AU595479B2 - Determination of fibrin using fibrin-specific antibodies - Google Patents

Abstract

1. A highly specific antibody against fibrin, obtainable by immunization using a peptide which contains an amino acid sequence of fibrin which is exposed by splitting off fibrinopeptide A from the fibrin molecule.

Description

COMMONW~EALTH OF AUSTRALIA5 i PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class I nt. Class Application Number: _3 7So52//? Lodged: Complete Specification Lodged: Accepted: Published: woi 4 rity 0 t r Related Art: Tsdocument co-ntains the~ 111m n-ien Is made unc!-r S~ect1Q1149 and is correct for printhungj Name of Applicant: Adhress of Applicant 0 0 0 Av~Qc~al Inventor: Address for Service: MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSOHAFTEN e.V. and BEHRINGWERKE AKTIENGESELLSCHAFT Bunsenstrasse 10, D-3400 Gottingen and D-3550 Marburg 1, Federal Republic of Germany, respectively URSULA SCHEEFERS-BORCHEL, GERT MULLER-BERCHAUS, REINHARD EBERLE, PETER FUHGE and NORBERT HEIMBURGER EDWD. WATERS SONS, 50 QUEEN STREET, MELBOUP E, AUSTRALIA, 8000.

Complete Specification for the invention entitled: DETERMINATION OF FIBRIN USING FIBRIN-SPECIFIC ANTIB3ODIES The following statement is a full description of this invention, including the best method of performing it known to us 1 2 HOE 84/B 750 The invention relates to a procedure for the selective determination of fibrin in plasma or in tissue by use of highly specific antibodies, to the antibodies required for this purpose, to a process for their preparation, and to the immunogens used for this purpose.

It is known that soluble fibrin is found in the circulating blood of patients with disseminated intravascular coagulation (consumptive coagulopathy). This fibrin has been produced from the soluble plasma protein fibrinogen by the action of proteolytic enzymes. 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: MGller- Berghaus, Seminars in Thrombosis and Hemostasis 3: 209 246, 1977). Moreover, in inflammatory disorders, a material derived from fibrinogen can be detected in tissue, within or outside vessels. Thus, the object was to develop a reagent and a procedure to be able to differentiate between fibrin and fibrinogen and to determine fibrin specifically and quantitatively.

ii It is known that proteolytic enzymes, such as, for example, thrombin, both within and outside vessels, split off peptides from the fibrinogen molecule, so that fibrin monomer is produced. Several fibrin monomers can associate to form a fibrin polymer, a fibrin clot, which then, if this takes place within a vessel, leads to deleterious consequences for the body. Accordingly, it S-is desirable to detect fibrin at a very early stage of a Spathological process.

To date, there are only qualitative 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 addition of ethanol (Godal, Abildgaard, Scand. J. Haematol.

3 3: 342 350, 1966), protamine sulfate (Niewiiarowski, S., Gurewich, J. Lab. Clin. Med. 77: 665-676, 1971) or agglutination of coated erythrocytes or latex particles (Largo, Heller, Straub, Blood 47: 991-1002, 1976). These cited methods do not permit differentiation between fibrin and fibrinogen. Other methods attempt to separate fibrin from fibrinogen by chromatography (Alkjaersig, Fletcher, Burstein, Am. J.

Obstet. Gynecol. 122: 199 209, 1975; Heene, D.L., Matthias, Thromb. Res. 2: 137 154, 1973). These methods are likewise not specific, since it is not possible to separate fibrin completely from fibrinogen.

Fibrinogen is necessary to keep fibrin in solution (Krell, Mahn, Multer-Berghaus, Thromb. Res.

14: 299 310, 1979).

A possible method of differentiating fibrin from 4 fibrinogen in a purified system is the determination of the N-terminal glycine (Kierulf, Godal, Scand.

J. Haematol. 9: 370 372, 1972). However, this method is complicated and requires large amounts of starting material, for which reason it has not found wide use as a test. Thus, to date it is not possible to differentiate fibrin from fibrinogen in a small sample of plasma or tissue.

This object is achieved according to the invention by determining fibrin using highly specific anti- S'.bodies. This determination can also be carried out not only qualitatively but even quantitatively in the pre- S sence of comparatively high concentrations of fibrinogen.

It has been found, surprisingly, that the antibodies according to the invention are formed when the antigens used are peptides which mimic the aminoacid sequence of certain molecular segments of the fibrin molecule. This was all the more surprising since earlier attempts at immunization against the complete fibrin molecule had not led to fibrin-specific antibodies.

Antisera which contain the antibodies according to the invention are obtained by introducing peptides as antigen into th- body of experimental animals. These pepa 0q I *r

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*l C 0* .5 *a 0 41 *at -4 tides represent part of the aminoacid sequence of fibrin.

