwise, and the solution ;s stirred at room temperature for 3 hours~ To spl;t off ~he protect;ve group, 15 ml of 0.2 N NaOH are added, and the m;xture is stirred at room temperature for 30 minutes. After neutral;~ation, the solut;on ;s di-alyzed against d;stilled water and freeze-dried.
Preparation_of heterologous antibodies Example 5 A peptide unidirectionally coupled to a carrier 20~ protein (~SA,KLH) is d;ssolved in P9S and mixed with equal parts of Freund's adjuvant. 200 y~ port;ons of th;s m;xture are injected iOp~ and s.c. ;nto BaLb/c mice 6 - 8 ~eeks old. These injections are repeated twice at ;ntervals of 3 - 4 weeks. Antisera of the majority of the animals thus treated show, in an ELISA assay, a posi-tive reaction to fibr;n monomers and the unconjugated peptide, but no ;mmune reaction ~ith fibrinogen~
Des-AA- and des-AABB-fibrin monomers are prepared by treatment of a fibrinogen solution ~;th batroxobin (2.5 U/mg fibr;nogen) or thrombin (5 Ulmg fibr;nogen).
The resulting clots ~re isolated and dissolved in 0~05 M
tr;s buffer (pH 7.4) ~ith the add;t;on of 3-molar urea~
Repolymerization is brought about by removing the urea by d;alys;s. The clot is again dissolved in the tris/urea buffer described above.
The presence in the ~ouse serum of antibodies against certain antigens (fibrinogen~ des-A-f;brin ~fibrin type I), des AB-fibrin ~fibrin type II) and un~
3 ~ 3 ~
., .;
conjugated pept;de) is determined by ELISA~ The pr;n-c;ple is based on a serum dilution being incubated ~ith ant;gens immobilized on a carr;er. After a wash;ng step followed by add;tion of an ant;-~ouse Ig solution labelled w;th an enzyme such as phosphatase or perox;dase, on addit;on of a substrate a change i~ the optical density is measured only ;n the reaction vessels in ~hich the serum antibodies have reacted with the corresponding ; antigens.
Using an ELISA, the following optical dens;ties were measured using antisera of mice ~hich had been immunized against a synthetic hexapept;de:
A n t i g e n 5 u s e d Mouse Fibrinogen Des-AA-fibrin Des-AABB-fibr;n Unconjugated hexa~ePtide ., . . . _ .
1 0 1.45 1.45 0.691 2 0.046 1.35 1.û8 0.51 3 0.017 1.64 1~25 0.49 4 0.03 2.0 1.55 0.51 Th;s result shows that heterologous antibodies in sera of m;ce wh;ch have been ;mmun;zed against conjugated pep-tide can differentiate unambi~uously between fibrinogen and fibrin.
Preparation of monoclonal antibodies - 25 Example 6 M;ce immunized ~ith conjugated peptide ~BSA, KLH) in accordance ~ith the abovementioned scheme receive, 3 days before removal of the spleen, an i.v. injection of 100 ~g of conjugated peptide dissolved in PBS.
About 108 cells from the spleen of an immunized mouse are fused ~ith 5 x 107 myeloma cells (x63-Sp8-653, a line which synthesizes no immunoglobulin; obtainable from the Salk Inst;tute, Cell Distribution Center, San D;ego CA 92112, USA) in the presence of polyethylene 35 glycol (MW 3,400). Fused cells are d;str;buted over 4 plates each of which contains 24 ~ells. Each o~ these wells contains 5 x 107 spleen cells of non-;mmuni~ed syngeneic mice in a nutrient medium which contains hypox-....
anth;ne~ aminopterin and thymidine~
The supernatants of these fused cells (hybridoma) containing the antibodies are'assayed, using the ELISA
assay described above, 10 - 14 days later in respect of 5 the;r specif;c;ty toward the following ant;gens: f;brino gen, des AA-fibrin, des-AABB-~ibrin and unconjugated peptide.
In order to obta;n monocLonal antibodies which ; are directed only against fibrin monomer and against the 10 synthetic peptide, hybridoma cells whose supernatants contain no antibodies directed against fibrinogen are cloned tw;ce.
