U.S. patent application number 10/767200 was filed with the patent office on 2005-05-05 for anti-hirudin polyclonal antibodies and their use for the identification, immunopurification and quantitative determination of hirudin.
This patent application is currently assigned to Farmitalia Carlo Erba S.R.L.. Invention is credited to Gerna, Marco, Giorgetti, Carla, Lansen, Jacqueline, Molinari, Antonio, Roncucci, Romeo.
Application Number | 20050095685 10/767200 |
Document ID | / |
Family ID | 32045695 |
Filed Date | 2005-05-05 |
United States Patent
Application |
20050095685 |
Kind Code |
A1 |
Molinari, Antonio ; et
al. |
May 5, 2005 |
Anti-hirudin polyclonal antibodies and their use for the
identification, immunopurification and quantitative determination
of hirudin
Abstract
A method of producing an immunogen comprising polymerizing a
hirudin and/or a hirudin-like protein is provided. A method of
producing an antibody specific for a hirudin or a hirudin-like
protein is also provided.
Inventors: |
Molinari, Antonio; (Milano,
IT) ; Gerna, Marco; (Milano, IT) ; Giorgetti,
Carla; (Stradella (Pavia), IT) ; Lansen,
Jacqueline; (San Villore Olona (Milano), IT) ;
Roncucci, Romeo; (Milano, IT) |
Correspondence
Address: |
ARENT FOX KINTNER PLOTKIN & KAHN
1050 CONNECTICUT AVENUE, N.W.
SUITE 400
WASHINGTON
DC
20036
US
|
Assignee: |
Farmitalia Carlo Erba
S.R.L.
|
Family ID: |
32045695 |
Appl. No.: |
10/767200 |
Filed: |
January 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10767200 |
Jan 30, 2004 |
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08603182 |
Feb 20, 1996 |
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6719975 |
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08603182 |
Feb 20, 1996 |
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08205764 |
Mar 4, 1994 |
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08205764 |
Mar 4, 1994 |
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07903797 |
Jun 24, 1992 |
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Current U.S.
Class: |
435/70.21 ;
530/388.1; 530/403 |
Current CPC
Class: |
C07K 14/815 20130101;
Y10S 530/855 20130101; C07K 16/38 20130101 |
Class at
Publication: |
435/070.21 ;
530/388.1; 530/403 |
International
Class: |
C07K 016/18; C12P
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 1991 |
IT |
MI 91A001767 |
Claims
1. A method of producing an immunogen, which method comprises
polymerising a hirudin and/or a hirudin-like protein.
2. A method according to claim 1 wherein the hirudin and/or the
hirudin-like protein are/is polymerized with glutaraldehyde.
3. A method according to claim 1 or 2 wherein the hirudin is HV1
(SEQ ID NO: 1), HV2 (SEQ ID NO: 2), HV3 (SEQ ID NO: 3), P1 (SEQ ID
NO: 4) or P2 (SEQ ID NO: 5).
4. A method according to claim 1 or 2 wherein the hirudin-like
protein is a derivative of HV1 (SEQ ID NO: 1), HV2 (SEQ ID NO: 2),
HV3 (SEQ ID NO: 3), P1 (SEQ ID NO: 4) or P2 (SEQ ID NO: 5) by way
of amino acid substitution, deletion, insertion, extension,
functionalisation or chemical modification, said derivative having
anti-thrombin activity.
5. An immunogen comprising a polymerised hirudin and/or
hirudin-like protein.
6. An immunogen according to claim 5 wherein the hirudin is HV1
(SEQ ID NO: 1), HV2 (SEQ ID NO: 2), HV3 (SEQ ID NO: 3), P1 (SEQ ID
NO: 4) or P2 (SEQ ID NO: 5).
7. An immunogen according to claim 5 wherein the hirudin-like
protein is a derivative of HV1 (SEQ ID NO: 1), HV2 (SEQ ID NO: 2),
HV3 (SEQ ID NO: 3), P1 (SEQ ID NO: 4) or P2 (SEQ ID NO: 5) by way
of amino acid substitution, deletion, insertion, extension,
functionalisation or chemical modification, said derivative having
anti-thrombin activity.
8. An immunogen according to claim 5, 6 or 7 obtainable by
polymerising the hirudin and/or the hirudin-like protein with
glutaraldehyde.
9. A method of producing an antibody capable of binding to a
hirudin or a hirudin-like protein, said method comprising raising
antibody against an immunogen as claimed in claims 5 to 8.
