KR20120024280A - Monoclonal antibody selectively recognizing homocysteine, hybridoma producing the antibody, test kit comprising the antibody and detection method using the antibody - Google Patents

Abstract

PURPOSE: A monoclonal antibody which seletively recognizes homocystein and a method for measuring the concentration of homocystein in blood using the same are provided to early diagnose and prevent vascular diseases. CONSTITUTION: A monoclonal antibody which specifcially binds to homocysteine protein is prepared by hybridoma of deposit number KCTC 11709BP. An antibody-antigen complex is used in detection by RIA, ELISA, immunofluorescence, or chemiluminescence binding method. A test kit for ELISA for detecting homocysteine in blood contains the monoclonal antibody.

Description

Monoclonal antibody that selectively recognizes homocysteine, hybridomas producing the same, test kits comprising the same, and detection methods using the same (monoclonal antibody selectively recognizing homocysteine, hybridoma producing the antibody, test kit comprising the antibody and detection method using the antibody}

The present invention relates to a monoclonal antibody that selectively recognizes homocysteine that is a causative agent of vascular disease, and a technique for measuring the concentration of homocysteine in the blood using the same, and specifically, to a monoclonal antibody that selectively recognizes homocysteine. To produce a monoclonal antibody through a hybridoma to produce a, and relates to a technique for accurately and easily diagnose the concentration of homocysteine in the blood using the same.

Homocysteine is a metabolite produced in the process of protein metabolism, but in small amounts it is harmless to the human body.However, if the blood levels increase due to metabolic abnormalities, the endothelial cells of the blood vessel walls are damaged and cardiovascular disease and cerebrovascular disease ( It is a causative agent that can cause vascular diseases such as cerebrovascular disease. Elevated levels of homocysteine are independent risk factors that increase the risk of cardiovascular disease in stages, and at the same time increase the effects of other risk factors, so it is known that mortality increases with higher homocysteine levels in patients with coronary artery disease. . On the other hand, the increase in blood homocysteine can be controlled by the administration of vitamin preparation, so measurement of blood homocysteine plays an important role in the prevention of vascular disease. Therefore, the blood concentration of homocysteine may be usefully used as a diagnostic and prophylactic indicator of coronary arteriosclerosis and cerebrovascular disease.

Homocysteine methods include radioenzyme assay, gas chromatography-mass spectrometry, high performance liquid chromatography (HPLC) by fluorescence detection, HPLC by electrochemical detection, automated ion exchange chromatography , Fluorescence polarization immunoassay (FPIA) and the like can be used. Among them, HPLC method has been used as a standard method, but internal standards should be used, precision and accuracy are very high, but skilled personnel are required, and a lot of time and labor is required in the analysis process. There is a disadvantage that cannot be reported. Therefore, efforts have been made to develop new methods that are more accurate and simple to overcome these problems, and an easy method of measuring homocysteine using an automated chemiluminescence immunoassay has been introduced. The principle of this method is to convert all forms of homocysteine into free form by a reducing agent, then convert it back to SAH using the S-adenosyl homocysteine (SAH) hydrolase enzyme, and then to the homocysteine concentration in the blood using a monoclonal antibody against it. To quantify. However, even in this method, the homocysteine reduction and enzymatic conversion process was required, which was complicated and inconvenient.

Therefore, the present inventors studied a method for quantifying the concentration of homocysteine in the blood without undergoing such reduction and enzymatic conversion, and by producing a monoclonal antibody that recognizes specific antigenic determinants of homocysteine, by using the reduction and enzymatic conversion process The present invention was completed to enable early diagnosis and prevention of vascular disease by measuring the concentration of homocysteine simply and accurately.

Therefore, an object of the present invention is to provide a monoclonal antibody that specifically binds to a homocysteine protein.

It is also an object of the present invention to provide a hybridoma cell producing the monoclonal antibody.

It is another object of the present invention to provide a method for measuring the concentration of homocysteine in blood simply and accurately by contacting the monoclonal antibody with a sample sample to examine the presence of the antigen-antibody complex of the homocysteine protein and the monoclonal antibody.

In addition, an object of the present invention is to provide a test kit for detecting homocysteine comprising the monoclonal antibody.

According to the present invention for achieving the above object, there is provided a monoclonal antibody that specifically binds to homocysteine, produced by hybridoma of Accession No. KCTC 11709BP.