, :,.. . .. .
~ ~ 3~3S3;2 Synthetic scheme I
h U C~ ~ P C r~ ~ ~, ~ C P , W ~ ~ ~ _ __ ~
H P..
U ,~ æ ~ ~ ~ ~ ~
N ~:C U N N N N N N N
O ~ O O O 8 o m C7 1 ~ \~ _ \ _ __ ~Y7 ~) H
I ) U
U o R r . g t4 U
,C~ ,U ,~C C~ , , , , C7 PC~ p C ~ ~ ~ P ~: U
pO :C V
: ~C?
~ ~. , , , ~ ~
p~ U
~, U C~
m m m 13~J3~2 ~ 13 --Synthetic scheme I I
u u u u o u O u m m Pq m ~ I , :
lll ~1 ~ ~
u m æ ~ m m m m m l$ C,.) l l l ~ l l ~o a~ ~ ~ ~ ~ ~
o I o o o o o o o , c \ ~ ~ \ \ _ P~
H
f P I __ _ ~ ~ C~ ~
a E~
: p-- f ' ~_ ,'~
Z
P: ~ :C
~t~
P~ .
N
:
~r~ C~
U
O O O
Claims (6)
1. A procedure for the determination of fibrin, which comprises the use of highly specific antibodies against fibrin, which do not react with fibrinogen, wherein said antibodies are obtainable by immunization using a peptide which contains an aminoacid sequence of the 3 to 12 terminal amino acids of fibrin which are exposed by splitting off fibrinopeptide A from the fibrinogen molecule.
2. The procedure as claimed in claim 1, wherein monoclonal antibodies are used.
3. Highly specific antibodies against fibrin, which do not react with fibrinogen, obtainable by immunization using a peptide which contains an aminoacid sequence of the 3 to 12 terminal aminoacids of fibrin which are exposed by splitting off fibrinopeptide A from the fibrinogen molecule.
4. The antibodies as claimed in claim 3, wherein the immunogen contains the sequence Gly-Pro-Arg-Val-Val-Glu.
5. The antibodies as claimed in claim 3, wherein the immunogen contains the aminoacid sequence bound to a carrier peptide via a spacer group.
6. The antibodies as claimed in claim 3, which are monoclonal.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3400434.3 | 1984-01-09 | ||
| DE19843400434 DE3400434A1 (en) | 1984-01-09 | 1984-01-09 | DETERMINATION OF FIBRINE WITH FIBRINE-SPECIFIC ANTIBODY |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1303532C true CA1303532C (en) | 1992-06-16 |
Family
ID=6224505
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000471712A Expired - Lifetime CA1303532C (en) | 1984-01-09 | 1985-01-08 | Determination of fibrin using fibrin-specific antibodies |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0152612B1 (en) |
| JP (1) | JPS60158353A (en) |
| AT (1) | ATE50458T1 (en) |
| AU (1) | AU595479B2 (en) |
| CA (1) | CA1303532C (en) |
| DE (2) | DE3400434A1 (en) |
| ES (1) | ES8606411A1 (en) |
| NO (1) | NO166591B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4916070A (en) * | 1986-04-14 | 1990-04-10 | The General Hospital Corporation | Fibrin-specific antibodies and method of screening for the antibodies |
| DE3632343A1 (en) * | 1986-09-24 | 1988-04-07 | Battelle Institut E V | METHOD FOR DETECTING 1,2,2-TRIMETHYLPROPYLMETHYLPHOSPHOFLUORIDAD |
| DE3701812A1 (en) * | 1987-01-22 | 1988-08-04 | Max Planck Gesellschaft | FIBRINE-SPECIFIC ANTIBODY, METHOD FOR THE PRODUCTION THEREOF AND ITS USE |
| SE8701437L (en) * | 1987-04-06 | 1988-10-07 | Rolf Kenth Sigvard Wallin | IN VITRO DIAGNOSTIC PROCEDURE AND MEASURES THAT CAN BE USED IN THE PROCEDURE |
| DE3730059A1 (en) * | 1987-09-08 | 1989-03-30 | Behringwerke Ag | METHOD FOR DETERMINING "SOLUBLE" FIBRINE |
