Monoclonal antibody and hybridoma producing its

FIELD: biotechnology, immunology.

SUBSTANCE: invention proposes hybridomas producing monoclonal antibodies showing specificity to human epiregulin. Invention discloses monoclonal antibody recognizing specifically human epiregulin with the sensitivity limit 10 pg/ml. Also, invention describes methods for specific detection of human epiregulin in a sample in vitro and a method for detection of cells expressing human epiregulin in extracellular fluid in vitro by using monoclonal antibody to human epiregulin. Invention provides a simple and highly sensitive method for detection of human epiregulin that can be used in diagnosis of human epiregulin-expressing tumors.

EFFECT: valuable biological and medicinal properties of antibody and hybridoma.

8 cl, 7 dwg, 8 ex

 

The technical field to which the invention relates

The present invention relates to a monoclonal antibody (MoAb), which specifically recognizes human epiregulin (hEPR), hybridoma, which produces MoAb, and to the immunological method of measurement for specific detection hEPR in samples using hEPR-polyclonal antibodies (hEPR-PoAb), which recognizes hEPR and MoAb.

Prior art

It was known that there are certain genetic abnormalities that occur with specificity and high frequency in cancer cells, and if the detected protein (proteins), which are caused by genetic anomaly, this protein may represent a molecule as a target for cancer diagnosis and treatment. Among them are a group of molecules that belong to the family of epidermal growth factor (EGF), and molecules of the family of their ErbB receptors were by target molecules for cancer diagnosis and treatment, because they participate in the growth of cancer cells and are associated with malignancy (J. Clin. Oncol., 17, 2639-2648 (1999); Biochem. Biophys. Acta., 1198, 165-184 (1994); Science, 244, 707-712 (1989); Anticancer Res., 20:91-95 (2000); Front. Biosci., 1;6:D685-707 (2001)).

It was reported that epiregulin (EPR), a member of the EGF family, is bifunctional in nature in the sense that it promotes the growth of normal cells and inhibits the growth of certain cancer cells in vitro, and that ur the level of expression of EPR (mRNA) in normal cells is very low, with the exception of the placenta and monocytes, but high in certain cancer cells (see, for example, The Journal of Biological Chemistry, 1995, Vol. 270, p. 7495-7500; The Biochemical Journal, 1997, Vol. 326, p. 69-75).

Further, in the clinical field indicates that epiregulin may be a useful marker for the diagnosis of cancer because of its high expression in bladder cancer and cancer of the pancreas (see, for example, Biochemical and Biophysical Research Communications, 2000, Vol.14 273 (3), p.1019-1024; AntiCancer Research, 2000, Jan-Feb, 20 1A9: p.91-95; Cancer Research, 2001, Vol.61, p.6227-6233).

To date there have been reports that the polypeptide EPR detected using polyclonal antibodies (hEPR-PoAb)that recognize human epiregulin (hEPR) (see, for example, The Journal of Biological Chemistry, 1995, Vol.270, p.7495-7500; The Biochemical Journal, 1997, Vol.326, p.69-75), but they do not represent methods with high sensitivity and high performance because they require concentrations, labels, radioisotopes and similar labels, and there are no reports of the detection of the polypeptide EPR in vivo. Thus, the development of a simple and highly sensitive method of detection hEPR can provide a useful tool as a diagnostic method for cancer early detection.

Description of the invention

It is known that the homology of amino acid sequence between EPR and other proteins-a family members GF is from 24 to 50% (see The Journal of Biological Chemistry, 1995, Vol. 270, p.7495-7500). In addition, the homology sequence of the EPR between species is very high, for example, EPR human and mouse differ in only 6 of the 46 amino acid residues. How to detect EPR using the above-described polyclonal antibodies requires complicated procedures and cannot differentiate between EPR human and mouse. In addition, the sensitivity of conventional methods for detecting amounts to a maximum of 1 ng/ml and therefore it is impossible to detect EPR in vivo.

The aim of the present invention is to provide a simple and highly sensitive method of detection hEPR and use of the method for detection of human cancers. In particular, the present invention provides a simple way of identifying hEPR on pilgrammage level, which can be used to detect tumors and other similar purposes.

Applicants have conducted a thorough research to solve the above described problems and eventually got 2 hybridoma (IC3, 3E8)producing monoclonal antibodies (MoAb), which specifically recognize hEPR. In addition, the developed sandwich ELISA assay (S-ELISA) by combining MoAb and hEPR-PolyAb and developed a detection system which is highly sensitive and high-performance.

Thus, the invention provides the following (1) to (9).

(1) Hybridoma, producer the expansion of monoclonal antibody which specifically recognizes human epiregulin hEPR.

(2) Hybridoma under item(1), which has the access number FERM BP-08647.

(3) Hybridoma under item(1), which has the access number FERM BP-08647.

(4) the Monoclonal antibody produced by hybridomas according to any one of paragraphs.(1)-(3).

(5) a Monoclonal antibody that recognizes the epitope which is recognized by the monoclonal antibody produced by hybridomas on PP.(2) or (3), and specifically recognizes hEPR.

(6) Method for specific detection hEPR in a sample in vitro, characterized in that it uses a monoclonal antibody according to p.(4) and a polyclonal antibody that recognizes hEPR (hEPR-PoAb).

(7) a Method of identifying cells expressing hEPR in vitro, characterized in that is used monoclonal antibody under item(4).

(8) the Method according to p.(7), in which cells expressing hEPR represent a tumor of the person.

(9) a Kit for identifying a human tumor comprising the monoclonal antibody according to p.(4).

The contents of the description and/or drawings of the patent application of Japan No. 2003-070864, which is the basis of priority of the present application, and fully incorporated into the present description by reference.

Brief description of drawings

Figure 1 shows the results of measurement of the titer of monoclonal antibodies against hEPR, which is produced by hybridomas of the present invention, methods for the om ELISA.