In addition, according to the present invention there is provided a hybridoma cell of Accession No. KCTC 11709BP for producing the monoclonal antibody.

In addition, according to the present invention there is provided a test kit for detecting ELISA concentration of homocysteine in blood comprising the monoclonal antibody specifically binding to homocysteine.

According to the present invention, the concentration of homocysteine in the blood is characterized by examining the presence of the antigen-antibody complex of the homocysteine protein and the monoclonal antibody after contacting the monoclonal antibody specifically binding to the homocysteine protein with the sample sample. A measurement method is provided. In this method, the antigen-antibody complex is preferably detected by RIA, ELISA, immunofluorescence, color particle binding or chemiluminescent binding.

Since monoclonal antibodies of the present invention selectively recognize homocysteine, simple and accurate measurement of homocysteine levels in blood using such antibodies enables early diagnosis and prevention of vascular disease. Moreover, it is economical because it can be measured without the reduction and enzymatic conversion process required in the conventional method.

FIG. 1 shows homocysteine in 20 amino acids and two forms (type D? L) homocysteine through ELISA test using hybridomas producing four selected monoclonal antibodies; selenocysteine, glutaraldehyde. This is a picture that specifically selects bays.
2 is a test result of a competition ELISA to verify the binding specificity of monoclonal antibodies to homocysteine.
3 is an indirect ELISA test result to determine the homocysteine recognition site of the monoclonal antibody.
Figure 4 is a graph verifying the selective recognition of homocysteine using a 5,5-dithiobis-2-nitrobenzoic acid reagent (DTNB) to verify the presence of a siol group.
5 is a visualization of the recognition of homocysteine on bovine vascular endothelial cells using anti-homocysteine monoclonal antibody as a capture antibody and an anti-rat polyclonal antibody fluorescent as a second antibody. It is a photograph.

The present invention relates to monoclonal antibodies that specifically detect homocysteine in the blood. Specifically, it relates to a novel monoclonal antibody produced by hybridoma of Accession No. KCTC 11709BP, which specifically binds to a specific site of homocysteine protein to selectively recognize only homocysteine in blood.

The term 'monoclonal antibody' as used in the present invention refers to a highly specific antibody directed against a single antigenic site as known in the art. Unlike polyclonal antibodies, which typically include different antibodies directed against different epitopes, a monoclonal antibody is directed against a single determinant on an antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding and also have the further advantage that they are not contaminated by other immunoglobulins because they are synthesized by hybridoma culture.

The monoclonal antibody of the present invention immunizes an animal using homocysteine as an immunogen, and then, B cells obtained from lymph nodes of the immunized animal are fused with myeloma cells to generate hybridomas, and specificity analysis for homocysteine is performed. It is an antibody prepared by selecting hybridoma clones that have passed through the same.

In the present invention, L-homocysteine was used as an immunogen for monoclonal antibody preparation. As an embodiment of the present invention, L-homocysteine was conjugated to a carrier protein, BSA, and a glutaraldehyde coupling method. The prepared immunogen and adjuvant were mixed at a ratio of 1: 1 (v / v), and an emulsion of the antigen and the adjuvant was prepared using ultrasonic waves (ultrasonication).

Thereafter, the emulsion was used for immunization according to the rat lymph node method. Specifically, the emulsion was injected once with a total of 1 ml of 500 μl each into the hind paw of the rat, and a large number of immune cells were generated and swollen in the knee iliac lymph nodes by this injection. This is called the immunization process. B cells were extracted from the lymph nodes and used for the preparation of hybridomas.

Hybridomas were prepared by fusing the B cells with mouse myeloma cells. Mouse myeloma cells used for cell fusion have been depleted of HGPRT (Hypoxanthine-Guanidine-Phosphoribosyl-Transferase). Mammalian cells use two pathways to synthesize DNA: the de novo pathway and the salvage pathway using HGPRT. In the de novo pathway, phosphoribosyl pyrophosphate receives methyl or formyl groups by THF (tetrahydrofolate) and binds to uridine to form the basic structure of the nucleic acid base. At this time, aminopterin contained in the HAT medium has a structure similar to THF, which inhibits this process, thereby causing cell death from inhibiting the nucleic acid synthesis of myeloma cells. Among the myeloma cells, HGPRT-repaired cells, which had been removed by fusion with B cells, could survive in the HAT medium through the salvage pathway. If this property is used, only hybridomas can be grown. Therefore, they are grown in HAT medium until hybridomas are established. The limiting dilution method was used as a method of selecting only those producing the desired antibody among the hybridomas. This method selects clones propagated from one cell so as to be 1 cell or less per 96 wells. Repeating this operation at least twice can not only completely eliminate unnecessary cells that are competing opponents in the cells producing the desired antibody, but also can select clones with excellent proliferative capacity and high antibody production capacity.