10. A method according to claim 9, wherein the said antibody is a
polyclonal antibody.
11. A method according to claim 9, wherein the said antibody is a
monoclonal antibody.
12. A method for the quantitative determination, purification or
identification of a hirudin or hirudin-like protein, said method
comprising raising antibody against an immunogen as claimed in
claims 5 to 8, and using the antibody thereby obtained in said
quantitative determination, purification or identification.
Description
[0001] The invention relates to a method of producing an antibody
specific for a hirudin or a hirudin-like protein.
[0002] Hirudin is a polypeptide which may be isolated in small
quantities from the salivary glands of the leech Hirudo medicinalis
(Markwardt F: Untersuchungem uber Hirudin. Naturwissenschaften 41:
537-538, 1955).
[0003] Hirudin is a potent and specific inhibitor of thrombin
(Chang J Y.: The functional domain of hirudin, a thrombin-specific
inhibitor. FEBS Lett. 164: 307-313, 1983). Hirudin binds to
thrombin, inhibiting the transformation of soluble fibrinogen into
insoluble fibrin and preventing the thrombin mediated activation of
factors V, VIII and XIII (Markwardt F.: Pharmacology of hirudin:
One hundred years after the first report of the anticoagulant agent
in the medicinal leeches. Biomed. Biochem. Acta 44: 1007-1013,
1985).
[0004] Thrombin has a greater affinity for hirudin than for the
platelet thrombin receptors (Fenton II J W, Landis B. H., Walz D.
A., Bing D. H., Feinman R. D., Zabinski M. P., Sonder S. A.,
Berliner L. J., and Finlayson J. S.: Human thrombin: Preparative
evaluation, structural properties and enzymic specificity. In: DH
Bind. Ed.: The Chemistry and Physiology of Human Plasma, Pergamon
Press, New York, pp. 151-182, 1979) and hence hirudin is able to
induce the dissociation of the thrombin-platelet receptor complex
(Tam S. W., Fenton J. W. and Detwiler T. C.; Dissociation of
thrombin from platelets by hirudin: Evidence for receptor
processing. J. Biol. Chem., 254: 8723-8725, 1979), thereby
inhibiting the release reaction and platelet aggregation (Hoffmann
A., and Markwardt F.: Inhibition of the thrombin-platelet reaction
by hirudin. Hemostasis, 14: 164-169, 1984, Reinhold D. S. and
Gershman H. Hirudin insensitives thrombin-stimulated platelet
release. Thromb. Res., 37: 513-527, 1985).
[0005] With a similar mechanism hirudin interferes with the binding
of thrombin to the receptors present on endothelial cells and on
fibroblasts (Fenton II J. W., Landis B. H., Walz D. A., Bing D. H.,
Feinman R. D., Zabinski M. P., Sonder S. A., Berliner L. J. and
Finlayson J. S.: Human thrombin: Preparative evaluation, structural
properties and enzymic specificity. In: DH Bind. Ed.: The Chemistry
and Physiology of Human Plasma, Pergamon Press, New-York, pp.
151-182, 1979).
[0006] Hirudin also inhibits interaction between thrombin and
thrombomodulin, which binds to a common site on thrombin, distinct
from the catalytic site (Holfsteenge J., Taguechi H. and Stone S.
R.: Effect of thrombomodulin on the kinetics of the interaction of
thrombin with substrates and inhibitors. Biochem. J. 237:243-251,
1986).
[0007] Hirudin therefore reduces the capacity of thrombin to
activate protein C, a serine-protease able to inactivate factors V
and VIII, necessary for the generation of thrombin itself.
[0008] Hirudin is also able to inhibit clot-bound thrombin which is
protected from inhibition by the heparin-antithrombin III complex
(Weitz II., Hudoba M., Massel D., Maraganore J. and Hirsh J.:
Clot-bound thrombin is protected from inhibition by
heparin-antithrombin III but is susceptible to inactivation by
antithrombin III independent inhibitors. J. Clin. Invest., 86:
385-391, 1990).
[0009] In experimental studies in animals, hirudin proved to be
effective in preventing venous and arterial thrombosis (Markwardt
F.: Development of hirudin as an antithrombotic Agent. Seminars in
Thrombosis and Haemostasis, 15 (3): 269-282, 1989; Heras M.,
Chesebro J. H., Webster M., Mruk J. S., Grill D. E., Penny W. J.,
Bowied E. J. W., Badimon L. and Fuster V.: Hirudin, Heparin and
Placebo during arterial injury in the Pig. The in vivo role of
Thrombin in Platelet. Mediated Thrombosis. Circulation 82:
1476-1484, 1990), vascular shunt occlusions (Kelly A. R., Marzek U.