Figure 2 shows the results of measurement of the titer of the biotinylated polyclonal antibody against hEPR.

Figure 3 shows the sensitivity of the detection system by sandwich ELISA (S-ELISA) using a stepwise method and one-step method.

Figure 4 shows the results of the test of the specificity of the present method using monoclonal antibodies S as the first antibody. In this system, the monoclonal antibody does not react with molecules of the family of EGF or mouse EPR and interacts only with hEPR.

Figure 5 shows the result of analysis of Western blot testing hEPR in human serum. The position of molecular weight markers (kDa) are shown on the left.

Figure 6 shows the detection of hEPR in human serum S-ELISA.

7 shows the detection of hEPR in various media cell cultures.

The best way of carrying out the invention

Although the present invention is described in detail below, the specialists in the field can implement the present invention in various embodiments, implementation, using methods known in this field, and the present invention is not limited to the following options implementation.

Used in the present description, the term "hEPR" in the absence of other guidance means the precursor and Mature form or fragment of a human epiregulin. "Before estwanik means associated with the membrane form before cleavage enzyme and the "Mature form" means a polypeptide with a 46 amino acid residues (SEQ ID NO:1), released from the cell membrane. The fragment contains a 20 amino acid residues or more, or preferably 30 or more amino acid residues of the polypeptide and must contain a sequence that is specific for human epiregulin. Sequence-specific human epiregulin, does not mean that it is limited, but includes, for example, a sequence containing the 2-th, 11-th, 26-29 th and 39 th amino acids amino acid sequence of SEQ ID NO:1, which are known to be different, when compared to human epiregulin and mouse epiregulin. The fact that amino acids in these various sites in human and mouse, described in patent application WO94/29340 of the same applicant. The fragments, having the amino acid sequence containing these sites take different tertiary structures when comparing fragments EPR obtained from human and mouse. "Human epiregulin" and "mouse epiregulin" are polypeptides with the same amino acid sequence as the EPR, naturally occurring, respectively, in human and mouse. However, they are not necessarily limited to proteins extracted from natural sources, but it is assumed that they include, for example, recombinants and C is synthetic polypeptides.

Used in the present description, the term "specifically recognizes" means that the detected (or associated) hEPR, but other proteins of the family of EGF or inhuman EPR, such as mouse EPR, and the like, essentially, are not recognized (bound). This means, for example, that is recognized (bound) predecessor, the Mature protein and fragmented polypeptides hEPR, but EGF and TGF-αmouse EPR and the like, essentially, are not recognized (not linked). In particular, this means that his affinity binding compared to the affinity of binding to other proteins of the family of EGF and inhuman EPR, such as mouse EPR, above 100 times, or more preferably 1000 times or more, preferably 10,000 times or more.

Used in the present description, the term "essentially not recognized (not linked)" means that the binding is not confirmed by means of identification, generally used in this field, or the present invention. In particular, it means that the precursor of the Mature protein and fragmented polypeptide hEPR - demonstrates a positive reaction to the way the sandwich ELISA (S-ELISA), Western blotting, or the like, but EGF and TGF-α EGF family, and EPR obtained in mammals, except humans, such as a mouse, show no reaction (not detected).

Used in the present description, the term shall antibody" means polyclonal antibody (PoAb) or monoclonal antibody (MoAb), which specifically binds to a molecule hEPR full length or a fragment of the hEPR, or a partial fragment of these antibodies (e.g., fragments (Fab or F(ab')2 or Fab')obtained by cleavage with papain or pepsin, or similar enzymes, and they can be obtained by the methods of production described below.

Hybridoma and monoclonal antibodies of the present invention can be obtained as follows. In addition, experts in this field can be used appropriately modified methods based on the following descriptions and techniques known in this field.

(1) Obtaining antigen

Amino acid sequence of the antigen hEPR and nucleotide sequence that encodes it, is described in WO94/29340. Amino acid sequence and the nucleotide sequence shown respectively in SEQ ID NO:1 and SEQ ID NO:2. In addition, EPR is described as a factor that inhibits the growth of tumor cells, in WO94/29340.

Recombinant human EPR can be obtained as described in WO94/29340, constructing expression vector that contains a DNA fragment (SEQ ID NO:2)that encodes a hEPR, and the introduction and expression of the vector in the cells of the host, for example, but not limited, but preferably Bacillus brevis. After expression using Bacillus brevis culture medium concentrated 20-fold using ultrafiltration the membrane (1000 kDa), the pH was adjusted to 7.4 with 1 M Tris-HCl (pH 8.0), and cleaning is performed using anion-exchange column, for example column of Q-sepharose. In this case, pH, passed through the membrane fraction was adjusted to 5.0 and then clean cation-exchange column, for example column of Q-sepharose. This is followed by a purification column chromatography in reversed-phase (column C4) in order to obtain a single band in electrophoresis.

hEPR can also be synthesized chemically by a solid phase method (Merrifield, J. Am. Chem. Soc., Vol. 85, p.2185 (1963)on the basis of the amino acid sequence shown in SEQ ID NO:1. Chemical synthesis by solid-phase method is usually carried out in a standard way, using an automatic peptide synthesizer.

(2) Obtaining polyclonal antibodies

A warm-blooded animal subjected to immunization is only 4-6 times one solution of peptide antigen or together with carriers and diluents. Examples of warm-blooded animals include, for example, rabbits, dogs, Guinea pigs, mice, rats, and similar animals. It is preferable to measure the antibody titer by taking blood samples after the third subcutaneous immunization. Measurement of the antibody titer in the serum spend immobilization of the peptide used as antigen in 96-well Mitrovice the microplate with the performance of the ELISA method. After confirming that the title and the antibodies raised high enough, take whole blood and the antibody is isolated and purified by a standard method. Cleaning methods include, for example, precipitation with ammonium sulfate, ion-exchange chromatography using the anion exchanger such as DEAE (Diethylaminoethanol)-cellulose and the like, gel filtration, affinity chromatography with an active adsorbent such as protein A/G and similar and other cleaning methods. In addition, the specificity in relation hEPR can be increased by purification using a column in which hEPR immobilized on a solid phase.

