KR101828939B1 - A monoclonal antibody for specifically binding heart-type fatty acid binding protein and hybridoma cell producing the same, and a method for preparing the same - Google Patents

Abstract

The present invention relates to a monoclonal antibody that specifically recognizes h-FABP (heart-type fatty acid binding protein), a hybridoma cell line that produces the monoclonal antibody, and a method of producing the same. The monoclonal antibody exhibits a high potency to h-FABP in the blood and specifically binds to it, so that it can be used in a diagnostic kit for detection of h-FABP.

Description

TECHNICAL FIELD [0001] The present invention relates to a monoclonal antibody specific for a heart-shaped fatty acid binding protein, a hybridoma cell producing the hybridoma cell, and a method for preparing the same. }

The present invention relates to a monoclonal antibody specifically binding to h-FABP (heart-type fatty acid binding protein), a hybridoma cell line producing the hybridoma, and a method for producing the same. More particularly, The present invention relates to a mouse monoclonal antibody that specifically recognizes the h-FABP protein, a hybridoma cell line producing the same, and a method for producing the same, and the monoclonal antibody described above is capable of inhibiting h-FABP eluted into blood And can be used to diagnose and treat acute myocardial infarction.

Acute myocardial infarction is a disease in which myocardial necrosis occurs because all three coronary arteries supplying oxygen to the myocardium are occluded by various causes and oxygen or nutrients are not supplied to all or part of the heart. Myocardial necrosis begins at the end of 30 minutes after coronary artery occlusion, and most of myocardial necrosis after 6 hours (Kim, Dong-Hwan, et al., Does acute myocardial infarction require simultaneous detection of CK-MB and troponin? Medical Journal, 2009; 20: 264-271).

Although the mortality rate of acute myocardial infarction is as high as 30%, it is difficult to distinguish between early diagnosis and precise differentiation because of similar early pain patterns such as chest pain (Kim, YoungJun et al., H-FABP diagnostic significance in acute myocardial infarction, There are cases in which the diagnosis is delayed and the treatment is not performed properly due to delayed treatment. Therefore, early and accurate diagnosis of acute myocardial infarction is important to increase the survival rate of patients and to expect a good prognosis of treatment.

Existing myocardial infarction markers include troponin, CK-MB, and myoglobin. Myoglobin is rapidly released from ischemic injured myocardial tissue, and its blood concentration begins to increase around 2 hours and then peaked at 6-9 hours. However, myoglobin is also abundant in the skeletal muscle, which may be due to other diseases or muscle damage Therefore, it is difficult to say that it is a marker specific to myocardial infarction. Troponin has three subtypes of I, T, and C, especially I and T, which are specific for myocardial tissues. However, it takes 3-6 hours to rise to the threshold value, there is a problem.

In contrast, h-FABP is a cytosolic protein with a molecular weight of 15 kDa first discovered by Ockner et al. In 1972, and is a fatty acid binding protein, mainly distributed in the cytoplasm of myocardial cells. Myocardial cell damage is progressively reduced from the initial stage of myocardial cell damage, and blood concentration begins to rise within 1.5 hours of symptom onset. Serum concentration reaches its maximum level at 6-8 hours after onset, and is detected faster than other myocardial infarction markers (Chan et al, A superior early myocardial infarction marker, human heart-type fatty acid-binding protein, Zeitschrift Fur Kardiologie , 2004; 93: 388-397).

[Prior Patent Literature]

Korean Patent Publication No. 10-2014-0015153

Disclosure of Invention Technical Problem [8] The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a monoclonal antibody capable of early diagnosis of myocardial infarction and appropriate medical treatment.

Another object of the present invention is to provide a hybridoma cell line that produces a monoclonal antibody that enables early detection of myocardial infarction and appropriate medical treatment.

In order to achieve the above object, the present invention provides a monoclonal antibody produced from hybridoma cells of accession number KCLRFBP 00361 or KCLRFBP 00362 and specific for heart-type fatty acid binding protein (h-FABP) do.

The present invention also provides a kit for diagnosing myocardial infarction comprising the antibody of the present invention.

The present invention also provides a myocardial infarction diagnostic strip comprising the monoclonal antibody according to the present invention.

In one embodiment of the present invention, the diagnostic strip is preferably immobilized on gold particles, nitrocellulose membrane, but not limited thereto.

The present invention also provides a latex antibody sensitization reagent for diagnosing myocardial infarction comprising the monoclonal antibody according to the present invention.