M., Krupski L., Bass A., Cadroy Y., Hauson S. R., and Harker L. A.:
Hirudin Interruption of Heparin-Resistant Arterial Thrombus
Formation in Baboons. Blood, 77 (5): 1006-1012, 1991) and
thrombin-induced disseminated intravascular coagulation (Markwardt
F., Nowak H. and Hoffmnan J.: The influence of drugs on
disseminated intravascular coagulation DIC. Effects of naturally
occurring and synthetic thrombin inhibitors. Thromb. Res. 11:
275-283, 1977).
[0010] Although the clinical application of hirudin as an
antithrombotic drug was proposed several years ago, this has been
severely limited because natural hirudin is not easily available.
Nowadays, with genetic engineering methodology and methods of
polypeptide purification available, it is possible to produce
sufficient amounts of hirudin for preclinical and clinical studies.
This fact has renewed interest in natural thrombin inhibitors.
[0011] The purification and characterization of different hirudins
from the leech Hirudo medicinalis was studied in detail and the
primary structures of these compounds were determined (Tripier D.:
Hirudin, A family of iso-proteins, isolation and sequence
determination of new hirudins. Folia Hematol. (Leipz) 115:30-35,
1988). In particular, a form of hirudin designated HV1 was
extracted from the whole body of the leech (Boskova I. P.,
Cherkesova D. V. and Mosolow V. V.: Hirudin from leech heads and
whole leechs and pseudo-hirudin from leech bodies. Thromb. Res.
30:459-467, 1983; Dodt J. P., Muller H. P., Seemuller V. and Chang
J. Y.: The complete amino acid sequence of hirudin, a
thrombin-specific inhibitor. FEBS Lett. 165:180-183, 1984) whereas
from the head another form was extracted with a slightly different
amino acid sequence, designated HV2 (Harvey R. P., Degryse E.,
Stefani L., Schamber F., Cazenave J. P., Courtney M., Ialstoshev P.
and Lecocq J. P.: Cloning and expression of cDNA coding for the
anticoagulant hirudin from the blood sucking leech, Hirudo
Medicinalis, Proc. Natl. Acad. Sci USA 83:1084-1088, 1986). A third
variant designated HV3 has been described (Harvey et al, Proc Natl.
Acad. Sci. USA (1986) 1084-1088).
[0012] The amino acid sequences of HV1, HV2 and HV3 are SEQ ID Nos.
1, 2 and 3. HV1 and HV2 consist of a single polypeptide chain of 65
amino acids in which the NH.sub.2-terminal apolar core and the
strongly acid C-terminal tail bind to the apolar binding site and
to the anion binding exosite of thrombin, thus preventing it from
interaction with the substrate (fibrinogen) (Rydel T. J.,
Ravichandran K. G., Tulinsky A., Bode W., Huber R., Roitsch C. and
Fenton II J. W.: The Structure of a Complex of Recombinant Hirudin
and Human .alpha.-Thrombin. Science, 249:277-280, 1990). In
addition, in position 63 a sulfated tyrosine is present; this
post-translational modification does not appear to be essential for
antithrombin activity (Stone S. R. and Hofsteenge J.: Kinetics of
the inhibition of thrombin by hirudin. Biochem., 25: 4622-4628,
1986).
[0013] HV3 is identical to HV2 from positions 1 to 32 and then
differs from HV1 in the following respects: Gln at position 33
instead of Asp, Lys at position 35 instead of Glu, Asp at position
36 instead of Lys, Gln at position 53 instead of Asp, Pro at
position 58 instead of Glu, Asp at position 62 instead of Glu, Ala
at position 63 instead of Tyr (SO.sub.3H), Asp at position 64
instead of Leu and Glu at position 65 instead of Gln.
[0014] The N-terminal domain (residues 1 to 39) of hirudin is
characterized by three disulfide bonds which stabilize the entire
conformation (Chang J. Y.: Production, Properties and Thrombin
Inhibitory Mechanism of Hirudin Amino-terminal Core Fragments. J.
Biol. Chem., 265 (36): 22159-22166, 1990).