(3) generation of monoclonal antibodies

Cells producing monoclonal antibodies, receive a selection from immunized warm-blooded animals an individual animal, which increased the titer of antibody, the capture of spleen or lymph node after 2-5 d after the last immunization, merge producing monoclonal antibodies of cells present in these organs, with myeloma cells and selection of hybridomas producing MoAb. The merger is carried out in accordance with the known method such as the method of Kohler at al. (Nature, 256, 495 (1975)). Myeloma cells include, but are not limited to, for example, PAI, P3U1 (Health Science Research Resources Bank; HSRRB), Japan, catalog numbers JCRB0113 and JCRB0708) and the like. Means of promoting the merger, include polyethylene glycol (PEG) and Sendai virus (HJB), but preferably with PEG molecules of the nuclear biological chemical (NBC weight of from 1000 to 6000. Efficient cell fusion can be achieved by adding the promoting means in a concentration of from about 10 to 80% and incubation at 20-40°C.

Selection of monoclonal antibodies can be carried out in accordance with the known method. In General it is held in the cell culture medium for animal cells with the addition of NAT (gipoksantin, aminopterin, thymidine). Environment for selection and growth may include, for example, the environment PRMI1640 containing from 10 to 20% fetal calf serum and the like. Cells are generally cultured in an atmosphere of 5% gaseous CO2at a temperature of 20-40°during the period from 5 d to 3 weeks.

The supernatant liquid is collected from the wells, which are cultivated cells hybridoma, and antibodies reacting with antigenic peptide can be selected by ELISA method. First antigenic peptide immobilized on 96-well tablets and then block calf serum. After interaction of the supernatant liquid hybrid mouse immunoglobulins/HRP (Amersham-Pharmacia) at 37°C for 1 h staining occurs when using tetramethylbenzidine microwell peroxidase substrate (TMB, Funakoshi) as substrate. After the reaction with the acid is measured spectral absorption ability 450/540. Selected antibodies with the spectral absorption capacity of about 3, and Clonie the Finance spend way end-breeding.

Thus obtained target cells of hybridoma cultivate, and monoclonal antibody can be obtained from the culture medium. Alternative cell hybridoma you can inoculate intraperitoneally, such as a mouse (Balb/c), and monoclonal antibody can be obtained from ascitic fluid.

Purification of monoclonal antibodies can be performed similarly as described above, conventional separation and purification PoAb.

In addition, applicants deposited 2 types of hybrid 25 September 2002, at the International Depository patented organisms National Institute of advanced industrial science and technology (AIST) (Center No. 6, 1-1-1 Hagashi, Tsukuba City, Ibaragi-Ken) with access numbers FERM P-19033 and FERM P-19034. Later they were moved on 27 February 2004 at the international Depositary under the Budapest Treaty on the International recognition of the Deposit of microorganisms for purposes of patent procedure, with access numbers FERM BP-08647 and FERM BP-08648.

In addition, the monoclonal antibodies of the present invention include monoclonal antibodies that specifically recognize an epitope which is recognized by the monoclonal antibody produced by the above-hybridomas, especially hybridomas with access numbers FERM BP-08647 and FERM BP-08648, and recognize hEPR. The epitope that is recognized by these monoclonal antibodies, contains minocyclinee residues, included in the unique sequence described above hEPR.

The present invention provides a method for specific detection hEPR, characterized in that uses the above-described monoclonal antibodies and polyclonal antibodies of the present invention that recognize hEPR (hEPR-PoAb). The sample includes blood, biological fluids, tissue extracts and the like materials taken from individuals. Although certain there are no restrictions, but the preferred method is a method of analysis S-ELISA, comprising the step of contact of the sample, which may contain the antigen hEPR, with a monoclonal antibody of the present invention and the phase contact of the complex antigen-antibody, which is generated on the ground, with a polyclonal antibody. The above steps can be performed sequentially (sequential method) or simultaneously (one way). Way S-ELISA described, for example, in the publication "a Monoclonal antibody, hybridoma and ELISA (Iwasaki, Tatsuo et al. Kodansha Scientific), and specialists in this field can carry out the method of the present invention on the basis of the present description. Because the method of detection of the present invention is very sensitive and can detect hEPR at a concentration of 10 PCG/ml or higher, the concentration of hEPR, expressed in a living organism (approximately 20-30 PCG/ml), rises the high to detect.

The present invention also provides a method of detecting in vitro cells expressing hEPR, in particular human cancers, characterized in that uses the above-described monoclonal antibodies of the present invention. Because hEPR are secreted from the cell membrane after expression in cells, hEPR may be detected in the extracellular fluids, and it is not necessary that the sample contained cells.

The above method includes the step of contact of the sample obtained from the man, and monoclonal antibodies of the present invention, and the step of detecting the presence of hEPR determining whether associated monoclonal antibody with hEPR as an indicator or not. Human tumor target include, for example, lung cancer, colon, bladder, uterus, colon and the like, but are not limited to.

Because the method of detection of the present invention is characterized by high sensitivity, detection can be done quickly and easily without the need for such manipulation with the sample concentration and the like. In this way samples containing hEPR at a concentration of 10 PCG/ml or more, you can identify the way S-ELISA and the like, but for reliable detection preferably, the sample contained 25 PCG/ml or more.

Using a way of identifying this is subramania, you can define, whether Express hEPR specific tumor cells or the like, or not, and if Yes, what is the level of expression. Further, it can contribute to the clarification of the relationship between expression of hEPR and mechanism of carcinogenesis. In addition, based on the tumor cells, which Express hEPR at a high level, and the level of expression in normal cells can detect the presence of a tumor in a specific individual.

The present invention also provides a kit for identifying a human tumor comprising the monoclonal antibody of the present invention.

