PL164991B1 - Method of producing polyclonal antibodies specifically reacting with protein of the human tissue inhibitor of plasminogen activator - Google Patents

Abstract

Process for producing polyclonal antibodies reacting specifically with the protein of the human tissue plasminogen activator inhibitor, by immunising animals, characterised in that the polypeptide equal to a fragment with formula (Y)a-A-(Z)b, is used for immunisation, where Y denotes amino acid sequence coded by the polylinker fragment, optimally by Glu, Ser, a=0 or 1; Z denotes sequence Glu, Ser and/or sequence coded by the polylinker fragment, b=0, 1 or 2, while A denotes sequence corresponding with fragment Asn172-Thr232 of the human tissue plasminogen activator inhibitor, while blood is taken and antibodies are isolated.

Description

The present invention relates to a method of producing polyclonal antibodies specifically reactive with a human tissue plasminogen activator inhibitor (PAI-1) protein by immunizing animals, collecting blood and isolating antibodies.

Plasminogen activators (PAI-1) play an important role in the process of fibrinolysis, as well as in various other physiological processes, including regeneration, ovulation, embryo implantation, tissue differentiation, and neoplastic transformation. Precise activity regulation

164,991 plasminogen activators are critical components of many biological systems. Such regulation can take place at the level of clot formation and dissolution, or at the level of interaction of plasminogen activators with cells, or by the action of specific inhibitors.

Most PAI activity determinations are two-step, depend on the presence of plasmin and do not distinguish between different types of inhibitors. Determination of the inhibitory activity of PAI is quantified by the ability to inhibit the lysis of ¹⁷⁵ J-labeled fibrin mediated by urokinase activation of plasminogen (Loskutoff and Edington, Proc. Nat. Acad. Sci., USA, 74, 3903-3909, 1977). In this method, one unit of PAI activity was defined as the amount of protein required to reduce the 21U UK activity to 50%. The method of binding the plasminogen activator to the inhibitor is used to quantify the amount of PAI (Schleef RR, Wagner NV, Loskkutoff DJ, J. of Cellular Physiology, 134, 269-274, 1989). The activity of PAI can also be determined using the Verheijen method, in which increasing concentrations of tPA (0-25 IU / ml), 1 (µM of plasminogen, and 1 μΜ of fibrinogen degraded by cyanogen bromide) are used. Under these conditions, 1 IU of PAI-1 determines the amount of the inhibitor. which inhibits 1 IU of tPA (Verheijen JH, Mullast D., Chang GTG, Kluft C., Wijngards G., Thromb. Haemostasis, 48, 266-269, 1982). based on the neutralization of tPA added to the plasma samples, and the residual activity of tPA is measured These methods are indirect methods and do not accurately determine the level of this inhibitor.

Recently, an enzyme-immunological method for the determination of human PAI-1 in ELIS A text using antibodies to this protein has been developed. In this test, the antibodies to PAI-1 are incubated with the test sample in which this antigen is determined, and after washing the non-specific bound proteins, the antigen-antibody complex is incubated with the antibodies to PAI-1 bound to horseradish peroxidase, and then the antigen is determined by detecting the label.

All these methods require the use of the native PAI-1 protein, both for the standard curve and for specific antibodies. PAI-1 is present in trace amounts in body fluids and cells, and obtaining this protein by classical methods by isolating it in amounts sufficient to prepare the Elisa test is extremely complicated and time-consuming. On the other hand, the human PAI-1 protein obtained so far is extremely expensive and not very accessible.

All the discussed methods of obtaining and determining the native PAI-1 are very complicated, time-consuming and expensive, which significantly increases the costs of the entire test.

Surprisingly, it turned out that it is possible to determine the native PAI-1 protein on the basis of the method in which the antibodies according to the invention are used. It has been found that a complete cross-reactivity can be used in the immunoassay of the antibodies obtained for the protein fragment and the native PAI-1 molecule.

In contrast to the known methods of PAI-1 determination, it was surprisingly found that when the antibodies obtained by the method according to the invention are used, the peptide fragment, and not the whole protein, serves to produce specific antibodies that recognize both the peptide fragment and the native PAI protein in the same way. -1. This fragment is also used to obtain the standard curve.

The method of producing polyclonal antibodies specifically reactive with the protein of the human tissue plasminogen activator inhibitor by immunizing the animals consists in that for immunization a polypeptide corresponding to the fragment of formula (Y) aA- (Z) b is used, wherein Y is an amino acid sequence encoded by a polylinker fragment, preferably Glu, Ser; a = 0 or 1; Z represents the sequence Glu, Ser and / or the sequence encoded by the polylinker fragment; b = 0, 1 or 2; and A is the sequence corresponding to the Asn172-Thr232 fragment of the human tissue plasminogen activator inhibitor, blood is drawn and antibodies are isolated.