As an embodiment of the present invention, the rat B cells and myeloma cells (myeloma cell p3) were mixed at a ratio of 2.5: 1, fused in polyethylene glycol, and cultured with HAT selective medium. Hybridomas produce antibodies and secrete them outside the cell during growth. Therefore, hybridoma cultures contain a large number of antibodies, which can be used without special purification. Specificity of the antibodies produced by the hybridomas was measured using the hybridoma culture solution. 65 wells containing hybridomas that survive in HAT selective medium and produce antibodies with homocysteine specificity from ELISA measurements were selected. Among them, single clones were isolated by limiting dilution method in the order of strong ELISA signal intensity, and specificity for homocysteine was measured by the culture of separated single clones. During this sequential process, 18 single clones were isolated from one well including 4 clones, and the selected clones were cultured. The culture supernatants were taken and screened by ELISA. As a result, six clones passed specificity analysis for homocysteine, and one cell with the strongest signal strength and easy antibody production was selected. The hybridomas were deposited with the accession number KCTC 11709BP to the Korean Collection for type Cultures (KCTC) on June 21, 2006.

As a result of the ELISA test using the culture supernatant of the hybridoma, the hybridoma selectively recognized homocysteine. The selected hybridomas were injected into the animal abdominal cavity, and monoclonal antibodies were isolated from the ascites of animals recovered after a certain period of time after injection.

Monoclonal antibodies were identified by direct indirect and competitive ELISA tests using purified monoclonal antibodies. As a result, the monoclonal antibody was identified as a monoclonal antibody that selectively recognizes L-homocysteine and D-homocysteine.

According to one aspect of the present invention, there is provided a method for measuring the concentration of homocysteine in blood using the monoclonal antibody which exhibits high specificity and sensitivity to blood homocysteine. Specifically, a method for detecting homocysteine concentration in blood is provided by contacting a sample sample with the monoclonal antibody that specifically binds homocysteine to examine the presence of an antigen-antibody complex of homocysteine and monoclonal antibody.

As used herein, the term 'antigen-antibody complex' refers to a combination of a homocysteine antigen and a monoclonal antibody that recognizes it to confirm the presence or absence of homocysteine in a sample. Detection of antigen-antibody complexes of the monoclonal antibodies and homocysteines of the invention can be carried out using methods as known in the art, for example spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbing, chemical, and other methods. Can be. For the purposes of the present invention, as an antigen-antibody complex detection method, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunofluorescence, color particle binding, chemiluminescent binding, etc. may be preferably used. .

Detection of antigen-antibody complexes involves the use of directly or indirectly labeled antibodies, and usable detection labels include, for example, biotin-streptavidin conjugates, fluorescent dyes, radiolabels, enzymes and color labels, e.g. For example, colloidal gold or colored glass or plastic beads. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241; Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene Oreg.)]

Particularly preferred method for detection of antigen-antibody complexes in the present invention is ELISA. According to the ELISA detection method, the sample sample is contacted with a monoclonal antibody of the present invention coated on a solid support such as a microtiter plate, membrane, test strip or the like. As a specific example, the wells of the microtiter plate are coated with the monoclonal antibody of the present invention and the unoccupied binding sites are blocked with BSA or the like, the wells of the coated plate are then incubated with the sample sample, followed by antigen-antibody complexes. Can be determined. The presence of the antigen-antibody complex can be confirmed using secondary antibodies specific for the antigen of the antigen-antibody complex. The specific secondary antibody may have a detection label, and may be identified by treating another tertiary antibody capable of detecting these specific antibodies in the absence of a detection label.

In the detection method of the present invention, a monoclonal antibody that can recognize an amino group or a carboxyl group may be preferably used as the second antibody that binds to the antigen bound to the monoclonal antibody that selectively recognizes homocysteine. Among them, many monoclonal antibodies that recognize all amino acids have been found and it is believed that these antibodies can be used. In addition, another method particularly preferable for the detection of an antigen-antibody complex in the present invention includes a gold particle binding method of binding a monoclonal antibody to colloidal gold particles.