The kit of the present invention may be advisable to include, in addition to the above-described monoclonal antibodies of the present invention, polyclonal antibodies, buffer, dye, aiming reagent, diluent and similar substances. Preferably the kit of the present invention is a kit for performing S-ELISA.

As will be shown below, the present invention is more specifically explained by the variations in implementation, but, as described above, the present invention is not limited to these examples.

Example 1 development of hybridomas

100 μl of recombinant hEPR (1 mg/ml) (obtained by the method described in WO94/29340) salt solution is mixed with an equal volume of complete adjuvant-blockers, emuleret and inocula the comfort in the back of a mouse (Balb/c, at the age of 6 weeks). After 2 weeks. the mouse repeatedly subjected to immunization with a mixture of 50 μl of saline antigenic peptide (hEPR, 1 mg/ml) and incomplete adjuvant-blockers, emuleret sonification and then weekly additional immunization. After 40 d after immunization remove the spleen, lymphocytes collect in the environment PRMI1640 (with the addition of penicillin and streptomycin) and treated with 0.17 M ammonium chloride to remove erythrocytes. The selected cells is drained from the strain P3U1 myeloma cells derived from a tumor of the bone marrow of mice polietilenglikoli way (PEG4000) to obtain cells hybridoma. Thus obtained cells hybridoma suspended in the NAT environment with feeder cells and distributed in 96-well plates and cultured for 15 days

Example 2. Screening for monoclonal antibodies

The supernatant culture medium is extracted from the wells, which were cultured cell hybridoma obtained in example 1, and the ELISA method chosen MoAb, which interact with the antigenic peptide.

First, 100 μl of antigenic peptide at a concentration of 10 μg/ml added to each well of 96-well plate, immobilized in the solid phase after keeping at 4°C overnight and blocked with 200 μl of 10% calf serum at 37°With during the night. 100 μl of nadosadocnaya culture medium of cells hybridoma added to each well, conduct the reaction at 37°C for 2 h, and then add antimurine antibody conjugated with horseradish peroxidase (HRP) (Amersham-Pharmacia), which was diluted in 1000 times, and conducting the reaction at 37°C for 1 h Staining receive using tetramethylbenzidine microwell peroxidase substrate (TMB, Funakoshi) as a substrate.

After stopping the reaction by adding 100 μl of 4N sulfuric acid was measured spectral absorption capacity at 450-540 nm and selected MoAb, IC3 and E that exhibit spectral absorption capacity of about 3, and clone way limited cultivation.

Mice (Balb/c), which for 7 d and 3 d before that was intraperitoneally injected with 0.5 ml of Pristina, intraperitoneally inoculant selected cells hybridoma IC3 and A producing MoAb, and the ascitic fluid is collected after about 10 days Collected ascitic fluid is kept at room temperature for 30 min at 4°C overnight and centrifuged at 15,000 rpm for 10 minutes, and then remove the supernatant.

The titles selected 2 types of MoAb measured by the ELISA method, and the results are shown in figure 1. The number of recombinant hEPR, shown on the horizontal axis, Immobiliser on microwell tablet add IC3 or A (1 μg/ml) and after the interaction occurs coloring when using the years of antimelanoma antibodies conjugated to horseradish peroxidase (HRP) and TMB. The results indicate that both MoAb interact dependent on concentration.

Next, hybridoma that produce MoAb IC3 and E were deposited on 25 September 2002 at the International Depository patented organisms National Institute of advanced industrial science and technology (AIST) (Center No. 6, 1-1-1 Hagashi, Tsukuba City, Ibaragi-Ken) with access numbers FERM P-19033 and FERM P-19034.

Example 3. Obtaining polyclonal antibodies

1 ml recombinant hEPR in saline solution (1 mg/ml) and 1 ml of complete adjuvant's adjuvant (Difco) mix and emuleret by sonication and subjected to immunization introduction back in rabbits (Japanese white, weight 2.7 kg, female, from Japan Clea) at 10 separate locations or more. After 1 month 0.5 ml recombinant hEPR in saline solution (1 mg/ml) and 0.5 ml incomplete adjuvant's adjuvant (Sigma) mixed and emuleret by sonication and subjected to immunization for the second time in a similar manner as in the first immunization. After the second immunization supplementary immunization conducted weekly 1 ml recombinant hEPR in saline solution (1 mg/ml) and 1 ml incomplete adjuvant's adjuvant, which emuleret sonification. Blood samples taken after 1 week. after immunization, keep at room temperature for 1 h after mixing the Pasteur pipette, who constitute at 4° With overnight and centrifuged at 5000×g for 10 min to obtain antisera.

Anticigarette precipitated with 40% ammonium sulfate, deleteroute against 50 mm Tris-HCl (pH 8.0) overnight on a column of Protein G (Amersham-Pharmacia) to obtain the IgG fraction. For further purification of the antibody column is prepared in accordance with the conventional method on the basis of manufacturer's description of the binding of 1 mg of recombinant hEPR approximately 1 ml of the activated NHS (normal human serum) Sepharose 4 Fast Flow (Amersham-Pharmacia). the pH of the IgG fraction obtained as described above was adjusted to 8.0 and cultivated through the column for 10 hours or longer, and antibodies associated with the column, elute with 50 mm glycine-HCl (pH 2,5)/0.15 M NaCl. pH buervenich antibodies immediately brought to neutral level of 1 M Tris and again deliberately against 50 mm phosphate buffer (pH 7.4)/0.15 M to obtain specific antibodies against hEPR.