In the method according to the invention, a polypeptide corresponding to a fragment of the PAI-1 molecule from 172 to 232 amino acids from the N-terminus with the sequence:

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172 177 198

Asn-Gly-Gln-T rp-Lys — Thr — Pro — Phe — Pro — Asp-Ser Ser-Thr-His-Arg 187 192 177

Arg-Leu-Phe — His — Lys — Ser — Asp — Gly-Ser Thr-Val-Ser — Val — Pro — Met— 202 207 212

Met-Ala-Glu-Thr-Asn-Lys-Phe-Asn-Tyr-Thr-Glu-Phe-Thr-Thr-ProAsp — Gly — His — Tyr — Tyr — Asp — Ile — Leu-Glu — Leu-Pro— Tyr — His — Gly — Asp—

Thr.

The method according to the invention uses a polypeptide obtained by expressing recombinant DNA, preferably chemically synthesized cDNA with the sequence:

(X) n AACGGCCAGTGGAAGACACCATTCCCAGACAGTAGCACCCACCGCCGCCTCTT CCACAAATCAGACGGAAGCACTGTCTCTGTGCCAATGATGGCTGAGACCAACAAATT

CAACTATACTGAATTCACCACGCCAGATGGACATTACTACGAGATCCTGGAACTGCC

ATACCACGGAGACACT (X) m, where X is GAATCT, n is 1 or 0 and m is 0, 1 or 2.

The polypeptide used in the method according to the invention is preferably obtained by using a recombinant cloning vector produced by linking the vector by known methods to a DNA fragment encoding a polypeptide of formula (Y) _a -A- (Z) b, in which the symbols have the above meanings. The polypeptide fragment generated in this way contains antigenic epitopes recognized by antibodies also in the native PAI-1 molecule.

For example, the gene used to produce the polypeptide encoding a polypeptide fragment corresponding to a fragment of a human tissue plasminogen activator inhibitor can be obtained from in vitro ligated oligonucleotide fragments.

Oligonucleotide fragments can be chemically synthesized on the support by sequentially adding blocked active group nucleosides. Preferably, an isopropoxyacetyl group is used to protect the amine function, as described in the Polish patent specification No. 159,234.

Chemically synthesized oligonucleotide fragments are subjected to an enzymatic kinase reaction in which phosphate is introduced at the 5 'ends using polynucleotide kinase and ATP. The kinased oligonucleotides are ligated in vitro into double-stranded stretches delimited by restriction enzyme recognition sequences. The ligated segments are cloned into a helper vector using the appropriate polylinker restriction site. The correctness of the sequence of the ligation and cloning products of these fragments is checked by sequencing according to the Maxam-Gilbert method. Various helper vectors, e.g. plasmid or phage vectors, can be used, with plasmids, e.g. pUC19 being preferred. DNA stretches cloned in the vector are multiplied in host cells, which can be bacteria, yeast and others. Vectors with cloned sections are isolated, and the sections are then recovered after digestion with restrictionases by polyacrylamide gel electrophoresis. The recovered segments are incorporated into the expression vector after digestion with the appropriate restriction enzymes. Various expression vectors, preferably plasmid or phage vectors, are used in this step. For example, the pWR450.1 expression plasmid. contains the gene encoding β-galactosidase. Host cells, preferably bacteria, transformed with the recombinant vector, are the source of the human PAI-1 protein fragment after amplification.

The fragment of the PAI-1 protein produced by this method can be used for the production of antibodies according to the invention as well as by various known methods and for the drawing of a standard curve necessary for the quantification of PAI-1.

The antibodies obtained by the method of the invention can be used to determine the antigen of a human tissue plasminogen activator inhibitor and to obtain a diagnostic test for the determination of this antigen.

An exemplary method of determining the antigen of a human tissue plasminogen activator inhibitor is that the test sample, preferably of biological material, is contacted with the antibodies obtained by the method according to the invention for the polypeptide corresponding to the fragment of formula (Y) aA- (Z) b, in which the symbols have the above-mentioned symbols. the meaning followed by determining the antigen-antibody complex produced.

In another exemplary method, the PAI-1 antigen binary complex in the sample is first produced with a first antibody capable of reacting with said antigen, and then the ternary complex is formed by contacting the binary complex with a second antibody capable of reacting with said antigen, one of which is of these antibodies, the antibodies according to the invention are used for the polypeptide corresponding to the fragment of formula (Y) aA- (Z) b in which the symbols have the above meanings. The presence of the ternary complex is detected by a signal generated by an analytically measurable reagent or a labeled anti-immunoglobulin antibody.

A test sample, in which the PAI-1 antigen is detected and determined, is contacted and incubated with antibodies reactive specifically with the antigen. The formation of a binary antigen-antibody complex is detected and determined by an immunological detection and signaling agent such as, for example, an antibody reactive specifically with PAI-1 labeled with an analytically determinable reagent.