In one embodiment of the present invention, the diagnostic latex antibody sensitization reagent preferably has the antibody of the present invention immobilized on insoluble polystyrene latex particles,

The latex particle size of the diagnostic latex antibody sensitization reagent is preferably 0.01 um-0.5 um,

The diagnostic latex antibody sensitization reagent preferably measures an increase in turbidity due to the agglutination reaction of the latex particles sensitized with the antibody of the first aspect by an antigen to be measured, but is not limited thereto.

The present invention also provides a method for providing information on myocardial infarction using the monoclonal antibody of the present invention.

The present invention also provides a method for providing information on myocardial infarction using the diagnostic kit of the present invention.

The present invention also provides a method for providing information on myocardial infarction using the diagnostic strip of the present invention.

The present invention also provides a method for providing information on myocardial infarction using the diagnostic latex antibody sensitization reagent of the present invention.

The present invention also provides a composition for detecting heart-type fatty acid binding protein (h-FABP) comprising the monoclonal antibody of the present invention as an active ingredient.

The present invention also provides a method for detecting heart-type fatty acid binding protein (h-FABP) in whole blood, serum, or blood plasma samples using the monoclonal antibody of the present invention.

The diagnostic strip uses immunochromatography to diagnose a disease using an immune response to which an antigen-antibody binds. A test strip is prepared using a strip of immobilized antibody on a nitrocellulose membrane and a conjugate pad prepared by binding gold particles to another antibody. The test strip is then absorbed on the sample pad and moved along the nitrocellulose membrane. Acute myocardial infarction can be diagnosed using the fact that the antibody-gold particle conjugate of the conjugate pad binds to the antigen and then migrates the nitrocellulose membrane and binds to the antibody immobilized on the test line, have.

The diagnostic latex antibody sensitization reagent uses an immunoassay method by an antigen-antibody reaction. When the reagent (R2) sensitized with the antibody of the present invention is prepared to insoluble latex particles and then added to the stabilizing solution (R1) mixed with the sample, it is optically measured that the latex particles aggregate and turbidity increases when the antigen is present Acute myocardial infarction can be diagnosed.

Hereinafter, the present invention will be described.

The present inventors have found that a monoclonal antibody specifically detecting h-FABP that can be useful for early diagnosis of myocardial infarction and a single monoclonal antibody that is specific for myocardial injury and shorter in time to a detectable concentration in blood than the above- Clone antibody. ≪ / RTI >

The h-FABP of the present invention can be a full-length protein of h-FABP, a fragment thereof and mutants thereof, which can be usefully used for the early diagnosis of myocardial infarction. For example, h-FABP may be a protein having the amino acid sequence of SEQ ID NO:

The present invention relates to a hybridoma cell line producing a monoclonal antibody that specifically binds to h-FABP through a process of immunizing h-FABP to a mouse, and a mouse monoclonal antibody specific to h-FABP produced from the hybridoma cell line Lt; / RTI > antibody.

First, mice are immunized with recombinant h-FABP, and splenocytes extracted from the cells are fused with mouse myeloma cells SP2 / 0 to select h-FABP-specific cell clones by ELISA (enzyme-linked immunosorbent assay) .

The mouse hybridoma cells of the present invention selected by the above process were named 1G4 and 3F2 and were deposited at KCLRFBP 00361 and KCLRFBP 00362 at the Korean Cell Line Research Foundation (KCLRF) in Cancer Research Institute, Seoul National University Cancer Research Institute, And deposited on March 21, 2016.

In order to confirm whether the monoclonal antibody produced from the hybridoma cells of the present invention had antigen specificity for selectively recognizing h-FABP, recombinant h-FABP and negative control BSA and GST fusion protein The reactivity was confirmed by ELISA.

As a result, it was confirmed that the monoclonal antibody had high antigen recognition ability specifically for h-FABP.

The monoclonal antibody produced by the mouse hybridoma cells of the present invention can be used for early diagnosis of myocardial infarction by detecting h-FABP present in human blood.

The present invention includes a method for producing a hybridoma cell line that produces a monoclonal antibody and a monoclonal antibody that specifically binds to h-FABP rapidly forming a high concentration in blood immediately after myocardial injury. In the diagnosis of acute myocardial infarction, it is well known that the shorter the time from diagnosis to the diagnosis is, the better the prognosis of treatment. Therefore, the monoclonal antibody specific for h-FABP of the present invention is useful for accurate early diagnosis of acute myocardial infarction Can be used.