Specific antibodies biotinylated the usual way, using the reagent for biotinidase (Amersham-Pharmacia). Title biotinylated hEPR-PoAb is measured in the following way. The solution hEPR the highest concentration of 200 ng/ml diluted in 5 times and immobilized on 96-well-microplate (100 μl/well). After blocking 25% Block Ace (Dainippon Pharmaceutical Co., 200 µl/well) add biotinylated antibody hEPR-PoAb (0.125 mg/ml (diluted 50% Block Ace (Dainippon Pharmaceutical Co.), 100 µl/l of the GCC) and conducting the reaction at room temperature for 2 h, and then add Avidin-HRP (Amersham-Pharmacia)diluted 1000 times in a concentration of 100 μl/well, and conduct the reaction at room temperature for 30 minutes After adding the ET reaction is carried out at room temperature for 30 min and stop 4N sulfuric acid. Spectral absorption capacity measured at 450-540 nm, and the titer against the antigen immobilized on the solid phase, are given as in example 2. The results indicate that all of them show a reaction, dependent on the concentration of the antigen, confirming that they have titles that you can use (2)

Example 4. System sandwich ELISA

The ELISA system design using monoclonal antibody obtained in example 2 and polyclonal antibodies obtained in example 3.

Monoclonal antibody, IC3, at a concentration of 1 μg/ml added to microplasma in the amount of 100 µl/plate and incubated at 4°C for 24 h for immobilization on the solid phase. The wells are washed 3 times with 200 ál/plate 50 mm Tris-HCl, pH 7.5, containing 0.1% Tween 20 (TBST). Blocking is performed by adding 25% Block Ace in the amount of 200 μl/plate and incubated at 4°within 24 hours

In a phased way the wells are washed 3 times in TBST 200 ál/plate, add hEPR, serially diluted 2-fold, starting with 1 ng/ml, and incubated at room temperature for 2 h, and C is the add biotinylated antibody hEPR-PoAb (1 µg/ml 100 μl/well) and incubated at room temperature for 2 hours

In a single-step method, the antigen hEPR and biotinylated antibody hEPR-PoAb add at the same time and conduct the reaction at room temperature for 2 hours

After washing the wells 5 times with TBST add avidin-HPR (Amersham-Pharmacia)diluted 1000 times in the amount of 100 μl/well and incubated at room temperature for 30 minutes the Reaction is stopped by addition of 4N sulfuric acid and the spectral absorption capacity measured at 450/540 nm. The results indicate that there is no difference in the sensitivity of detection between a phased manner and the instant method, and both methods have sufficient sensitivity (25 PCG/ml) for measuring in vivo (figure 3). In a similar test conducted using E as the primary antibodies, the results were the same.

Example 5. The specificity of the system sandwich ELISA

The specificity of this system was confirmed by the method shown in example 4 (one-stage method).

MoAb IC3 (1 μg/ml, 100 PC/ml) immobilized to the wells microwell tablets as the primary antibody and after blocking 25% Block Ace (200 µg/well) add a group of molecules member of the EGF family, to which EPR (6 factors: epidermal growth factor (EGF), transforming growth factor - α (TGF-1 ), heparin binding growth factor that is similar to EGF (HB-EGF), betacellulin (BTC), amphiregulin (AR), heregulin-α (HRG-α), all purchased from R&D Co. or Sigma Co.), mouse EPR (obtained in the same way as human EPR), or human (EPR) in the amount shown on the horizontal axis, together with biotinylated hEPR-PoAb (2 μg/ml, 500 μl/well; the final concentration of 1 μg/ml). 6 members of the family of EGF and mouse EPR serially diluted in 4 stages, 5 times on each stage, starting with a concentration of 200 ng/ml, and add 200 µl/well. Similarly human EPR plant in 4 stages, 5 times on each stage, starting with a concentration of 200 ng/ml After reaction with Avidya-HPR staining receive using TMB.

As shown in figure 4, hEPR was detected with high sensitivity, but other molecules, which consisted of a molecule of the EGF family, and the mouse EPR were not detectable at concentrations above 1000 times, confirming the specificity of the present system by sandwich ELISA. The same results were obtained in a similar test using E as the primary antibody.

Example 6. Analysis of Western blot testing human EPR in human serum

Human serum (Rockland INC.) diluted 10 times using 50 mm buffer Tris-HCl at pH 7.4 (Gibco BRL). Recombinant hEPR add to diluted human serum floor for the treatment of tested samples in 5-fold serial dilution, so the final concentration was in the band 1:0 PCG/ml, lane 2:8 PCG/ml, lane 3:40 PCG/ml, lane 4:200 PCG/ml, lane 5:1000 PCG/ml Obtained in this way test samples are subjected to electrophoresis on SDS (sodium dodecyl sulphate) polyacrylamide gel (SDS-PAGE), transferred to a PVDF membrane (Daiichi Pure Chemical Co.), and then analyze the way immuno-blotting (J. Biol. Chem., 270, 7459-7500), using antibodies, such as anti-hEPR-PoAb, MoAb1C3 and E (PCG/ml)that recognize hEPR. The results indicate that each antibody specifically detects hEPR in the field from 3.7 to 8.2 kDa (figure 5). Although the band of about 28 kDa was detected PoAb and MoAb IC3, it was considered as non-specific band, because the same band was detected in the band antigen (-).

Example 7. Detection of human EPR method ELISA

Monoclonal antibody S or E, adds to the wells of the microplate in an amount of 100 μl/well and immobilizerpower the solid phase by incubation at 4°within 24 hours the plate is washed 3 times with 200 μl/well of 50 mm Tris-HCl at pH 7.5, containing 0.1% Tween 20 (TBST)and blocked by adding 200 μl/well of Block Ace and incubated at 4°within 24 hours After washing the wells 3 times with 200 μl/well TBST twice in diluted human serum hEPR serially diluted 2 times, starting with 400 PCG/ml, and add 50 ál/well and incubated at room temperature for 2 h After washing 5 times with TBST was added and diluted to 1000 times avidin-HPR (Amersham-Pharmacia) in an amount of 100 μl/well and conduct the reaction at room temperature for 30 minutes The reaction is stopped by addition of 4N sulfuric acid staining receive using TMB, and spectral absorption capacity measured at 450/540 nm. The results are shown in Fig.6, which indicate that hEPR at a concentration of 25 pkg/ml can sufficiently identify any of S and E.