According to another aspect of the present invention, there is provided a test kit for detecting homocysteine concentration in blood, comprising a monoclonal antibody that specifically binds to a homocysteine protein as described above. The test kit for detecting homocysteine concentration of the present invention may include reagents commonly used for immunological analysis in addition to monoclonal antibodies that selectively recognize homocysteine. Reagents used in immunological assays include suitable carriers, labels capable of producing detectable signals, solubilizers, detergents and the like. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

Examples of suitable carriers are soluble carriers, ie buffers such as physiologically acceptable PBS known in the art, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, polyacrylonitrile, fluororesins, crosslinked dextran , Polysaccharides, polymers such as latex-plated magnetic fine particles, paper, glass, metals, agarose and combinations thereof can be used.

Examples of assay systems for use in the homocysteine concentration detection methods and kits of the invention include ELISA plates, dip-stick devices, immunochromatography test strips and radiation split immunoassay devices, and flow-through devices. Etc. can be mentioned. As a test kit for detecting homocysteine of the present invention, preferably, an ELISA kit can be used.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited by the following Examples.

[Example]

Example  One: Hybridoma  Cell manufacturing

Preparation of the antigen

L-homocysteine used as an immunogen of the present invention was prepared by purchasing Sigma Aldrich products. L-homocysteine was used by conjugate with the carrier protein (BSA) and glutaraldehyde coupling (glutaraldehyde coupling) method. The prepared immunogen and adjuvant were mixed 1: 1, followed by ultrasonication to prepare an emulsion of the antigen and adjuvant.

immune

Thereafter, the emulsion was used for immunization according to the rat lymph node method. Specifically, the emulsion was injected once with a total of 1 ml of 500 μl each into the hind paws of 6-week-old female rats, followed by a first booster injection three days after the first antigen injection, and a second booster injection one week later. It was. The injection swells the lymph nodes, which means that the rats have been immunized, resulting in an increase in B cells against the antigen. All rats were housed in selected areas to avoid contamination.

Hybridoma manufacturing

Knee lymph nodes extracted at the sacrifice of immunized rats were pulverized, B cells were extracted, mixed with myeloma cells (myeloma cell P3) at a ratio of 2.5: 1, and treated with polyethylene glycol 4000 and fused. Cell precipitation was suspended with 100 ml of HAT selective medium, and 100 μl per well was dispensed into 96 well cell culture plates and incubated for 1 week in a 37 ° C., 5% CO ₂ incubator.

Screening and Isolation of Hybridomas

Hybridomas fused in the above process were first identified cells survived in the HAT selective medium through a microscope, and the antibody generating the antibody that recognizes the target antigen by ELISA using the medium of the wells containing the viable cells. Hybridomas were selected secondarily. As a result, 65 wells were selected from 10 96 well plates (960 wells). Selected wells were separated into single clones using the limiting dilution method in the order of strong ELISA signal intensity, and then subjected to specificity test for homocysteine by ELISA method to select monoclonal hybridomas that produce antibodies that recognize only homocysteine. Could.

Example 2: Antibody Production

Antibodies were obtained from the culture supernatants of the hybridomas selected in Example 1. Incubated at 37 ° C., 5% CO ₂ , using HT medium (RPI 1640 containing 10% heated inactivated FBS, 1% Gentamycin, 1% HT). HT is dissolved in 10 mM Hypoxanthine, 1.6 mM Thymidine in distilled water, heated at 70 ~ 80 ℃ for about 1 hour and completely dissolved, filter sterilization. After centrifuging the hybridoma culture solution obtained at this condition for 5 minutes at 1000 to 1500 rpm, the supernatant was aliquoted, and the 10 X PBS solution was added at a final concentration of 1 X and stored at 4 ° C. Used as antibody.

The test methods used in the test examples of the present invention are as follows.