An agent for detecting and signaling an immune response may be present when the antigen is incubated with the antibody. For example, the antibodies contacted with the test sample can be directly labeled and signal is obtained after washing the reaction product. The center is then the marker itself. The detection agent can also be added only after the formation of the two-component complex and removal of unreacted material. Detection and determination then proceed as follows.

After washing, the two-component antigen-antibody complex is contacted with a second antibody specifically reactive with PAI-1, one of these antibodies being the antibody to the polypeptide corresponding to the fragment of formula (Y) aA- (Z) b, in which the symbols have the above given meaning. The residual (first or second) antibody can be the antibody to the entire PAI-1 molecule. The ternary antibody-antigen-antibody complex is then obtained. Second antibodies may be directly labeled with an analytically determinable reagent. Alternatively, the agent is a two-component system containing second antibodies forming a ternary complex that is detected by an anti-immunoglobulin antibody.

An enzyme, biotin, radioisotope, for example ¹²⁵ J, or other known reagents can be used for labeling. Detection and determination are carried out by means of the signal produced by the label.

In such a determination or detection process, the test sample is marked with an antigen or one of the antibodies, preferably the first, is immobilized on a solid support, such as a plastic plate, nitrocellulose paper or other.

Detection and determination is conveniently carried out by a diagnostic test. An exemplary diagnostic test comprises antibodies to a polypeptide corresponding to the fragment of formula (Y) a-A- (Z) b, wherein the symbols have the above meanings, optionally

164,991 immobilized on a solid support or dissolved in the liquid phase. The antibodies are bound to an analytically detectable reagent.

Alternatively, the diagnostic assay comprises first antibodies reactive specifically with a human tissue plasminogen activator inhibitor protein, and an antigen-antibody complex detection system comprising second antibodies capable of specifically reacting with a human tissue plasminogen activator inhibitor, and optionally anti-immunoglobulin antibodies, one of these antibodies being antibodies to the polypeptide. corresponding to the fragment of formula (Y) _a -A- (Z) b, wherein the symbols are as defined above. Preferably the first antibodies are bound to a solid or a liquid phase.

The test may additionally contain a solid or a liquid carrier to bind the sample containing the antigen.

The system for detecting an antigen-antibody complex comprises second antibodies directly linked to an analytically detectable reagent, or the system comprises third anti-immunoglobulin antibodies bound to a label.

In the method and assays for determining a human tissue plasminogen activator inhibitor, antibodies to a polypeptide corresponding to a fragment of formula (Y) a-A- (Z) b, wherein the symbols have the above meanings made by various methods.

For simplicity, the following abbreviations are used in the description and examples:

A: adenine

C: cytosine

G: guanine

T: thymine

Ala: alanine

Arg: arginia

Asn: asparagine

Asp: aspartic acid

Cys: cysteine

Gin: glutamine

Glu: glutamic acid

Gly: glycine

His: Histidine

How much: isoleucine

Leu: leucitin

Lys: lysine

Met: methionine

Phe: phenylalanine

Pro: proiina

Cheese: serine

Thr: threonma

Trp: rryptoann

Tyr: yy / rosina

Val: waHna

DNA: deoxyribonucleic acid cDNA: complementary DNA

Tris-HCl: trihydroxymrtrloaminomethane hydrochloride

EDTA: ethylenediaminetroacetic acid

10x Eco R1 buffer 100 mM Tris-HCl pH 7.5

M NaCl mM β-mercaptoethanol

10x kinase buffer 0.7 M Tris HCl pH 7.6

0.1 MMgCl2 50 mM dithiothreitol

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10x ligation buffer: 660 mM Tris-HCl pH 7.6 50 mM MgCl ₂ 50 mM dithiothreitol 10 mM ATP Extraction buffer: 50 mM Tris-HCl pH 8.0 300 mM sodium acetate 0.2% SDS 4mM EDTA LB medium 10 g Bacto Trypton 5 g of Yeast Extract 5 g of NaCl per liter of solution TE buffer 10 mM Tris-HCl pH 8.0 1mM EDTA SDS buffer 10 g Tris-HCl pH 8.0 77 g of glycine 1 g of SDS per liter of solution Cell lysis buffer 150 mg of Tris 400 mg of SDS 1 ml of β-mercaptoethanol 2 ml of gl and ic ^ and ^ c ^ lu 7 ml of water Buffer R I 50 mM glucose 10mM EDTA 25 mM Tris-HCl pH 8.0 4 mg / ml lysozyme (Pharmacia) Buffer R II 0.2 M NaOH 1 <% SDS Buffer R III 60 ml of 5M potassium acetate 11.5 ml glacial acetic acid 28.5 ml of H20