Brief Description of the Drawings Fig. 1 is a diagram showing a process for producing a monoclonal antibody that specifically binds to h-FABP of the present invention.
FIG. 2 is a result of mass culturing of 1G4 and 3F2 clones producing h-FABP-specific monoclonal antibodies in the abdominal cavity of mice, followed by separation and purification using affinity column chromatography.
FIG. 3 is a graph showing ELISA results confirming the binding specificity and potency of purified monoclonal antibody (1G4, 3F2) to h-FABP.
FIG. 4 shows the results of the immunochromatographic rapid diagnostic kit prepared and tested using a monoclonal antibody that specifically binds to the produced h-FABP as a raw material.
FIG. 5 is a quantitative curve of a latex reagent sensitized with h-FABP-specific monoclonal antibody.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example  1: Recombinant h- FABP  Produce

PCR was carried out using Biorad (USA) apparatus in 35 cycles of 95 ° C for 30 seconds, 53 ° C for 30 seconds, and 72 ° C for 1 minute using a primer of Table 1 in a patient with cardiac-related patients through IRB with a B hospital in Seongnam city Respectively.

PCR was performed using 0.4 uM primer of Hot Start Taq PCR MasterMix (Bioquest, USA) and electrophoresis on agarose gel to confirm that target DNA of about 400 bp size was obtained. After cloning using pGEX-4T1 vector with GST fusion, FABP3-pGEX-BL21 (codon +) was selected after transformation with BL21 (DE3) codon plus strain.

The transformed recombinant strain (FABP3-pGEX-BL21 (codon +)) was inoculated into LB liquid medium containing 50 μg / ml of ampicillin and cultured with shaking at 37 ° C. for 16 hours or more. Similarly, in a LB liquid medium containing ampicillin, the flask having a volume of at least 2 times the volume of the culture medium was inoculated in a 1/50 volume ratio and cultured at 37 占 폚. When the absorbance at 600 nm reached 0.7, the IPTG final concentration Overexpression induction was carried out at 1 mM, and 4 hours each were further cultured from the induction time point. The expressed culture was centrifuged at 16,000 x g for 3 minutes at 4 ° C to collect the cells. The separated cells were suspended in 5 ml of Bugbuster (Novagen, USA) protein extraction solution per 1 g of cells, and the supernatant was purified using FPLC to obtain the starting material.

Forward primer GC GGA TCC ATG GTG GAC GCT TTC (SEQ ID NO: 2) Reverse primer CG GTC GAG TCA TGC CTC TTT CTC (SEQ ID NO: 3)

Example  2: Immunization of mouse

To obtain immunized mice required for hybridoma cell preparation, 100 占 퐂 of recombinant h-FABP was diluted in 100 占 퐇 of phosphate buffered saline (PBS) and mixed with an equal volume of CFA (complete Freund's adjuvant, Sigma) . The emulsion was injected intraperitoneally into female BALB / c mice at 5 weeks of age to perform primary immunization. Fourteen days after the first immunization, the same amount of recombinant h-FABP was mixed with IFA (Incomplete Freund's adjuvant, Sigma) and immunized with the mouse by intraperitoneal injection three times at intervals of two weeks after emulsion preparation . Three days before the preparation of the fusion cell, 100 占 퐂 of recombinant h-FABP was diluted in 100 占 퐇 of phosphate buffered saline (PBS) and injected into the tail vein of the mouse.

Example  3: h- FABP  To produce a monoclonal antibody specific for Hybridoma  Cell Manufacturing

SP2 / 0 mouse myeloma cell line before cell fusion was first cultured in Dankook University, Department of Life Science and Biochemistry Lab.). Then, spleen cells were extracted from the spleen of the immunized mouse, and mixed so that the number of cells of the mouse myeloma cell line SP2 / 0 prepared earlier was 2: 1. Then, the mixed cell solution was centrifuged to remove supernatant, and 1 ml of PEG-1500 (polyethylene glycol-1500) was added for 1 minute to perform cell fusion. Fused cells were cultured in 96-well plates in HAT selection medium and cell colonies were formed and cultured in HT medium.

Example  4 : Monoclonal antibody  Produce Hybridoma  Cell sorting and Cloning

When the hybridoma cells prepared in Example 3 were continuously observed using a microscope to grow cells at a concentration of 10-20% at the bottom of each well, it was confirmed whether or not an antibody specifically reactive to h-FABP was produced And hybridoma cells were selected by performing an enzyme-linked immunosorbent assay (ELISA).