Indirect ELISA Test

100 μl of glutaraldehyde diluted to 5% final concentration with 3 M sodium acetate on an amino group coated plate was dispensed into each well and incubated for 2 hours. Each well was washed three times with PBS solution to remove unreacted glutaraldehyde. Each amino acid was made into 10 mM using 3 M sodium acetate, 50 μl was dispensed per well and incubated for 2 hours. Instead of discarding the treated amino acid, 50 μl of a solution containing 1 M or more of sodium borohydride in sodium acetate was added to each well and incubated for 30 minutes to reduce the double bond of the glutaraldehyde amino group to a single bond. Each well was washed three times with PBS solution and then dried. 5% skim milk was made using PBS solution, 400 μl was added to each well, and incubated at room temperature for 1 hour to block nonspecific binding of the antibody. Each well was washed three times with PBS solution. Hybridoma culture medium containing the antibody as the primary antibody was dispensed into each well 50 [mu] l and incubated for 1 hour at room temperature. Each well was washed three times with TBS solution to remove antibodies that did not bind antigen. As a secondary antibody, anti-rat antibody was diluted 10,000-fold with TBS solution, and 100 μl of each well was added and reacted for 1 hour. Washed three times with TBS solution. To each well, 10 ml of the color reagent buffer solution was mixed with 5 mg of ABTS (2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) and 0.35 μl of 30% hydrogen peroxide. Color development was observed by dispensing the solution in μl and incubating at room temperature As a color reagent, 0.1 M citric acid (mono hydrate) and 0.2 M disodium hydrogen phosphate were prepared, and 0.2 M disodium hydrogen phosphate was used. Citric acid was prepared by titrating to pH 4.6.

Competitive ELISA Exam

ELISA measurement plates were prepared in the same manner as in the indirect ELISA test above and 5% skim milk was used to block nonspecific binding of the antibodies. Different concentrations of homocysteine at room temperature (control 0 mM, 1 × 10 ⁻³ mM, ¹ × 10 ⁻² mM, ¹ × 10 ⁻¹ mM, 10 mM) were preincubated for 2 hours at room temperature with the culture medium containing the antibody. 50 µl of each of the antibody cultures preincubated with homocysteine was aliquoted and incubated at room temperature for 1 hour. Each well was washed three times with TBS solution to remove antibodies that did not bind antigen. As a secondary antibody, anti-rat antibodies were diluted 10,000-fold with TBS solution, aliquoted at 100 μl per well and incubated for 1 hour. By washing three times with TBS solution, the secondary antibody that did not bind to the primary antibody was removed. The plate was developed in the same manner as above, and the absorbance was observed at 405 nm using an ELISA reader.

Staining Cells Using Immunofluorescence

The cover glass was placed inside the cell culture dish so that the cells could be attached to the cover glass upper surface, and the small vessel endothelial cells were incubated. At this time, RPMI 1640 medium containing 10% FBS, 1%, Gentamycin was used, and incubated at 37 ° C. and 5% CO ₂ . The surface of the cover glass to which the small blood vessel endothelial cells were attached was allowed to face upward and soaked in PBS solution. Intracellular free amino acids were fixed to surrounding proteins by treatment with 3 M sodium acetate buffer containing 5% glutalaldehyde. This method is called cell fixation method by glutaraldehyde. 1 M sodium borohydride was treated for 1 hour to reduce the double bond between glutaldehyde and the amino acid to a stable single bond. Reagents that did not participate in the reaction were washed three times with PBS buffer. 0.5% BSA was treated for 1 hour to block nonspecific bindings. As a primary antibody, hybridoma culture solution was diluted 100-fold and aliquoted and incubated at room temperature for 1 hour. Unbound antibodies were washed by washing three times with PBS buffer (5 minutes after PBS dispensing, 2 minutes after High Salt PBS dispensing, and 5 minutes after PBS dispensing). High Salt PBS means PBS containing 23.38 g of sodium chloride per liter. The anti-rat secondary antibody which develops fluorescence in the light-blocked place was diluted 1,000-fold and incubated for 1 hour at room temperature. It was wetted with a mount (30% glycerol) solution, covered with a clean cover glass, and observed under a fluorescence microscope.

DTNB method for checking the presence of a siol group

A cysteine and homocysteine dissolved in a culture solution containing an antibody and a PBS solution were prepared. Cysteine and homocysteine were respectively added to the culture medium containing the antibody and incubated at room temperature for 2 hours. The blank of ultraviolet absorption was mixed with 50 μl of DTNB solution, 100 μl of Tris-HCl (pH 8.0) solution and 850 μl of distilled water, and the sample was 50 μl of DTNB solution, 100 μs of Tris-HCl (pH 8.0) solution. 10 μl of the homocysteine and cysteine, the culture medium containing the antibody and the incubation of the antibody and homocysteine, and the incubation of the antibody and cysteine, respectively, before reacting with 840 μl of the second distilled water. Was observed.