[0015] Recently, hirudins have also been detected in other species
of leeches. In particular, two polypeptides with antithrombin
properties similar to those of hirudin HV1 and having the amino
acid sequences depicted in SEQ ID NOs 4 and 5 (referred to
hereinafter as P1 and P2) were isolated from the leech Hirudinaria
manillensis, characterized and produced by genetic engineering
(European Application No. 92301721.4). P1 shows 60% homology with
hirudin HV1 and does not possess sulfated amino acids.
[0016] The isolation and purification of hirudin from natural
sources or from recombinant biological material is generally
carried out by ion exchange chromatography followed by a series of
separations through high pressure liquid chromatography (HPLC) on
reverse phase columns and on ion exchange columns (Courtney M.,
Loison G., Lemoine Y., Riehl-Bellon N., Degryse E., Brown S. W.,
Cazenave J. P., Defreyn G., Delebassee D., Bemat A., Maffrand J. P.
and Roitschl.: Seminars in Thrombosis and Haemostasis, 15(3):
288-292, 1989).
[0017] Natural and synthetic genes coding for hirudins have been
expressed in Escherichia coli and in yeast (Harvey R. P., Degryse
E., Stefani L., Schamber F., Cazenave J. P., Courtney M.,
Ialstoshev P. and Lecocq J. P.: Cloning and expression of cDNA
coding for the anticoagulant hirudin from the blood sucking leech,
Hirudo medicinalis, Proc. Natl. Acad. Sci USA 83:1084-1088, 1986;
Bergmann C., Dodt., Kohler S., Fink E. and Cjassen H. J.: Chemical
synthesis and expression of a gene coding for hirudin, the
thrombin-specific inhibitor from the leech Hirudo medicinalis.
Biol.-Chem. Hoppe Seyler, 367:731-740, 1986; Fortkamp E., Rieger
M., Heisterberg-Moutses G., Schweizer S., and Sonnmer R.: Cloning
and expression in Escherichia coli of a synthetic DNA for hirudin,
the blood coagulation inhibitor in the leech. DNA, 5:511-517,
1986).
[0018] The resulting unsulfated proteins show biochemical and
biological properties very similar to the natural proteins (Talbot
M.: Biology of Recombinant Hirudin (CGP 39393): A New Prospect in
the treatment of Thrombosis. Seminars in Thrombosis and
Haemostasis, 15 (3): 293-301, 1989; Courney M., Loison G., Lemoine
Y., Riehl-Bellon N., Degryse E., Brown S. W., Cazenave J. P.,
Defreyn G., Delebassee D., Bemat A., Maffrand J. P. and Roitschl.:
Seminars in Thrombosis and Haemostasis, 15 (3): 288-292, 1989).
[0019] With the recombinant proteins it was possible to carry out
more extensive preclinical and clinical studies which indicate a
low toxicity and immunogenicity of the protein.
[0020] The in vivo anticoagulant efficacy of hirudin depends mainly
on the maintenance and control of proper levels in the blood. The
knowledge of its pharmacokinetics is hence an essential
prerequisite for its use as an antithrombotic agent in human. In
the course of preliminary studies, Markwardt et al. were able to
measure hirudin in biological samples exclusively on the basis of
its antithrombin activity assessed in a coagulation test (Markwardt
F., Nowak G., Sturzebecher J., Griessbach V., Walsmann P. and Vogal
G.: Pharmacokinetics and anticoagulant effect of hirudin in man,
Throm. Hemost. 52:160-163, 1984) or using a thrombin-specific
chromogenic substrate (Griessbach V., Sturzebecher J. and Markwardt
F.: Assay of hirudin in plasma using a chromogenic thrombin
substrate. Thromb. Res. 37:347-350, 1985).
[0021] Because of the very low immunogenicity of hirudin, it is
extremely difficult to raise precipitating antibodies in
experimental animals (Bicher J., Germmerli R. and Fritz H.: Studies
for revealing a possible sensitization to hirudin after repeated
intravenous injections in baboons. Thromb. Res., 61:39-51,
1991).
[0022] After various attempts, Spinner et al. (Spinner S., Stoffler
G. and Fink E.: Quantitative Enzyme-Linked Immunosorbent Assay
(ELISA) for Hirudin. J. Immunol. Methods, 87:79-83, 1986) were able
to obtain antibodies, using natural hirudin, in 2 out of 11
immunized goats. These antibodies enabled the development of an
ELISA method for the determination of natural and recombinant
hirudin.