The preferred peptides have an aminoacid sequence of 3 to 12 aminoacids, which is exposed after splitting off fibrinopeptide A from the N-terminal end of the fibrinogen molecule. The hexapeptide of the formula Gly-Pro-Arg-Val-Val-GLu has proved to be advantageous. This is bound in a customary manner, advantageously 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. Wunsch in Houben-Weyl, "Methoden der organischen Chemie" (Methods of Organic Chemistry), Volume XV/l 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 (editors), 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 free carboxyl groups of aminoacid derivatives 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 synthesis of the partially protected oligopeptides Boc-Gly-Pro-Arg-Val-Val-GLu-E.-Capr-OH and Fmoc- Gly- Pro-Arg-Val-VaL-Glu-HMDA-H can be carried out stepwise from the C-terminal 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-

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tective groups,for the alpha-amino group of the aminoacids or peptide fragments are Boc, Fmoc, Z, 0cXx-dimethyl- 2-(4-biphenyLyL)-2-propyLoxycarbonyl and trityl. Boc, Fmoc and Z are preferably used.

Esterification with alkanols or araLiphatic alcohols is used to protect the alpha-carboxyL group of aminoacids and peptide fragments. The methyl, benzy and tert.-butyl esters are pieferred.

The side-chain groups of the partially protected oligopeptides are a :arboxyl group and a guanidino group.

These can be substituted by protective groups customary in peptide chemistry in accordance with their reactivity.

Esterification with aliphatic or araliphatic alcohols, such as, for example, methanol, tert.-butanol or benzyl alcohol, is preferably used to protect the carboxyl.group i in Glu. To protect the guanidino group in arginine, it can be protonated or'substituted by NG-tosyl, NG-nitro, NG-adamantyLoxycarbonyi and NG-benzyloxycarbonyL.

Protonation is preferred.

For an unambiguous course of reaction in the coupling to the carrier protein, the N-terminal of the oligopeptides used must be protected so that it is possible to split off the protective group on the peptideprotein conjugate without damaging the protein. Suitable for this purpose are the Fmoc protective group, which can be split off under alkaLine conditions, and the 2-(4biphenylyL)-2-propyLoxy.carbonyL, oC4-dimethyL-3,5-dimethoxybenzyLoxycarbonyl, Boc and trityl groups, which can be split off under relatively mild acid conditions.

Suitable "spacer" groups are the bridge forming reagents customarily used inpeptide chemistry, such as are also used, for example, for the immobilization of enzymes. The appropriate reagents contain two identical or different reactive groups at the ends of an alkylene group having, for example, 2 to 8 carbon atoms, suitable reactive groups being those which can undergo covalent bonding with the functional groups of the aminroacids. Suitable -6examples are dialdehydes such as glutaraldehyde, diisocyanates such as 1,6-hexamethylene diisocyanate, diamines such as 1,6-hexamethylenediamine, or tL-aminocarboxylic acids such as 4-aminocaproic acid. These bifunctional reagents are, where appropriate after activation, linked in a known manner with, on the one hand, the oLigopeptide and, on the other hand, the carrier.

In the case of Boc-GLy-Pro-Arg-Val-Val-GLu- -Capr- OH, the reaction of the partially protected oligopeptides with the carrier protein takes place via activation of the carboxyl group using a water-soluble carbodiimide, such as, for example, N-ethyl-N'-(3-dimethylaminopropyL)carbodiimide, or with DCCI/hydroxysuccinimide. Fmoc-GLy- Pro-Arg-Val-Val-Glu-HMDA-H is bonded via the free amino group using a bifunctionaL reagent such as, for example, MBS. The Boc protective group on the peptide-protein conjugate can be split off with 1.2 N HCL/HAc only in the case of BSA. With many carrier proteins, such as, for example, BSA or KLH, it is possible to split off the Fmoc 20 protective group using 0.2 N NaOH.

Any desired protein is suitable as a carrier.

S .It is advantageous to select those carrier proteins which cause no cross-reaction. Suitable examples are albumins, such as bovine serum albumin, or hemocyanins, such as 25 keyhole limpet hemocyanins.

Using the antigens thus obtained, experimental animals, such as mice, rats, rabbits or goats, are immunized in a known manner, and thus antisera containing polyclonal antibodies are obtained.

In a preferred embodiment, monoclonal antibodies are produced in an appropriate manner by the method of G. K"hler and C. Milstein, Nature 256, 495 497 (1975).

The known mouse myeloma cell lines can be used for this purpose. It has proved to be particularly favorable to use a cell line which itself produces no immunoglobulin.