DDT: dithiothreitol

PMSF: phenylmethylsulfonic acid fluoride

SDS: sodium dodecyl sulfate

BSA: Bovine Serum Albumin

PBS: buffered saline solution

ATP: adosine triphosphate

HPLC: high pressure liquid chromatography

EDTA: ethylenediaminetetraacetate

In the accompanying drawings, Figure 1 is a graph of the hydrophobicity of the PAI-1 polypeptide chain. Fig. 2 shows the sequence of the human PAI-1 gene fragment divided into chemically synthesized fragments. Fig. 3 shows a construction diagram of a gene fragment for PAI-1 cloned in the recombinant pUC-19 and expression vector pWR 450. Fig. 4 shows the SDS-polyacrylamide gel electrophoresis of protein extracts obtained from E. coli cells and the purified PAI-1 protein complex from β-galactosidase: 1 - inclusion bodies dissolved in a buffered solution of 6-molar urea, 2 - purified hydrophobic protein β-galactosidase-PAI-1 peptide fragment. Figure 5 shows a scheme for the disruption of the protein complex and the purification of the Asn172-Thr232 fragment of PAI-1. The β-galactosidase stretches shown are 4 to 182 amino acids long. The PAI-1 fragment after disruption of the complex consists of 61 amino acids.

The invention is explained in more detail in non-limiting embodiments.

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Example I.

Level 1.

A. Oligonucleotide synthesis.

The synthesis of 10 oligonucleotide fragments (Fig. 2) is carried out by the phosphoramidite method on a solid support. A controlled pore glass, LCA-CPG type, bonded to the first nucleoside unit is used as the support. This unit has an acid-labile blocking group dimethoxytrityl (DMT) attached to the 5'-OH deoxyribose terminus, which is removed in an acidic environment. The thus exposed 5'-OH group of the nucleoside unit joins the precursor of the next unit: the 3'-phosphoramidite of the corresponding nucleoside. With the help of iodine in the presence of water, the phosphite group is oxidized to phosphate. The DMT is then removed from the attached unit - thereby starting the cycle all over again. To remove unreacted units with free 5'-OH groups, the so-called capping, ie blocking of wrong sequences by acetylation with acetic anhydride in the presence of activators. After formation of the desired oligonucleotide, it is cleaved from the bed and other groups are removed with aqueous ammonia, purified by electrophoresis, then desalted by HPLC. (Waters C-18 RP, 10 gm). Oligonucleotides are stored in aliquots of approx. 0.5 OD.

B. The kinase and ligation process.

The individual fragments of the DNA strand are dissolved in distilled water so that there is 1 gg of the fragment in 5 gL. 5 g of the solution of fragments 1 to 5 (1st strand of DNA) and 5 to ΊΟ (2nd strand of DNA) are mixed. 0.1 volumes of kinase buffer (50 mM Tris-HCl, 5 gM MgCk), 5 gM of 20 gM ATP, 1 g of polynucleotide kinase B (T4 polynucleotide kinase) are added to the fragment mixture. The reaction is carried out for 4 hours at 37 ° C. Both strands are mixed and incubated for 20 min. at 100 ° C. After slow cooling, 0.1 volume of ligation buffer, 1 g and 200 mM ATP, 1 g of T4 ligase are added to each sample. After mixing thoroughly, the reaction is run for 12 hours at 4 ° C.

C. Hydrolysis with enzymes.

To the dissolved DNA (80 µl) is added 10 × 10 µl of EcoRi buffer, 2U of EcoRi restrictionase (from Pharmacia) and water to a volume of 100 µl. The mixture is incubated at 37 ° C for 2 hours. After this time, the DNA was precipitated with ethanol by adding 3 volumes of ethyl alcohol (99.8%), frozen at -20 ° C for 5 minutes, and then centrifuged at 15,000. rotate for 5 minutes. The pellet is dissolved in 20 gL of TE buffer. The same is done for the hydrolysis with Pst I-restrictionase.

D. Electrophoretic separation.

In order to check that the ligated DNA fragments are of the correct length, they are separated on an 8% SDS polyacrylamide gel in TBE. The separation is carried out at a voltage of 130 V for 3 hours. The gel is stained with ethidium bromide (0.5 g / ml - by Serva) for 20 minutes at room temperature, then rinsed 2x with distilled water. The separated products are observed under a UV lamp. The following standards were used to estimate the size of the DNA fragments during electrophoresis: pUC digested with Msp, pUC digested with Bsp RI. The ligated fragments are of the expected length. After checking electrophoresis, preparative electrophoresis is performed, and a fragment is obtained this way, which is then ligated with the pUC plasmid digested with Eco RI and Pst I as described above.