[0023] Recently, monoclonal antibodies were obtained directed
against recombinant hirudin (HV1 variant) and against synthetic
peptides representing the C-terminal sequence of HV1 (Schlaeppi J.
M., Vekemans J., Rink H. and Chang J. Y.: Preparation of monoclonal
antibodies to hirudin and hirudin peptides. Eur. J. Biochem.,
188:463-470, 1990; Mao S. J. T., Yates M. T., Owen T. J. and
Krstenansky J. L.: Preparation of antibodies to a synthetic
C-terminus of Hirudin and identification of an antigenic site. J.
Immunol. Methods 120: 45-50, 1989). These antibodies were
especially used to identify the regions of hirudin involved in the
interaction with thrombin. However, their low or even absent
reactivity with natural or recombinant hirudin may limit their use
in pharmacokinetic studies.
[0024] The immunopurification and characterization of the different
forms of hirudin has never been performed since specific antibodies
are not available in sufficient amounts. The development of
immunological methods for the identification, purification and
quantitative determination of hirudins and hirudin-like proteins
has been until now hindered by the extremely low immunogenicity of
these compounds.
[0025] Thus, the present invention seeks to provide an effective
immunogen capable of reliably raising antibodies against a hirudin
or a hirudin-like protein in an experimental animal. The invention
further seeks to provide a reproducible method of producing
antibodies specific for a hirudin or a hirudin-like protein.
[0026] Accordingly, the invention provides a method of producing an
immunogen, which method comprises a polymerising a hirudin and/or a
hirudin-like protein. The invention also provides an immunogen
comprising a polymerised hirudin and/or hirudin-like protein. The
invention further provides a method of producing an antibody
capable of binding to a hirudin or hirudin-like protein, said
method comprising raising antibody against such an immunogen.
[0027] The term "a hirudin" as used in herein includes any protein
having the sequence of a naturally occurring hirudin isoform, such
as HV1, HV2, HV3, P1 or P2. The term "hirudin-like protein"
includes any derivative of a hirudin, e.g. by way of amino acid
substitution, deletion, insertion, extension, functionalisation or
chemical modification, said derivative having anti-thrombin
activity. The term "hirudin-like protein" also includes hybrids of
more than one hirudin, which may be produced by genetic
engineering. For example, WO-A-91/17250 describes a hirudin-like
protein composed of the first 46 residues of HV1 followed by amino
acids 47 to 65 of HV2.
[0028] A hirudin-like protein may be a derivative of HV1, HV2, HV3,
P1 or P2 by way of amino acid substitution, deletion, insertion,
extension, functionalisation or chemical modification, said
derivative having anti-thrombin activity. A substitution, deletion,
insertion or extension may comprise one or more amino acid(s).
Typically there is a degree of homology of 60% or more between the
amino acid sequence of HV1, HV2, HV3, P1 or P2 and the amino acid
sequence of the derivative thereof. The degree of homology may be
75% or more, 85% or more, or 95% or more.
[0029] The physicochemical character of the original sequence can
be preserved, i.e. in terms of charge density,
hydrophobicity/hydrophilicity- , size and configuration. Candidate
substitutions are, based on the one-letter code (Eur. J. Biochem.
138, 9-37, 1984):
[0030] A for G and vice versa,
[0031] V by A, L or G;
[0032] K by R;
[0033] S by T and vice versa;
[0034] E for D and vice versa; and
[0035] Q by N and vice versa.
[0036] As far as extensions are concerned, a short sequence of up
to 50 amino acid residues may be provided at either or each
terminal. The sequence may have up to 30, for example up to 20 or
up to 10 amino acid residues.
[0037] The hirudin or hirudin-like protein may be subjected to one
or more chemical modification(s) (e.g. by post-translational
modification), such as glycosylation, sulphation, COOH-amidation or
acylation. The Tyr residue at position 63, for example, may be
sulphated. A recombinant hirudin or hirudin-like polypeptide would
not normally be sulphated at this position. Further, the invention
may be applied to lower molecular weight derivatives which do not
have the N-terminal or C-terminal portions of HV1, HV2HV3, P1 or
P2.
[0038] The immunogen of the invention is a polymer of a hirudin
and/or a hirudin-like protein. Thus, the immunogen may be a
homopolymer of a hirudin, a homopolymer of a hirudin-like protein
or a copolymer of a hirudin and a hirudin-like protein. The
immunogen may also be a copolymer of more than one hirudin isoform
or more than one hirudin-like protein.