The 96-well plate was coated with h-FABP antigen, and the culture solution of the fusion cell line was added at a rate of 100 μl / well to react with horse radish peroxidase (HRP). Subsequently, the substrate solution was added, and the absorbance at 450 nm was measured using an ELISA reader (BioRad) to firstly select a cell line having an absorbance of 1 or more. The first screening of hybridoma cells was carried out by limiting dilution and then ELISA was performed to select two hybridoma cells with high absorbance. Ultimately selected hybridoma cells were transferred to a 75T / C culture flask and cultured for 1G4 and 3F2, respectively.

Example  5: Screened Hybridoma  H- FABP  Specific to Monoclonal antibody  production

In order to obtain a monoclonal antibody specific to h-FABP, a mouse abdominal culture fluid was prepared using the hybridoma cells selected in Example 4, and the antibody was isolated therefrom.

First, to obtain antibody ascites, 500 쨉 l / mouse of pristane was intraperitoneally injected into BALB / c mice before hybridoma cells were injected. Hybridoma cells selected in Example 4 were collected in a 75T / C culture flask and injected into the abdominal cavity of mice 7-10 days after the administration of Pristane. When the ascites was filled in the abdominal cavity of the mice after 7 to 14 days, each mouse was disassembled with cervical vertebrae, and the plurality was recovered using a syringe. The recovered ascites was separated by centrifugation, and the supernatant was collected and stored at -20 ° C.

Example  6: h- FABP  Specific to Monoclonal antibody  refine

The ascites recovered in Example 5 were purified by affinity column chromatography using protein G column. Before purification, the ascites recovered by ammonium sulfate were fractionally precipitated and passed through a Protein G column (GE health care, HiTrap protein G HP). The bound antibody was eluted with elution buffer (100 mM glycine-HCl, pH 2.7). Finally, monoclonal antibody purification was completed. (Fig. 2)

Example  7: Hybridoma  Cell-produced Monoclonal antibody  h- On FABP  Identification of binding specificity

Monoclonal antibodies produced from the hybridoma cells (1G4, 3F2) of the present invention were confirmed by ELISA (Enzyme-linked immunosorbent assay) in order to confirm the binding specificity for selectively recognizing h-FABP.

BSA and GST fusion proteins were mixed well in a coating buffer (0.05M Na-cabonate, pH 9.5) as a negative control, and coated on a 96-well ELISA plate at a concentration of 1 ug / ml per well. Lt; / RTI > buffer.

Thereafter, the solution was removed and the monoclonal antibody produced from the hybridoma cells (1G4, 3F2) of the present invention was reacted, and the antigen-recognition ability of the monoclonal antibody against the recombinant h-FABP was measured in the same manner as in Example 4 Respectively.

As a result, the monoclonal antibody produced by the hybridoma cells (1G4, 3F2) of the present invention showed high antigen recognition ability selectively for the recombinant h-FABP3 without recognizing the negative control BSA and GST fusion proteins . (Fig. 3)

1G4 10000 1000 100 10 One h-FABP 1.728 1.691 1.234 0.564 0.232 BSA 0.090 0.080 0.080 0.082 0.080 GST 0.059 0.058 0.065 0.066 0.051

3F2 10000 1000 100 10 One h-FABP 1.637 1.605 1.324 0.638 0.378 BSA 0.092 0.076 0.095 0.086 0.070 GST 0.057 0.052 0.051 0.061 0.066

Tables 2 and 3 show binding specificity and potency for h-FABP of monoclonal antibodies (1G4, 3F2)

{Antibody concentration: ng / ml}

Example  8 : Monoclonal antibody  Used diagnostics Kit  Manufacturing and testing

The monoclonal antibody (1G4) and the anti-mouse IgG specific to h-FABP prepared in Example 6 were dispensed into a nitrocellulose membrane at a concentration of 1 mg / ml on a nitrocellulose membrane and then dried at 37 ° C for 10 minutes or more To prepare an antibody-immobilized strip.

The other monoclonal antibody (3F2) was reacted with gold particles at a concentration of 0.1 mg / ml for 30 minutes at room temperature. BSA was added to the reaction solution in an amount of 1%, followed by further reaction for 30 minutes. The supernatant was removed by centrifugation, and the precipitate was dissolved in 1% BSA buffer to prepare an antibody-gold particle conjugate. Immunochromatographic rapid diagnostic kits were prepared using antibody - immobilized strips and antibody - gold particle conjugates.