Example 8. Detection of EPR produced by cultured human cancer cells

Various culture cells derived from mouse (IC38, RAW264,7, NIH3T3, clone T7, MoAb3) and human (T-24, A-549, NST, colo205, colo210, HeLa, NB69, A431, SK-BR-3, MDA-MB-468, KB, TR-13), cultured for 3 d in culture medium containing 10% calf serum, ranging from 106cells/10 ml/plate, and the culture medium is subjected to detection of hEPR method S-ELISA.

MoAb IC3 (1 μg/ml) immobilizing the solid phase at a concentration of 100 μl/well, and after the lock in the hole at the same time add 50 ál of different media culture cells and 50 μl of biotinylated hEPR-PoAb (2 μg/ml) and incubated at room temperature for 2 hours After surgery were similar to the operations in example 4. As controls, similar operations were performed on sera with additional or without added hEPR (10% FBS (fetal calf serum, human serum, 0.1% of BSA (hEPR 1 ng/ml), human serum (EPR 1 ng/ml).

The results indicate that A450 nm is not observed in the culture media of isleton mice, such as NIH3T3, clone T7, producing murine EPR and others, while products hEPR confirmed in cancerous human cells, such as A-549 (lung cancer cells human), NST (colon cancer man), colo201 (colon cancer man), colo205 (colon cancer) and T-24 (bladder cancer) (7), confirming that hEPR can specifically be detected in the culture media. However, hEPR cannot be detected in such cancerous human cells like HeLa (cervical cancer man), NB69 (human neuroblastoma), KV (epidermal carcinoma (oral) person)), A431 (epidermal carcinoma (epidermis) person)), SK-BR-3 (breast cancer) and TR-13 (kidney cancer man).

Industrial applicability

In accordance with the present invention, it becomes possible to provide a system, which with high specificity and sensitivity reveals hEPR in the blood, tissues and other materials derived from the human body, and it can be used to diagnose cancer and other neoplastic diseases to identify tumor cells expressing hEPR. In addition, it is expected that the application of the method of the present invention will facilitate the detection of the presence or absence of expression of hEPR and the relation between expression of hEPR and mechanism of carcinogenesis.

All publications, patents, and p the patent application, some of which are referenced in this description are incorporated into it by reference.

1. Hybridoma producing a monoclonal antibody that specifically recognizes human epiregulin (hEPR), deposited in the International Depository patented organisms National Institute of advanced industrial science and technology (AIST) under the number FERM BP-08647.

2. Hybridoma producing a monoclonal antibody that specifically recognizes human epiregulin (hEPR), deposited in the International Depository patented organisms National Institute of advanced industrial science and technology (AIST) under the number FERM BP-08648.

3. A monoclonal antibody that specifically recognizes human epiregulin (hEPR) with a sensitivity of 10 PG/ml or more.

4. Monoclonal antibody according to claim 3, produced by hybridomas having a Depository number FERM BP-08647.

5. Monoclonal antibody according to claim 3, produced by hybridomas having a Depository number FERM BP-08648.

6. Method specific detection hEPR in a sample in vitro, characterized in that the monoclonal antibody according to claim 3 is used as the first antibody, and a polyclonal antibody that recognizes hEPR (hEPR-PoAb), is used as the second is nitela.

7. Detection of cells expressing hEPR or hEPR in the extracellular fluid in vitro, characterized in that uses a monoclonal antibody according to claim 3.

8. The method according to claim 7, in which cells expressing hEPR represent cancer cells human colon.



 

Same patents:

FIELD: medicine, neonatology.

SUBSTANCE: in umbilical cord blood at the moment of a child's birth one should detect the ratio of the parameters for relative content of CD3+HLA-DR+ against CD3+ lymphocytes and at its value being equal to 8.1% or above it one should diagnose perinatal hypoxic CNS lesion at accuracy of 89.28%. Application of the present method enables to diagnose perinatal hypoxic CNS lesion in mature neonatals in gestosis-suffering women at earlier terms.

EFFECT: higher accuracy of diagnostics.

3 ex, 1 tbl

FIELD: immunology, biotechnology.

SUBSTANCE: invention relates to antibodies showing specificity to anomalous processed form of human tau protein that differs by conformation from the normal tau protein and doesn't bind with normal tau protein. Also, invention relates to conformational distinctive tau proteins ("tauones") and diagnostic and therapeutic aspects related to Alzheimer's disease and related taupathies. Proposed antibodies are produced by hybridomas DC-11 or Dc-11/1 deposited in ECACC at numbers 00082215 and 00082216. Also, invention described truncated forms of human tau protein that are truncated by N- and/or C-end and comprise amino acid residues from amino acid 300 to amino acid 400 in the longest isoform of human tau protein (441 amino acids residues). Above mentioned truncated forms of human tau protein can be recognized specifically by antibodies described above. Also, invention describes a method for assay of truncated forms of tau protein in a patient biological sample using a set comprising a proposed antibody and suitable container. Using the proposed invention provides a suitable target for medicinal preparations with early therapeutic effect used in Alzheimer's disease and other taupathies.

EFFECT: valuable medicinal properties of proteins.

11 cl, 15 dwg, 10 ex

FIELD: medicine, laboratory diagnostics, pediatrics, neonatology.

SUBSTANCE: in neonatals born in mothers with endemic goiter it is necessary to detect the content of CD25+ and HLA-DR+ lymphocytes in peripheral venous blood on the 5th d of their life and at the value of the first parameter being either equal or below 6.9% and the second parameter being either equal or below 16.5% one should predict the onset of infectious-inflammatory diseases in the course of neonatal period.

EFFECT: higher accuracy, sensitivity and specificity of prediction.

3 ex, 1 tbl

FIELD: medicine, analytical immunology.