[0039] The hirudin and/or the hirudin-like protein are/is suitably
polymerised with glutaraldehyde. A molar excess of glutaraldehyde
is suitably used. A 1.1 to 5-fold molar excess can be employed.
[0040] The polymerization reaction is carried out in the presence
of an aqueous solvent. Suitable solvents include phosphate-buffered
saline (PBS). The time duration of the reaction is suitably 5 h to
30 h, preferably 15 h to 20 h. The temperature of the reaction is
suitably 15.degree. C. to 35.degree. C., for example from ambient
temperature to 30.degree. C. From the viewpoint of convenience,
ambient temperature is preferred.
[0041] Methods for obtaining the hirudin and/or the hirudin-like
starting material are known. These methods include extraction of
hirudins from leeches and production of recombinant hirudins and
hirudin-like proteins by genetic engineering. Hirudins and
hirudin-like proteins, particularly HV1, HV2, HV3 and derivatives
thereof, may be obtained in accordance with WO-A-91/17250. The
method of WO-A-91/17250 is based on chemical synthesis of a
nucleotide sequence encoding a hirudin or a hirudin-like
polypeptide, and expressing the polypeptide in a recombinant
organism. P1 and P2 may be isolated from tissue of the leech Hirudo
manillensis by obtaining a preparation according to WO-A-90/05143
and subjecting the preparation to high pressure liquid
chromatography (European Application No. 92301721.4).
[0042] The invention provides a method for producing an antibody
capable of binding to a hirudin or a hirudin-like protein, said
method comprising raising antibodies against an immunogen according
to the invention. The antibody produced may be monoclonal or
polyclonal.
[0043] Methods of producing monoclonal and polyclonal antibodies
are well known. A method for producing a polyclonal antibody
comprises immunising a suitable host animal, for example an
experimental animal, with an immunogen of the invention and
isolating immunoglobulins from the serum. The animal may therefore
be inoculated with the immunogen, blood subsequently removed from
the animal and the IgG fraction purified. A method for producing a
monoclonal antibody comprises immortalising cells which produce the
desired antibody. Hybridoma cells may be produced by fusing spleen
cells from an inoculated experimental animal with tumour cells
(Kohler and Milstein, Nature 256, 495-497, 1975).
[0044] An immortalized cell producing the desired antibody may be
selected by a conventional procedure. The hybridomas may be grown
in culture or injected intraperitoneally for formation of ascites
fluid or into the blood stream of an allogenic host or
immunocompromised host. Human antibody may be prepared by in vitro
immunisation of human lymphocytes, followed by transformation of
the lymphocytes with Epstein-Barr virus.
[0045] For the production of both monoclonal and polyclonal
antibodies, the experimental animal is suitably a goat, rabbit, rat
or mouse. If desired, the immunogen of the invention may be
administered as a conjugate in which the immunogen is coupled, for
example via a side chain of one of the amino acid residues, to a
suitable carrier. The carrier molecule is typically a
physiologically acceptable carrier. The antibody obtained may be
isolated and, if desired, purified.
[0046] The invention includes a method for the quantitative
determination, purification or identification of a hirudin or a
hirudin-like protein said method comprising raising antibodies
against an immunogen according to the invention and using the
antibody thereby obtained in said quantitative determination,
purification or identification.
[0047] Conventional methods for the quantitative determination of
an antigen in a sample using an antibody may be used. For example,
an antibody produced in accordance with the invention may be used
in an ELISA (enzyme-linked immunoassay) method such as a
non-competitive ELISA.
[0048] Typically, an ELISA method comprises the steps of:
[0049] (i) immobilising on a solid support an unlabelled antibody
produced in accordance with the invention,
[0050] (ii) adding a sample containing the hirudin and/or
hirudin-like protein to be determined such that the hirudin and/or
hirudin-like protein is captured by the unlabelled antibody,
[0051] (iii) adding an antibody produced in accordance with the
invention which has been labelled, and
[0052] (iv) determining the amount of bound labelled antibody.
[0053] Examples of suitable antibody labels include biotin (which
may be detected by avidin conjugated to peroxidase) and alkaline
phosphatase. The sample containing the hirudin and/or hirudin-like
proteins may be biological fluid (e.g. a leech extract), tissue
culture medium from a culture of either non-transferred or
transformed cells, or a sample produced by recombinant DNA
procedures.