Test Example 1: Homocysteine Recognition

ELISA and cell immunofluorescence tests were performed on the monoclonal antibody isolated in Example 2, and it was observed whether the antibody recognizes only homocysteine, and the results are shown in FIG. 1. Indirect ELISA analysis using anti-homocysteine monoclonal antibody as a capture antibody confirmed that only homocysteine was specifically selected from a total of 20 amino acids, selenocysteine, glutaraldehyde, and type D L-cytocysteine.

Test Example 2: Antibody Sensitivity

Competitive ELISAs were performed to determine the sensitivity of the antibodies using anti-homocysteine monoclonal antibodies as capture antibodies and anti-rat polyclonal antibodies as the second antibody. The results are shown in FIG. The binding of free amino acids and antibodies during pretreatment no longer binds amino acids immobilized on the bottom of the ELISA plate. The y-axis of the graph compares the ELISA signal intensity (Bo) when the free amino acid treatment was not performed with the ELISA signal intensity (B) according to the concentration of the pre-treated free amino acid. A decrease in B / Bo values means that the free amino acid and antibody are bound and the resulting free homocysteine can also be recognized. From the graph, homocysteine cyolactone, methionine, and cysteine showed no change in B / Bo value, whereas homocysteine decreased B / Bo value.

Test Example 3: Antigen Recognizer

Indirect ELISA was performed to determine the epitope of the anti-homocysteine monoclonal antibody, and the results are shown in FIG. 3. First, the results of color development according to the treatment of the iodoacetamide reagent blocking the siol group were confirmed. When blocking the siol group, no signal was detected, indicating that the monoclonal antibody recognizes the siol group of homocysteine. Second, since the monoclonal antibody recognizes CH ₂ CH ₂ SH of homocysteine without recognizing CH ₂ SH of cysteine, it can be seen that ethane siol (CH ₂ CH ₂ SH) is a recognition site. Since the antibody does not distinguish between D-type and L-type, it can be seen that the position of the amino group or the carboxyl group of homocysteine is not related to the recognition of the antibody.

Test Example 4: Selective Recognition of Homocysteine

The 5,5-dithiobis-2-nitrobenzoic acid (DTNB) reagent for verifying the presence of a siol group was used to verify whether the monoclonal antibody selectively recognizes homocysteine, and the results are shown in FIG. 4. . According to the contents of FIG. 3, since the antibody recognizes a siol group when it binds to homocysteine, if the antibody and homocysteine bind, the siol group is blocked and is not detected by the DTNB reagent. In the case of cysteine, the absorbance value was similar regardless of the reaction with the antibody, and in the case of homocysteine, the absorbance value decreased after the antibody reaction, so that the antibody specifically binds homocysteine and blocks its siol group. As a result, it was observed that the siol group detected by the DTNB reagent was reduced.

Anti-homocysteine monoclonal antibodies were used as capture antibodies and fluorescent anti-rat polyclonal antibodies as second antibodies were used to visualize homocysteine recognition on bovine vascular endothelial cells. The results are shown in Fig. The control group was treated with iodoacetamide blocking the siol group, or monoclonal antibody was preincubated with homocysteine and then treated with cells. As a result, fluorescence of the cells was observed only in the experimental group that was not treated with cells and antibodies. This is the same result as the above experiment, and this can be evidence that the antibody can recognize and bind the homocysteine fixed in the cell, and visualize the location and amount of homocysteine in the cell and further in the tissue.

Korea Research Institute of Bioscience and Biotechnology KCTC11709BP 20100621

Claims (5)

A monoclonal antibody that specifically binds to a homocysteine protein, produced by hybridoma of Accession No. KCTC 11709BP. A hybridoma cell of Accession No. KCTC 11709BP which produces the monoclonal antibody of claim 1. A method for detecting the concentration of homocysteine in blood, characterized by contacting a monoclonal antibody of claim 1 with a sample sample to examine the presence of an antigen-antibody complex of a homocysteine protein and a monoclonal antibody. The method for detecting the concentration of homocysteine in blood according to claim 3, wherein the antigen-antibody complex is detected by RIA, ELISA, immunofluorescence, color particle binding or chemiluminescence binding. A test kit for detecting ELISA concentration of homocysteine in blood comprising the monoclonal antibody specifically binding to homocysteine of claim 1.

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