| NL8801227A (en) * | 1988-05-10 | 1989-12-01 | Tno | ANTIBODIES AGAINST FIBRINE; IMMUNOGEN, METHOD FOR DETERMINING FIBRIN WITH THE ANTIBODIES AND PHARMACEUTICAL PREPARATION BASED ON THE ANTIBODIES FOR THE PREPARATION OF THE ANTIBODIES. |
| US5609869A (en) * | 1988-08-19 | 1997-03-11 | The General Hospital Corporation | Hybrid immunoglobulin-thrombolytic enzyme molecules which specifically bind a thrombus, and methods of their production and use |
| US5811265A (en) * | 1988-08-19 | 1998-09-22 | The General Hospital Corporation | Hybrid immunoglobulin-thrombolytic enzyme molecules which specifically bind a thrombus, and methods of their production and use |
| DE4134833A1 (en) * | 1991-09-25 | 1993-04-01 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING FIBRINE |
| DK0556906T3 (en) * | 1992-02-17 | 1996-09-02 | Akzo Nobel Nv | Calibrator and its use for an immunoassay |
| DE4420742A1 (en) * | 1993-10-20 | 1995-04-27 | Boehringer Mannheim Gmbh | Synthetic standard for immunoassays |
| US5780255A (en) * | 1995-06-09 | 1998-07-14 | Instrumentation Laboratory, S.P.A. | Protein C pathway screening test |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2532151C3 (en) * | 1975-07-18 | 1979-06-13 | Biotest-Serum-Institut Gmbh, 6000 Frankfurt | Antisenim for the quantitative determination of the breakdown products of fibrin and fibrinogen «and process for its production |
| JPS5811856A (en) * | 1981-07-16 | 1983-01-22 | Mihama Hisaharu | Preparation of antifragment dgamma-2 dimer blood serum |
| US4438209A (en) * | 1981-07-17 | 1984-03-20 | Mallinckrodt, Inc. | Radioimmunoassay for fibrinopeptide A |
| AU572125B2 (en) * | 1983-03-17 | 1988-05-05 | Mabco Limited | Monoclonal antibodies with specificity for crosslinked fibrin and their diagnotic uses |
| US4722903A (en) * | 1983-11-14 | 1988-02-02 | New York Blood Center, Inc. | Monoclonal antibodies specific to in vivo fragments derived from human fibrinogen, human fiberin I or human fibrin II |
-
1984
- 1984-01-09 DE DE19843400434 patent/DE3400434A1/en active Granted
- 1984-12-20 AT AT84116008T patent/ATE50458T1/en not_active IP Right Cessation
- 1984-12-20 EP EP84116008A patent/EP0152612B1/en not_active Expired - Lifetime
- 1984-12-20 DE DE8484116008T patent/DE3481386D1/en not_active Expired - Lifetime
-
1985
- 1985-01-07 ES ES539382A patent/ES8606411A1/en not_active Expired
- 1985-01-08 CA CA000471712A patent/CA1303532C/en not_active Expired - Lifetime
- 1985-01-08 AU AU37521/85A patent/AU595479B2/en not_active Ceased
- 1985-01-08 NO NO850087A patent/NO166591B/en unknown
- 1985-01-08 JP JP60000471A patent/JPS60158353A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DE3481386D1 (en) | 1990-03-29 |
| DE3400434C2 (en) | 1992-11-05 |
| JPS60158353A (en) | 1985-08-19 |
| NO166591B (en) | 1991-05-06 |
| EP0152612B1 (en) | 1990-02-21 |
| AU595479B2 (en) | 1990-04-05 |
| EP0152612A3 (en) | 1986-04-16 |
| ES539382A0 (en) | 1986-04-01 |
| ES8606411A1 (en) | 1986-04-01 |
| AU3752185A (en) | 1985-07-18 |
| DE3400434A1 (en) | 1985-09-26 |
| EP0152612A2 (en) | 1985-08-28 |
| ATE50458T1 (en) | 1990-03-15 |
| NO850087L (en) | 1985-07-10 |
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