[0054] Conventional methods of purifying an antigen using an
antibody may be used. Such methods include immunoprecipitation and
immunoaffinity column methods. In an immunoaffinity column method,
an antibody produced in accordance with the invention is coupled to
the inert matrix of the column and a sample containing the hirudin
and/or hirudin-like protein to be purified is passed down the
column, such that the hirudin and/or hirudin-like protein is
retained. The hirudin and/or hirudin-like protein is then
eluted.
[0055] The sample containing the hirudin or hirudin-like protein
may be biological fluid (e.g. leech extract) tissue culture medium
from a culture of transformed or non-transformed cells, or a sample
produced by recombinant DNA procedures.
[0056] Conventional methods for identifying an antigen with an
antibody may be used. For example, a Western blotting method may be
used. Such a method can comprise the steps of:
[0057] (i) subjecting a sample containing a hirudin and/or a
hirudin-like protein to gel electrophoresis,
[0058] (ii) transferring the separated proteins in the gel onto a
solid support (e.g. a nitrocellulose support) by blotting, and
[0059] (iii) allowing an antibody produced in accordance with the
invention and which has been labelled to bind to the hirudin and/or
hirudin-like protein.
[0060] The sample containing the hirudin and/or hirudin-like
protein may be biological fluid, (e.g. leech extract), tissue
culture medium from a culture of transformed or non-transformed
cells, or a sample produced as a result of recombinant DNA
procedures.
[0061] The following Examples illustrate but do not in any way
limit the invention. In the accompanying drawings:
[0062] FIG. 1 shows a sodium dodecyl sulfate polyacrylamide
electrophoresis (SDS-PAGE) gel of hirudin HV1 polymers obtained
after reaction of HV1 and glutaraldehyde. Lane A is loaded with
hirudin (20 .mu.g) and lane B is loaded with the HV1 polymers (20
.mu.g). The numbers on the left of the gel are molecular weights
(kD).
[0063] FIG. 2 shows an immunodiffusion agarose gel according to
Ouchterlony. Well A contains immune serum diluted 1:16, well B
contains buffer, and wells C, D and E contain 2.5, 5 and 10 .mu.g
of hirudin respectively. The gel demonstrates the presence of
precipitating antibodies in blood obtained form inoculated
rabbits.
[0064] FIG. 3 shows a standard curve for HV1 hirudin of absorbence
at 490 nm (.times.1000) (x-axis) versus concentration of hirudin
(ng/ml) in buffer (--.quadrature.--), plasma (--.diamond-solid.--)
and urine (--.box-solid.--) (y-axis). Each point on the curve
represents the mean of five measurements of absorbance at 490 nm
and the bars represent standard deviations.
[0065] FIG. 4 shows the specificity of the anti-hirudin antibodies
obtained in Example 2 for natural HV1 (--.quadrature.--) and
recombinant HV2 (--.smallcircle.--) but not P1 (--.box-solid.--).
The x-axis is concentration of hirudin (ng/ml) and the y-axis is
absorbance at 490 mn.
EXAMPLE 1
[0066] Procedure for Making the Antigen Immunogenic
[0067] HV1 hirudin, obtained in Escherichia coli by recombinant DNA
technology, was dissolved at a concentration of 6 mg/ml in PBS pH
7.3 containing 1.25% glutaraldehyde and incubated for 18 hours at
room temperature. After dialysis against 0.9% NaCl the formation of
hirudin polymers was checked by 15% polyacrylamide gel
electrophoresis (SDS-PAGE).
[0068] FIG. 1 shows the polymers formed after the reaction. The
band corresponding to hirudin (A) disappears whereas 7 compounds
(B) appear with molecular weights corresponding to multiples of
that of the starting hirudin.
[0069] With a similar procedure it is possible to prepare
immunogenic polymers of other forms of hirudin, for example HV2 and
P1.
EXAMPLE 2
[0070] Induction and Isolation of the Antibody
[0071] 5 New Zealand white, male rabbits were inoculated
subcutaneously with 250 .mu.g of HV1 hirudin polymer re-suspended
in 2 ml of Freund's complete adjuvant. Booster injections with
Freund's incomplete adjuvant were give after 4, 6, 8 and 10 weeks.