The polyclonal and monoclonal antibodies according to the invention do not react with fibrinogen, but do react with des-A-fibrin (fibrin type des-AB- -7fibrin (fibrin type II), the peptide conjugate and the synthetic peptide. This binding takes pLace with such great sensitivity and specificity that nanogram amounts of monoclonal antibodies are abLe specificaLLy to detect nanogram amounts of antigens (des-A-fibrin and/or des-ABfibrin) even in the presence of fibrinogen in the physiological concentration (3 mg/mI).

In the above text and in the examples which follow, the abbreviations shown below have been used: Arg L-arginine Boc t-butyloxycarbonyL E-Capr C-aminocaproic acid (or radicaL derived therefrom) DCCI dicyclohexyLcarbodiimide DCU dicycLohexyLurea des-AA-fibrin type I des-AABB-fibrin type II DMF dimethyLformamide ELISA enzyme Linked immunosorbent assay Fmoc fLuorenylmethoxycarbonyl GLu L-gLutamic acid Gly glycine HAc acetic acid HMDA 1,6-hexamethylenediamine (or radical derived therefrom) HOBt 1-hydroxy-1H-benzotriazoLe Ig immunoglobutLin i.p. intraperitoneaL i.v. intravenous 30 KLH keyhole limpet hemocyanins MBS m-maLeinimidobenzoyL-N-hydroxysuccinimide ester OBzL benzyl ester OtBu t-butyl ester PBS phosphate buffered saline Pro L-proLine BSA bovine serum albumin s.c. subcutaneous 8 TFA trifluoroacetic acid Vat L-vaLine Z benzyloxycarbonyl Preparation of the synthetic peptides The peptides of Examples 1 and 2 are characterized by elemental analysis, aminoacid analysis and thin-layer chromatography. The chemical purity is established by thin-layer chromatography in a variety of solvent mixtures. The racemization is checked by gas chromatography using a glass capillary column coated with RCHIRASIL-Val by the method of J. Chromat. Sci. 15, 174 (1977), and is below 2 per cent for each aminoacid.

Example 1 0 9 t U tf

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o o 0 90 0 0 it The partially protected peptide Boc-Gly-Pro-Arg- Val-Val-Glu-E.-Capr-OH is synthesized in accordance with synthetic scheme I, including -aminocaproic acid as a spacer.

Example 2 The partially protected peptide Fmoc-Gly-Pro-Arg- Val-VaL-GLu-HMDA-H is synthesized in accordance with synthetic scheme II, including hexamethylenediamine "as a spacer.

Example 3 mg of Boc-Gly-Pro-Arg-Val-Val-Glu--Capr-OH (Example 1) are dissolved in 0.4 ml of ethanol, and 4 mg of DCCI and 2.5 mg of hydroxysuccinimide are added.

After stirring at room temperature for 2 hours, the peptide hydroxysuccinimide ester is slowly added dropwise to a solution of 760 mg of BSA (extra pure) in 7 ml of buffer (0.25 M HEPES, 0.2 M CaCi 2 pH After hours at room temperature, the DCU is filtered off, and the filtrate is dialyzed against distilled water and freeze-dried.

300 mg of the peptide-BSA conjugate thus obtained are dissolved in 1 ml of 1.2 N HCL/HAc, and the solution is stirred at room temperature for 20 minutes. The solvent is removed by distillation in vacuo, and the residue is dissolved in water and freeze-dried.

9 Example 4 mg of KLH are dissolved in 1 ml of 0.05 M sodium phosphate buffer (pH Then 3 mg of MBS, dissolved in 0.2 ml of DMF, are added, and the mixture is stirred at room temperature for 30 minutes. Low molecular weight constituents are removed by gel filtration on RSEPHADEX G 25. The protein fractions are combined and concentrated to 4 ml by ultrafiltration. After addition of 10 ml of sodium borate buffer (pH 5 mg of Fmoc-GLy-Pro-Arg-Val-Val-GLu-HMDA-H '(Example dissolved in 0.5 ml of DMF, are added dropwise, and the solution is stirred at room temperature for 3 hours. To split off the protective group, 15 ml of 0.2 N NaOH are added, and the mixture is stirred at room temperature for o 15 30 minutes. After neutralization, the solution is dialyzed against distilled water and freeze-dried.

SPreparation of heterologous antibodies Example t A peptide unidirectionally coupled to a carrier protein (BSA,KLH) is dissolved in PBS and mixed with equal parts of Freund's adjuvant. 200 pg portions of this mixture are injected i.p. and s.c. into Balb/c mice 6 8 weeks old. These injections are repeated twice at intervals of 3 4 weeks. Antisera of the majority of a.o 25 the animals thus treated show, in an ELISA assay, a positive reaction to fibrin monomers and the unconjugated peptide, but no immune reaction with fibrinogen.