E. Preparation of competent cells.

One colony is picked from the DH5 plate and inoculated onto 5 ml of Lb medium. The bacteria are grown at 37 ° C for 18 hours. 2 ml of the obtained culture are added to 100 ml of LB medium and the culture is cultivated to an optical density of OD 600 = 0.3. The bacterial suspension is centrifuged (5,000 rotations, 20 minutes), and the bacterial pellet is cooled and suspended in 50 ml of SB buffer and placed at 0 ° C. It is centrifuged as above and the pellet is suspended in 1/12 of the original volume in SB buffer. Incubation is made for 20 minutes at 0 ° C. After this time, 200 µl of the cell suspension are withdrawn from the suspended cells into sterile tubes and incubated for 30 minutes at 0 ° C. 7 µl of DTT and plasmids (20 µl) are added. Incubation on ice (0 ° C) for 30 minutes followed by 45 seconds at 45 ° C. It is cooled in ice and added 800 g

164,991 LB media, then incubated for 1 hour at 37 ° C. After this time, it is seeded into plates. 200 µL of the ligation mixture is poured onto the plate and incubated at 37 ° C for 18 hours.

F. Analysis of the obtained transformants.

Single colonies are observed on the plate with the inoculated transformation mixture after 18 hours. 10 colonies are selected for further analysis. 5 ml of medium is inoculated with single colonies and cultured at 37 ° C for 18 hours, followed by isolation of plasmid DNA. Preparation of plasmid DNA from 5 ml of medium is as follows.

The bacteria are swirled (5 minutes 5 rpm). The bacterial pellet is resuspended in 200 µΐ RI buffer and kept for 5 minutes at 0 ° C. 400 µΐ RII (0 ° C) is added followed by 300 µΐ RIII (0 ° C). The whole thing is spinning (15,000 rpm, 5 minutes). The supernatant is transferred from the pellet to a new tube and 2.5 volumes of isopropanol are added. It is frozen at -20 ° C, centrifuged, rinsed with 70% ethanol and dried in a vacuum. The pellet is dissolved in 200 µΐ TE and 20 µl of a 1 mg / ml RNase solution is added. Incubation is 30 minutes at 37 ° C. 200 µl of phenol are added. The aqueous phase is collected and 200 µl of a mixture of chloroform / isoamyl alcohol (24: 1, v / v) are added. The supernatant is swirled, the activity is repeated once more. 0.1 volumes of 3M sodium acetate (pH = 5.2) and 2.5 volumes of ethanol are added to the aqueous phase and frozen for 30 minutes at -20 ° C. It is swirled at (15,000 rpm), and the precipitate is washed with 70% ethanol and dried. The plasmid DNA pellet is dissolved in 40 µl TE buffer.

In order to select a clone containing the pUC 19 plasmid with the cloned double-stranded fragment, analysis of the obtained plasmid dNa is performed with the restriction enzymes EcoRI and Pst I, and the digests are separated on a polyacrylamide gel. Etching is performed as in point C and electrophoretic separation as in D.

G. Check the sequence of cloned fragments.

In order to ascertain whether the sequence of the cloned fragment is correct, the fragments are sequenced by the method of Maxam and Gilbert (Maxam & Gilbert, Methods in Enzymology, 1980, 65).

H. Construction of the expression vector.

A verified double-stranded DNA fragment encoding the Asnm-Thr232 polypeptide is ligated into the pWR.450 expression vector digested with the restriction enzymes EcoRI and Pst I. A recombinant expression plasmid pW-PAI-1 is obtained. The transformation of bacterial cells is carried out as in point E.

I. Purification of inclusion bodies.

Bacteria from the bacterial colony containing the recombinant plasmid are transferred to a tube containing 1 ml LB medium + 5 μl ampicillin at a concentration of 100 pg / ml and incubated for