At week 12, 30 ml of blood were taken from each animal and the
presence of precipitating antibodies was determined by the double
diffusion technique in agarose gel according to Outcherlony
(Outcherlony O.: Diffusion in gel methods for immunological
analysis. Progr. Allergy, 5.1, 1958). Five wells were obtained form
a 1% agarose gel in 0.15 M PBS pH 7.4 as shown in FIG. 2.
[0072] 35 .mu.l of immune serum diluted 1:16 in PBS was loaded in
well A, whereas in wells B, C, D, E, buffer, 2.5, 5 and 10 .mu.g of
HV1 hirudin, respectively were loaded. After 48 hours incubation at
room temperature in a moist chamber the gel was washed for 24 hours
with normal saline and distilled water, dried at 40.degree. C. and
stained with Coomassie blue 0.2% in 50% trichloroacetic acid.
[0073] FIG. 2 shows well-defined precipitation bands between the
central well A and wells C, D, E in which hirudin was placed.
[0074] In a parallel experiment in which non immune rabbit serum
was placed in well A, no precipitation bands were observed, whereas
in this experiment four out of the 5 immunized rabbits showed the
pressure of precipitating antibodies.
[0075] The IgG fraction was prepared by protein A-sepharose
chromatography. Serum diluted 1:2 in PBS pH 7.4 was loaded on a
protein A-sepharose column equilibrated with PBS pH 7.4. The IgG
were subsequently eluted with 0.1 M citrate buffer pH 3. Using a
similar procedure it is possible to prepare polyclonal antibodies
for other forms of hirudin, for example HV2 and P1.
EXAMPLE 3
[0076] Immunoenzymatic Determination of the Antigen using the ELISA
Method
[0077] A part of the anti HV1 IgG isolated in Example 2 was
biotinylated according to the method of Guesdon et al (Guesdon J.
L. et al.: The use of avidin-biotin interaction in immunoenzymatic
techniques. J. Histochem. Cytochem., 27: 1131-1139, 1975).
[0078] The following scheme was followed for the ELISA test. Rabbit
polyclonal anti HV1 IgG were immobilized in the solid phase for
capturing the antigen (HV1) contained in the samples which was then
tagged with rabbit polyclonal biotinylated IgG anti HV1. Detection
was carried out with avidin conjugated to peroxidase. More
precisely: 150 .mu.l of polyclonal IgG, 10 .mu.l/ml in 0.1 M
Na.sub.2CO.sub.3 pH 9.6 were placed in microplate wells and
incubated for 16 hours at 4 .degree. C. The concentration of IgG
was chosen so as to optimize ELISA and obtain high sensitivity and
low background.
[0079] The plates were washed and the remaining binding sites
blocked by incubation for one hour with PBS pH 7.4 containing 3%
bovine albumin. After washing, the plates were incubated for 60
minutes at 37.degree. C. either with PBS-Tween pH 7.4 or with
plasma or urine, containing increasing concentrations of standard
hirudin or samples at unknown concentrations. After washing with
PBS-Tween pH 7.4 the plates were incubated for 60 minutes at
37.degree. C. with 5 .mu.g/ml of biotinylated IgG, washed again and
incubated for 60 minutes at 37.degree. C. with 158 ng/ml of
avidin-peroxidase. The plates were then washed three times with
PBS-Tween pH 7.4 and once with bidistilled water. Peroxidase was
allowed to react for 15 minutes at room temperature with 0.1%
o-phenylendiamine, 0.015% H.sub.2O.sub.2 in 0.1 M citrate buffer pH
5. The reaction was stopped by adding 4.5 M H.sub.2SO.sub.4 and
absorbence was measured at 490 nm by a microplate reader.
[0080] A dose-dependent linear relationship was obtained between
0.61 and 10 ng/ml with a limit of detection of 0.3 ng/ml (FIG. 3).
The coefficient of variation between the dosages was established on
the basis of 7 different doses and ranged between 3.89% and
6.28%.
[0081] The specificity of the anti HV1 antibodies isolated in
Example 2 was tested by ELISA against natural HV1 hirudin,
recombinant HV2 hirudin and P1 (FIG. 4).
[0082] The antibodies recognize natural HV1 hirudin and the HV2
variant but not P1 which shows 60% homology with HV1 hirudin.
Antibodies able to recognize hirudins, in particular P1, can be
obtained, as already said, by a procedure similar to that described
in Example 2 and used, for example in an ELISA method similar to
that described in Example 3, for the determination of hirudins, in
particular P1.
Sequence CWU 1
1
* * * * *