4 Des-AA- and des-AABB-fibrin monomers are prepared by treatment of a fibrinogen solution with batroxobin (2.5 U/mg fibrinogen) or thrombin (5 U/mg fibrinogen).

S The resulting clots are isolated and dissolved in 0.05 M tris buffer (pH 7.4) with the addition of 3-molar urea.

Repolymerization is brought about by removing the urea by dialysis. The clot is again dissolved in the tris/urea buffer described above.

The presence in th mouse serum of antibodies against certain antigens (fibrinogen, des-A-fibrin (fibrin type des-AB-fibrin (fibrin type II) and uni 10 conjugated peptide) is determined by ELISA. The principle is based on a serum dilution being incubated with antigens immobilized on a carrier. After a washing step followed by addition of an anti-mouse Ig solution labelled with an enzyme such as phosphatase or peroxidase, on addition of a substrate a change in the optical density is measured only in the reaction vessels in which the serum antibodies have reacted with the corresponding antigens.

More specifically, the wells of polystyrol microtestplates (Nunc Immunoplate IFF) are incubated i0 overnight at 40 C with antigen (10 pg/ml, 50 pl/well). If the antigen is des-AA or des AABB the incubation buffer contains 3M urea.

Then the wells are washed 5 times with PBS S containing 0.05% Tween 20. Into the coated wells 50 pl plasma (diluted 1:1000 with PBS) of immunized mice or 50 pl culture medium (see example 6) are pipetted and for 1 hour incubated at room temperature. After further washing (see i above) 50 pl of a POD conjugated rabbit anti-mouse IgG Santiserum (diluted 1:400 with PBS/0.05 Tween 20) are added and incubated for another hour. The wells .re then washed again and 150 ul of POD-substrate (2mM o-Phenylendiamin (Merck) in 100 mM Na-acetate/50 NaH 2 PO containing 0.003 H 2 0 2 are added.

The reactiGn was terminated after 30 min by the addition of 0.5 N HSO 4 The absorbance was determined at 492 nm.

The following optical densities were measured using antisera of mice which had been immunized against a S3 synethetic hexapeptide: c30 SAntigens used SMouse Fibrinogen Des-AA-fibrin Des-AABB-fibrin Unconjugated hexapeptide 1 0 1.45 1.45 0.691 2 0.046 1.35 1.08 0.51 3 0.017 1.64 1.25 0.49 4 0.03 2.0 1.55 0.51 I 10a This result shows that heterologous antibodies in sera of mice which have been immunized against conjugated peptide can differentiate unambiguously between fibrinogen and fibrin.

Preparation of monoclonal antibodies Example 6 Mice immunized with conjugated peptide (BSA, KLH) in accordance with the abovementioned scheme receive, 3 days before removal of the spleen, an i.v. injection of 100 pg of Sconjugated peptide dissolved in PBS.

8 About 10 cells from the spleen of an immunized mouse are fused with 5 x 10 7 myeloma cells (x63-Sp8-653, a line which synthesizes no immunoglobulin; obtainable from teh Salk Institute, Cell Distribution Center, San Diago CA 92112, in the presence of polyethylene glycol (MW 3,400). Fused cells are distributed over 4 plates each of which contains 24 wells. Each of these wells contains 5 x spleen cells of non-ii~munized syngeneic mice in a nutrient medium which contains hypox- I V L t 1 4 4 11 anthine, 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 their specificity toward the following antigens: fibrinogen, des-AA-fibrin, des-AABB-fibrin and unconjugated peptide.

In order to obtain monoclonal antibodies which are directed only against fibrin monomer and against the synthetic peptide, hybridoma cells whose supernatants contain no antibodies directed against fibrinogen are cloned twice.

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Claims (4)

1. Antibodies which are highly specific against fibrin characterized in that they are produced by immunization with a peptide which contains an amino acid sequence of 3-12 amino acids which is exposed by splitting off fibrinopeptide A from the N-terminus of the fibrinogen molecule.

2. Antibodies according to claim 1 characterized in that the immunogen contains the sequence Gly Pro Arg Val Val- Glu

3. Antibodies according to claim 1 characterized in that the peptide is bound to a carrier protein via a spacer group.

4. Antibodies according to claim 1 characterized in ,r that they are monoclonal antibodies. t Use of antibodies according to claim 1 for the specific detection and determination of fibrin. SDATED this 7th day of December, 1989 MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN E.V. and BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS, 2nd Floor, "The Atrium" 290 Burwood Road, HAWTHORN VIC. 3122 (LN 7.47) 1