2-5 hours at 37 ° C with constant shaking. The suspension of bacteria grown in this tube is transferred to a flask containing 1 L of LB medium + 1 ml of ampicillin at a concentration of 100 μg / ml. The cultivation is carried out for 14 hours at 37 ° C with constant shaking. The bacteria are then centrifuged (15 min, 5000 rpm) and the pellet resuspended in 200 ml 0.1 M CaCl2 (Tris buffered, pH = 7.5). After incubating for 20 minutes in an ice bath, the suspension is centrifuged for 20 minutes at 12,000 rpm. at + 4 ° C and the pellet is resuspended in 200 ml 0.1 M CaCl2. After another incubation for 2 hours in an ice bath, the suspension is centrifuged (in two centrifuge tubes) as above. The pellet is suspended in 20 ml (20 ml are added to both tubes) of phosphate buffer, pH 6.2, and then 200 μΐ 1 M glycerol and 400 μΐ 1% glycerol are added to both tubes. They are warmed to room temperature, 40 µL each of 0.5 M EDTA, pH 8.0 is added and, after a short incubation, cooled in an ice bath to about 0 ° C. The test tubes are then placed in a 42 ° C bath (thermal shock) for 1 minute. Whirls as above. After cooling, the pellet is resuspended in 100 ml of 100 mM Tris-HCl, pH 8.0, containing 10 mM EDTA, incubated for 5 minutes at 0 ° C and centrifuged. The pellet is resuspended in 35 ml of 100mM Tris-HCl, 10mM EDTA, 1θ0mM β-ME, 1mM PMSF, 1% Triton Χ-100, pH 8.0. It is subjected to ultrasound - 10 times for 30 seconds, and then centrifuged (20,000 rpm, 30 minutes, temperature + 4 ° C), thus obtaining a sediment of inclusion bodies, which are then washed by suspending them in 35 ml of 50 mM Tris-HCl buffer, pH 7.4, 1 mM PMSF, 1 mM EDTA. After they are centrifuged again, they are dissolved in 9 M urea. In order to get rid of the rest of the impurities (undissolved bodies

164,991 inclusion), the suspension is centrifuged again (20,000 rpm, 30 min., Temperature + 4 ° C). The supernatant should contain the major part of the protein. Further purifications of the hybrid protein containing the PAI-1 fragment are performed on a DEAESephacel chromatography column. The proteins contained in the inclusion bodies dissolved in 9 M urea are dialyzed into a solution of 6 M urea, 50 mM Tris-HCl, pH 7.5, containing 10 mM EDTA, 1 mM PMSF. The same buffer is used to equilibrate two DEAE-Sephacel columns (2.5 x 10 cm). On one of them a protein solution is applied. The part of the protein which will not bind to the matrix after passing through the column is applied (after 2-3 fold dilution with the same buffer) to the second column. Pure conjugate is eluted from 0.1 M NaCl bed.

J. Disruption of the complex by chemical reaction.

The purified hybrid protein consisting of the β-galactosidase fragment and the Asnr72-Thr232 PAI-1 fragment is dialyzed against 10 mM Tris-HCl buffer, pH 7.5 to remove urea, and then lyophilized. Cleavage of the Asn-Gly bond is performed using

M hydroxylamine in 6 M guanidine hydrochloride, pH 9.3. The reaction is carried out at a temperature of 45 ° C. The breakdown of the conjugate was checked by electrophoresis.

The products resulting from this reaction are analyzed in a polyacrylamide gel after electrophoresis.

K. Method for isolation of the Asnn2-Thr232 (PAI-1) fragment of the human tissue plasminogen activator inhibitor by HPLC.

The polypeptide fragment corresponding to the AsnmOTh1 fragment (PAI-1) of the human tissue plasminogen activator inhibitor is isolated from the hydrolyzate prepared as described in point I by HPLC. The chromatographic separation was performed on a TSK G3000SW semi-preparative column from LKB, Sweden.

Stage 2.

A. Obtaining antibodies by the method of the invention using a polypeptide fragment.

In order to obtain a polyclonal serum for a fragment of the human PAI-1 protein, rabbits are immunized with this protein. The intradermal route is 30-40 injections of a dose of 50 µg of antigen in 0.5 ml of PBS and mixed with 0.5 ml of Freud's complete adjuvant. The ear vein of rabbits is blood collected 7 days after each vaccination. After clotting at 37 ° C for 0.5-1.0 hours, the clot is detached from the walls of the tube and left at 4 ° C for 12-24 hours. The serum is then withdrawn into sterile centrifuge tubes and centrifuged at 3000 rpm for 20 minutes. The serum is inactivated by incubation for 30 minutes at 56 ° C. The titer of individual sera samples is determined by radioimmunoassay, determining their ability to bind the labeled antigen. Sera with the highest antibody titer are stored at -20 ° C.

B. Purification of antibodies.

Antibodies to the PAI-1 fragment are purified from the antiserum by affinity chromatography. The immunoadsorbent contains a PAI-1 protein fragment bound to CNBr-Sepharose 4B resin (Pharmacia). To prepare the immunoadsorbent, 1 g of the bed is washed with 200 ml of 0.001 mol / l HCl solution and then suspended in a borate buffer (pH 8.3). 20 mg of the PAI-1 protein fragment dissolved in PBS is immediately added and gently stirred at 4 ° C for 2 h. After centrifugation, the protein concentration in the supernatant is checked. The bed is then suspended in a glycine solution (2 mol / l) in borate buffer pH 8.3. After incubation for 1 hour, they are centrifuged (10 min, 4000 rpm) and washed x with borate buffer (pH 8.3), 1 x with distilled water, 3 x with borate buffer (pH 8.3). The resin obtained in this way is incubated with 15 ml of serum obtained after immunization with the PAI-1 protein fragment, with gentle agitation for 12-14 hours at 4 ° C. 1 mM PMSF (Sigma) and 1 mM benzamidine chloride (Biochemical) are added to the mixture. After centrifugation (10 min, 3000 rpm), the media is washed with buffer (PBS with 1% Tween 20) followed by 4 x 1 M buffer (1 NaCl 21% Tween 20 in PBS). After each centrifugation, the protein content of the supernatant is checked by measuring the light adsorption at 400 nm. The bed is then placed in a column (0.7 x 10 cm). Antibodies specifically bound to the immobilized protein on the bed are eluted with 0.5 mol / L acetic acid and collected in tubes containing 100 µL of 2 mol / L Tris. High protein fractions are pooled and dialyzed against PBS for 24 hours at 4 ° C.