SUBSTANCE: invention relates to a set of reagents used in quantitative determination of secretory immunoglobulin A (sIgA) that provides assaying status of topical immunity and immunodeficient states in carrying out analysis of serum and secrets of human body. Proposed set comprises a plate with immobilized monoclonal antibodies, five calibrating samples containing 0; 1.0; 5.0; 10.0 or 20.0 mcg of sIgA/ml, conjugate of murine monoclonal antibodies with horse radish peroxidase and a reagent for carrying out the enzymatic reaction. Murine monoclonal antibodies produced by hybrid strain of animal Mus musculus L., № PKKK (P) 676D cultured cells, are immobilized on a carrier. Conjugate comprises murine monoclonal antibodies produced by another strain - Mus musculus L., № PKKK (P) 677D. The advantage of invention involves enhancing sensitivity in assay of sIgA.

EFFECT: improved assay method.

3 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: method involves determining availability of antibodies to basic myelin protein at the first and the third week after burn, by applying immunoenzyme analysis approach. The third week values belonging to the range of 0.1-0.19 ODU, vegetovascular dystonia syndrome is predicted to develop. The values being within the 0.2-0.274 ODU limits, disseminated cerebral microsymptoms are to be predicted. The values being within the 0.275-0.3 ODU limits, focal symptom manifestations syndrome is to be predicted.

EFFECT: high accuracy in predicting cerebral disorders severity degree.

FIELD: medicine.

SUBSTANCE: method involves determining relative content of CD16+ monocytes in monocytic gate in peripheral venous blood of barren woman. The value being equal to or less than 45%, external genital endometriosis diagnosis is to be set.

EFFECT: high accuracy, specificity and sensitivity of diagnosis method.

1 tbl

FIELD: medicine; microbiology.

SUBSTANCE: method an be used for specific identification and evaluation of potential ability of non-cultivated forms of vibrio cholerae O1 and O139 serous groups to reversion, which vibrios take place in water solutions. Method is based upon preliminary preparation of bacterial suspension of non-cultivated forms of vibrio cholerae O1 and O139 serous groups and bacterial suspension of reference culture of Vibrio Cholerae O1 and O139 serous groups. The working dilution of bacterial suspensions and monoclonal antibodies conjugate is determined and specific monoclonal antibodies are selected for conservative and labile lactose-peptone medium epitopes followed by dot-immune-enzyme analysis or solid phase immune-enzyme analysis reaction. Then appearance of color point or optical density, being higher than 0,2-0,65, is registered and conclusion is made on presence of non-cultivated forms of Vibrio Cholerae O1 and O139 serous groups in sample and their potential reversion ability. Preliminary preparation of non-cultivated forms is performed due to increase in their concentration up to 106-107 mxkl/ml followed by inactivation. For the purpose 20 mkl of 4% sterile gentamicine solution is added to 1 ml of microbe suspension followed by subsequent cooling of samples down to 3-4 deg C during 2 hours. Bacterial suspension of control culture Vibrio Cholerae O1 or Vibrio Cholerae O31 is made from 18-hours culture, grown onto Martin's agar. After that the suspension is used for preparation of bacterial suspension in physiological solution, pH 7,6 till final concentration of 107 mxkl/ml. Method uses set of specific monoclonal antibodies, got to different epitopes of O-antigen of lactose-peptone medium cultures of Vibrio Cholerae O1 and O139 serous groups: 3A9, D11, 3E4, 4H5, D8D6, C3B4, F8G12, F11E5.

EFFECT: ability of getting express-answer at low concentration of tested cells.

5 cl, 2 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: it is necessary to study saliva due to carrying out immunoenzymatic assay by applying mycobacterial antigen specifically binding with human secretory immunoglobulin A only, and specific monoclonal antibodies directed against human secretory immunoglobulin A. The present method is considered to be of high efficiency, noninvasive, simple in application, of high information value, specificity and sensitivity to tuberculosis.

EFFECT: higher accuracy of diagnostics.

3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: method involves determining concentrations of carcinogens like aniline, toluyline, diethyl alanine, diphenyl and 1-naphtholamine in urine and urodynamic disorders and proliferative urothelium activity from Ki-67 expression degree data. High carcinogen concentrations and urodynamic disorders being detected apart from the fact of tumor removal and unchanged mucosa biopsy, transurethral prostate resection or urinary bladder neck resection is carried out. Next, liquid consumption increase in postoperative period and carcinogen contact reduction are advised. Three months later, control study is carried out. Carcinogen concentration level remaining high and no proliferative urothelium activity reduction being observed on the background of urodynamic disorders, cystectomy is to be repeatedly carried out.

EFFECT: enhanced effectiveness of treatment; high accuracy of diagnosis.

FIELD: medicine.

SUBSTANCE: method involves studying superficial lymphocyte receptors in heparinized venous peripheral blood sample with indirect immunofluorescence method using monoclonal antibodies of ICO series with relative CD3+ cells content being below 30%, CD4+ cells content being below 12.5%, and CD95+ cells content being below 7.7%, prognosis is considered to be unfavorable.

EFFECT: high accuracy of prognosis.

FIELD: microbiological and immunological methods.

SUBSTANCE: invention is intended for use to study structural and functional arrangement of nucleolus and mechanisms of action of pharmacological preparations on human cells. Mouse hybridoma cell strain, called A3, is obtained through fusing mouse splenocytes immunized by coarse fraction of nuclei of human cell line RAMOS with cells of mouse myeloma line P30X63-Ag8.653. Resulting hybridoma secretes monoclonal antibodies against antigen, called A3 antigen, localized inhuman cell nucleoli irrespective of their tissue or line origin. In cases of different cell fixation ways. A3 antigen is revealed as incorporated in discrete (typically several tens) foci located exclusively in the zone of nucleoli. Unique property of A3 antigen is its high sensitivity to the action of various protein synthesis inhibitors, e.g. emetine, anisomicyne, cycloheximide, and puromicyne. During incubation of cells in presence of above-listed substances, A3 antigen migrates from nucleoli to numerous foci located in nuclear nucleoplasma. This A3 antigen migration precedes apoptotic death of cells. Monoclonal antibodies A3 produced by the strain A3 are recommended to reveal nucleoli and to estimate total level of protein synthesis in human cells by cell biology techniques. Antibodies can be used to reveal possible contamination of human cell cultures with another-species cells as well as to investigate biological activity of known and novel protein synthesis inhibitors, including pharmacological preparations in human in vitro cells.