The detection limit of h-FABP using recombinant h-FABP and negative sera was found to be about 7 ng / ml. (Figure 4)

Example  9: The stabilizing solution (hereinafter referred to as R1)  Produce

BSA was added to 75 mM HEPES buffer (pH 8.0) so as to be 0.5% to obtain R1.

Example  10: Insoluble On the carrier  Fixed antibody Sensitization  Preparation of reagent (hereinafter R2)

A 2% latex solution and 750 ug of the monoclonal antibody of the present invention per 10 mg of latex were stirred in MES buffer (pH 5.8) for 3 hours. BSA was added to 2%, the mixture was stirred for 1 hour to block, and the resulting latex was suspended in Tris-HCl buffer (pH 8.5) to obtain R2.

Example  11: h- FABP's  dose

The coagulation reaction with h-FABP was measured by a biochemical automatic analyzer (Beckman Coulter AU680) using R1 and R2 prepared in Examples 9 and 10. The measurement results at 450 nm wavelength were measurable from h-FABP concentration of 0 to 160 ng / mL as shown in Fig.

Korea Cell Line Research Foundation KCLRFBP00361 20160321 Korea Cell Line Research Foundation KCLRFBP00362 20160321

<110> HBI CO., LTD. <120> A monoclonal antibody specifically binding to heart-type fatty          acid binding protein and hybridoma cell producing the same, and a          method for preparing the same <130> P16-0232HS <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 133 <212> PRT <213> Homo sapiens <400> 1 Met Val Asp Ala Phe Leu Gly Thr Trp Lys Leu Val Asp Ser Lys Asn   1 5 10 15 Phe Asp Asp Tyr Met Lys Ser Leu Gly Val Gly Phe Ala Thr Arg Gln              20 25 30 Val Ala Ser Met Thr Lys Pro Thr Thr Ile Ile Glu Lys Asn Gly Asp          35 40 45 Ile Leu Thr Leu Lys Thr His Ser Thr Phe Lys Asn Thr Glu Ile Ser      50 55 60 Phe Lys Leu Gly Val Glu Phe Asp Glu Thr Thr Ala Asp Asp Arg Lys  65 70 75 80 Val Lys Ser Ile Val Thr Leu Asp Gly Gly Lys Leu Val His Leu Gln                  85 90 95 Lys Trp Asp Gly Gln Glu Thr Thr Leu Val Arg Glu Leu Ile Asp Gly             100 105 110 Lys Leu Ile Leu Thr Leu Thr His Gly Thr Ala Val Cys Thr Arg Thr         115 120 125 Tyr Glu Lys Glu Ala     130 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gcggatccat ggtggacgct ttc 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cggtcgagtc atgcctcttt ctc 23

Claims (14)

Accession number KCLRFBP 00361 or KCLRFBP 00362 Monoclonal antibody produced from hybridoma cells and specific for heart-type fatty acid binding protein (h-FABP). A kit for diagnosing myocardial infarction comprising the antibody of claim 1. A diagnostic myocardial infarction strip comprising a monoclonal antibody according to claim 1. 4. The strip of myocardial infarction according to claim 3, wherein the diagnostic strip is immobilized on the gold particle, nitrocellulose membrane. A latex antibody sensitization reagent for diagnosing myocardial infarction comprising the monoclonal antibody according to claim 1. The latex antibody sensitization reagent according to claim 5, wherein the diagnostic latex antibody sensitization reagent is immobilized on the insoluble polystyrene latex particle with the antibody of the first aspect. The latex antibody sensitization reagent according to claim 5, wherein the latex particle size of the diagnostic latex antibody sensitization reagent is 0.01 um-0.5 um. The immunoassay according to claim 5, wherein the diagnostic latex antibody sensitization reagent is optically measured for an increase in turbidity due to the agglutination reaction of the latex particles sensitized with the antibody of the first aspect, Reagent, latex antibody sensitization reagent. A method for providing information on myocardial infarction using the monoclonal antibody of claim 1. A method for providing information on myocardial infarction using the diagnostic kit of claim 2. A method for providing information on myocardial infarction using the diagnostic strip of claim 3. A method for providing information on myocardial infarction using the diagnostic latex antibody sensitization reagent of claim 5. A composition for detecting heart-type fatty acid binding protein (h-FABP) comprising the monoclonal antibody of claim 1 as an active ingredient. A method for detecting heart-type fatty acid binding protein (h-FABP) in whole blood, serum, or plasma sample using the monoclonal antibody of claim 1.

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