C. Conjugation of antibodies to peroxidase.

The thus obtained antibodies to the PAI-1 protein fragment are conjugated to peroxidase. For this purpose, 5 mg of HRPO are dissolved in 1 ml of freshly prepared 0.3 M sodium bicarbonate (NaHCO3). pH 8.1. Then 0.1 ml of 1% FDNB in absolute alcohol is added. It is shaken gently for 1 hour at room temperature. 0.04-0.08 M NaJO 4 (dissolved in distilled water) is added, gently mixing for 30 min. in room temperature. The color of this solution should turn green-yellow. Then 1.0 ml of 0.16 M ethylene glycol (dissolved in distilled water) are added and the mixture is gently stirred at room temperature for 1 hour. After incubation, the solution is dialyzed against 0.01 M sodium carbonate, pH 9.5, temperature 4 ° C (at least 3 1 liter changes). 5 mg of IgG are added to 3 ml of the thus prepared peroxidase aldehyde (HRPO) solution and gently stirred for 2-3 hours at room temperature. IgG is dissolved in 1 ml of carbonate buffer before mixing or added as a salt-free lyophilized powder. After the addition of IgG, 5 mg of NaBH4 are introduced and the mixture is left at 4 ° C for 3 to 12 hours. The solution is then dialyzed at 4 ° C against PBS. If a precipitate is formed, it is removed by centrifugation. The sample is applied to a column (85 x 1.5 cm), Sephadex G-100, equilibrated in PBS. Fractions of 1.5 ml are collected and the protein level in the individual fractions is determined spectrophotometrically. The HRPO labeled IgG fraction comes down in the first peak. Solutions of this conjugate are stored in the presence of bovine serum albumin at a concentration of 10 mg / ml at -20 ° C.

Stage 3.

Coating the plate with antibodies and performing an ELISA test.

The wells of the ELISA plate are coated with polyclonal antibodies to the polypeptide fragment of the PAI-1 protein Asni72-Thr232 at a concentration of 10 µg / ml, suspended in 50 mM phosphate buffer at pH 7.5. Unbound protein is washed away and the surface of the wells is coated with serum albumin. The test plasma samples are diluted prior to determination with 50 mM phosphate buffer, pH 7.5, containing 0.15 M NaCl, 10 mM EDTA, 1% Tween 20 and 1% BSA, and 200 µl / well are added. Incubate for 2 hours at room temperature. After incubation, the plates are washed 5 times with 0.15 M NaCl containing 0.1% Tween 20. Then 200 μl of IgG-HRP conjugate (polyclonal antibodies for the whole PAI-1 molecule bound to peroxidase) are added at a concentration of 5 μg / ml. . It incubates for 2 hours at room temperature and then rinses 5 times. To promote the enzymatic reaction, 200 μl of substrate solution (0.4 mg / ml of orthophenyldiamine in 0.05 M citrate buffer, pH 5.0 containing 0.02% H2O2) are added. Color appears after 6 minutes of incubation and the reaction is stopped by adding 50 μl of 3M sulfuric acid. Adsorbance is measured at 490 nm.

Example II.

Detection of the PAI-1 antigen in the test sample using antibodies to the polypeptide fragment corresponding to the Asn 72-Thr232 fragment of the human tissue plasminogen activator inhibitor.

The peroxidase-labeled antibodies for the polypeptide fragment corresponding to the Asnr72-Thr232 fragment of the human tissue plasminogen activator inhibitor are added to the immobilized antigen and incubated for 2 hours at room temperature. To induce the enzymatic reaction, a substrate solution - orthophenyldiamine in 0.05 M citrate buffer, pH 5.0, containing 0.02% H 2 O 2, is used. Color appears after 15 minutes of incubation and the reaction is stopped by the addition of 50 μΐ 3 M sulfuric acid. Adsorbance is measured at 490 nm. The result can be read from the standard curve for the PAI-1 standard solution.

Example III.