EFFECT: expanded microbiological and immunological possibilities.

2 cl, 6 dwg

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma T2/S-6E11 is prepared by fusion of murine myeloma strain Sp-2/0 and murine BALB/c splenocytes immunized with the TOPC virus, strain CoD, purified preparation inactivated with concentrate formaldehyde solution and by using polyethylene glycol-1000 Da and the following cloning by method of limited dilution. Prepared hybridoma T2/S-6E11 produces monoclonal antibodies (MCAb) to epitopes of the abovementioned pathogen. Specificity of prepared MCAb is shown by absence of cross-reactions in IFA-test with Hantaan and Ebol viruses and with noninfected substrates accumulated by TOPC virus. IFA-test system based on MCAb provides carrying out the specific detection of TOPC virus in analyzed samples. The sensitivity of this test-system based on MCAb is estimated to be 2.5 x 103 plaque-forming units (virus) in cubic centimeter (PFU/cm3). Using the invention in immunological researches in creature of diagnosticum used for detection of TOPC virus in samples provides enhancing the specificity of analysis in detection of coronaviruses.

EFFECT: valuable properties of strain.

3 tbl, 1 ex

FIELD: biotechnology, virology.

SUBSTANCE: invention relates to preparing a new strain of hybrid cells of Mus musculus L., NIIMB-280 (9E2), as a producer of monoclonal antibodies to the West Nile virus (WNV) protein E. West Nile virus (strain WNV/LEIV-VIg99-27889) is isolated in Volgograd district in 1999 year from a patient. Producing monoclonal antibodies can be used effectively for detection of the strain WNV/LEIV-VIg99-27880 of WNV that causes human diseases in Russia territory. New hybrid strain of cells is obtained by fusion of murine myeloma cells p3-X63/Ag8.653 (NS0/1) with murine splenocytes BALB/c immunized with the purified and inactivated preparation WNV (strain WNV/LEIV-VIg99-27889). The strain of hybrid cells Mus musculus L., NIIMB-280 (9E2), is deposited in Collection of cellular cultures of NII cellular cultures GNTS VB "VEKTOR" at number № NIIMB-280. Author's name of hybridoma cellular strain is 9E2. Using hybridoma allows preparing specific monoclonal antibodies raised to the West Nile virus protein E that, in turn, gives a possibility for identification of WNV and to standardize the content of protein E in immunodiagnostics.

EFFECT: valuable properties of strain.

1 dwg, 3 ex

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma strain is prepared by fusion of murine plasmocytoma Sp2/0-Ag.8 and B-lymphocytes of murine spleen of the inbred strain BALB/c immunized with protein-polysaccharide complex from Y. enterocolitica. Hybridoma produces monoclonal antibodies of isotype IgG to Y. enterocolitica O3 and O9 serovars used as components of IFA-test-system for identification of indicated serovars that are isolated most often in European areas from sick humans, agricultural animals and from objects of environment. The usage of monoclonal antibodies producing by hybridoma allows carrying out the identification of Y. enterocolitica strains of indicated serovars representing the most epidemic danger among other intestine-persistent microorganisms. Invention can be used in the development of diagnostic test-systems for identification of Y. enterocolitica strains O3 and O9 serovars for aims laboratory diagnosis in the public health, veterinary science and in carrying out scientific investigations.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

FIELD: biology, hybridoma technology.

SUBSTANCE: invention represents a new strain of mammalian hybrid cells C3/S-3E5 of Mus musculus L. producing monoclonal antibodies (MCAb) to Bernet's coxiellas (strain "Grita") in cell cultures and abdominal cavity of syngenic animals. Hybridoma C3/S-3E5 producing MCAb to this pathogen is obtained by fusion of murine myeloma of strain Sp-2/0 and murine splenocytes of strain BALB/c immunized with the concentrated and purified Bernet's coxiella preparation (strain "Grita) inactivated with formalin using polyethylene glycol of molecular mass 1000 Da as a fusing agent and the following cloning by method of maximal dilutions. Specificity of prepared MCAb: absence of cross-reactions in IFA with Provacheck's rickettsia antigen and with the non-infected accumulation substrate. Using prepared MCAb it is possible to carry out specific detection of Bernet's coxiellas by method IFA (direct and indirect variants). IFA sensitivity based on these MCAb is 2.0 x 103 ID50 x cm-3 for white rats. Applying the present invention allows detecting and identifying pathogens of rickettsial etiology.

EFFECT: improved method preparing, valuable properties of strain.

3 tbl, 1 dwg, 1 ex

FIELD: biology, biotechnology, medicine.

SUBSTANCE: strain of murine hybridoma cells is prepared by fusion of murine splenocytes immunized with cell lysates of lymphoblastoid line RAMOS with cells of murine myeloma. Hybridoma secrets monoclonal antibodies directed to antigen of molecular mass 110 kDa located on microtubules in cell cytoplasm and detected in nuclear cells of different species. Using the invention allows studying pathogenesis of cell dividing and state of mitotic activity of health and malignant cells, and evaluation of anti-mitotic (anti-tumor) effect of different chemopreparations and substances. Invention can be used for identifying phase of mitotic activity of cells.

EFFECT: valuable properties of strain.

2 dwg

The invention relates to biotechnology, in particular hybridoma technology

The invention relates to immunology and can be used to generate antibodies, the diagnosis of autoimmune diseases and the treatment of rheumatoid arthritis
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