The diagnostic test kit for the determination of the human tissue plasminogen activator inhibitor level consists of: a container containing a buffered saline solution

164,991 physiological (PBS), pH 7.5, EDTA, Tween 20; a container with serum albumin; an antibody container for a polypeptide fragment corresponding to the AsnmThr232 fragment of a human tissue plasminogen activator inhibitor; a container with peroxidase labeled antibodies to PAI-1; a container with a substrate (orthophenyldiamine); a container with citrate buffer, pH 5.0 and a container with 3 M sulfuric acid.

GAATCTAACGGCCAGTGGAAGACACCATTCCCAGACAGTAGCACCCACCGCCGCCTCTTCCA

GATTGCCGGTCACCTTCTGTGGTAAGGGTCTGTCATCGTGGGTGGCGGCGGACAAGGT

CAAATCAGACGGAAGCACTGTCTCTGTGCCAATGATGGCTGAGACCAACAAATTCAACTATA

GTTTAGTCTGCCTTCGTGACAGAGACACGGTTACTACGGACTGTGGTTGITTAAGTTGATAT

CTGAATTCACCACGCCAGATGGACATTACTAC (} ACATCCTGGAACTGCCATACCACGGAGAC

GACTTAAGTGGTGCGGTCTACCTGTAATGATGCTGTAGGACCTTGACGGTATGGTGCCTCTG

ACT

TCAA7CT

3 '5'

1. CGGTCACCTTCTGTGGTAAGGGTCTGTC

2. AlCGTGGGTGGCGGCGGAGAAGGTGTTTAGTCTGCCTTCG lower thread

3. TGACAGAGACACOGTTACTACCGACTCTGGTTG

4. TTTAACTTGATATGACTTAAGTGGTGCGGTCTAC.CTGTAA

5. TGATGCTGTAGGACCTTG4CGGTATGGTGCCTCTGTGAATCT

6. TCACAGAGGCACCATACCGTCAAGGTCCTACAGCATCĄTTAC

7. AGGTAGACCGCACCACTTAAGTCATATCAACT

Θ.TAAACAACCAGAGTCGGTAGTAACCGTGTCTCTGTCACGA upper thread

9. AGCCAGACTAAACACCTTCTCCGCCGCCACCCAC

10. GATGACAGACCCTTACCACAGAAGGTGACCGGCAATCTAAG.

Fig. 2

164 991

Eco RI

Pst

Fig.3

164 991

Fig.4

164 991

BETA - CALACTGZYDAASE: PAI PROTEIN

beta-galactosidase protein PAI degradation products

purification of the hydrolysis products

Fig 5

164 991

C —ΟΛΛζ-WWW-wwi 1- \ w

Fig.1

Publishing Department of the UP RP. Circulation of 90 copies. Price: PLN 10,000

Claims (5)

Patent claims A method of producing polyclonal antibodies specifically reactive with the protein of a human tissue plasminogen activator inhibitor by immunizing animals, characterized in that a polypeptide corresponding to the fragment of formula (Y) aA- (Z) b is used for immunization, wherein Y is the amino acid sequence encoded via a polylinker fragment, preferably Glu, Ser; a = 0 or 1; Z represents the sequence Glu, Ser and / or the sequence encoded by the polylinker fragment; b = 0, 1 or 2; and A is the sequence corresponding to the Asn 172-Thr232 fragment of the human tissue plasminogen activator inhibitor, blood is drawn and antibodies are isolated. 2. The method according to p. A method according to claim 1, characterized in that the polypeptide of the sequence: Asn-Gly-Gln-Trp-Lys-Thr-Pra-Phe-Pro-Asp-Ser-Ser-Thr-His-ArgArg-Leu-Phe-His-Lys- is used Ser — Asp-Gly-Ser — Thr-Val -Ser-Val-Pro-MetMet-Ala-Glu-Thr — Asn-Lys-Phe-Asn-Tyr-Thr-Glu-Phe-Thr-Thr — ProAsp-Gly- His-Tyr-Tyr-Asp-I le-Leu-Glu-Leu-Pro-Tyr-His-Gly-AspThr. 3. The method according to p. The method of claim 1, wherein the polypeptide is obtained by expression of recombinant DNA. 4. The method according to p. The process of claim 3, wherein a chemically synthesized cDNA is used. 5. The method according to p. 4. The method of claim 4, characterized in that a chemically synthesized cDNA with the sequence: (X) nAACGGCCAGTGGAAGACACCATTCCCAGACAGTAGCACCCACCGCCGCCTCTT CCACAAATCAGACGGAAGCACTGTCTCTGTGCCAATGATGGCTGAGACCAACAAATT CAACTATACTGAATTCACCACGCCAGATGGACATTACTACGACATCCTGGAACTGCC ATACCACGGAGACACT (X) _m , where X is GAATCT, n is 1 or 0, and m is 0, 1 or 2.