Monoclonal antibody specifically binding to MRS
11518817 · 2022-12-06
Assignee
Inventors
Cpc classification
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/10
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to an anti-MRS monoclonal antibody and, more specifically, to an antibody or a fragment thereof characterized by specifically binding to a fragment represented by amino acid 861-900 of a human-derived methionyl-tRNA synthetase (MRS) protein set forth in SEQ ID NO:1, a method for producing the same, and a composition for diagnosing cancer comprising the same. The antibody or the fragment thereof of the present invention specifically binds to the human-derived MRS, and has no cross-reactivity with other proteins comprising the same ARS family. Therefore, as MRS detection is possible, the antibody or a fragment thereof can be effectively used for diagnosing MRS-related cancer.
Claims
1. An antibody or an antigen-binding fragment thereof which binds specifically to a peptide fragment consisting of the 861.sup.st to 900.sup.th amino acid residues of human-derived methionyl-tRNA synthetase (MRS) protein as set forth in SEQ ID NO:1, wherein the antibody or the fragment comprises: a light chain variable region (VL) comprising a light chain complementarity determining region 1 (CDR1) comprising an amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:15; a light chain complementarity determining region 2 (CDR2) comprising an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:17; and a light chain complementarity determining region 3 (CDR3) comprising an amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:19; and a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (CDR1) comprising an amino acid sequence as set forth in SEQ ID NO:9 or SEQ ID NO:21; a heavy chain complementarity determining region 2 (CDR2) comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:23; and a heavy chain complementarity determining region 3 (CDR3) comprising an amino acid sequence as set forth in SEQ ID NO:13 or SEQ ID NO:25.
2. The antibody or the fragment thereof of claim 1, wherein the antibody or the fragment comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: the light chain variable region (VL) comprising the light chain complementarity determining region 1 (CDR1) comprising the amino acid sequence as set forth in SEQ ID NO:3, the light chain complementarity determining region 2 (CDR2) comprising the amino acid sequence as set forth in SEQ ID NO:5, and the light chain complementarity determining region 3 (CDR3) comprising the amino acid sequence as set forth in SEQ ID NO:7, and the heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (CDR1) comprising an amino acid sequence as set forth in SEQ ID NO:9, the heavy chain complementarity determining region 2 (CDR2) comprising the amino acid sequence as set forth in SEQ ID NO:11, and the heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequence as set forth in SEQ ID NO:13; and the light chain variable region (VL) comprising the light chain complementarity determining region 1 (CDR1) comprising the amino acid sequence as set forth in SEQ ID NO:15, the light chain complementarity determining region 2 (CDR2) comprising the amino acid sequence as set forth in SEQ ID NO:17, and the light chain complementarity determining region 3 (CDR3) comprising the amino acid sequence as set forth in SEQ ID NO:19, and the heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (CDR1) comprising an amino acid sequence as set forth in SEQ ID NO:21, the heavy chain complementarity determining region 2 (CDR2) comprising the amino acid sequence as set forth in SEQ ID NO:23, and the heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequence as set forth in SEQ ID NO:25.
3. The antibody or the fragment thereof of claim 1, wherein the light chain variable region comprises a amino acid sequence as set forth in SEQ ID NO:27 or SEQ ID NO:31 and the heavy chain variable region comprises a amino acid sequence as set forth in SEQ ID NO:29 or SEQ ID NO:33.
4. The antibody or the fragment thereof of claim 1, wherein the antibody is selected from the group consisting of IgG, IgA, IgM, IgE and IgD, and the fragment thereof is selected from the group consisting of diabody, Fab, Fab′, F(ab)2, F(ab′)2, Fv and scFv.
Description
BRIEF DESCRIPTION OF DRAWINGS/FIGURES
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MODE FOR CARRYING OUT INVENTION
(7) Hereinafter, the present invention will be described in more detail with reference to examples, experimental examples and manufacturing examples. However, the following examples, experimental examples and preparation examples are illustrative of the present invention, and the present invention is not limited to the following examples, experimental examples and manufacturing examples.
(8) <Experimental Methods>
(9) Cell Culture
(10) Each of 293T, H460 and Panc-1 cells were cultured in DMEM medium and cells with passages 5 to 9 were used. Different cell lines were incubated in RPMI-1640 (Hyclone, GE lifesciences) and DMEM (Hyclone, GE lifesciences) media containing 10% FBS (fetal bovine serum, Hyclone, GE lifesciences), 1% penicillin (Hyclone, GE lifesciences). Cells were incubated in a condition of 5% CO.sub.2, 37° C.
(11) Animal Models
(12) 10-week-old BALB/c mice weighing 25˜30 g were purchased from Orient Bio Co (Sungnam, Kyunggido, Republic of Korea). They were used in this study after sufficient acclimation under constant conditions (temperature: 20±2° C., humidity: 40˜60%, 12 hour light/dark cycle) at the animal facility. Animal experiments were performed following the guidelines of the University Animal Care and Use Committee of Seoul National University.
Example 1
Selection of Cells Producing Monoclonal Antibodies Against MRS
(13) 1-1. Synthesis of MRS-AIMP3 Proteins
(14) In order to express and purify MRS-AIMP3 (aminoacyl tRNA synthetase complex-interacting multifunctional protein 3) protein using E. coli, the following experiments were carried out.
(15) BL21DE3 strain was used to transform with MRS (methionyl-tRNA synthetase, SEQ ID NO:1) and AIMP3 (NM_004280.4, SEQ ID NO:49) and a single colony was incubated in 5 ml LB media containing ampicillin until OD600 value reaches 0.6˜0.8. Afterwards, 1 mM of IPTG was added thereto, followed by incubation at 37° C. for 3 hours, and centrifugation for 10 minutes to collect only cells. SDS-PAGE was performed with the cell solution and the expression was confirmed using Coomassie stain.
(16) Subsequently, cell solution in which overexpression was induced by IPTG was collected and centrifuged to obtain cells. After resuspension with 1 ml DPBS, cells were lysed using an ultrasonicator, and centrifuged to isolate co-purified MRS-AIMP3 protein from the lysates.
(17) 1-2. Immunization of Mice
(18) In order to obtain immunized mice required for the production of hybridoma cells, the first injections of the co-purified MRS-AIMP3 proteins obtained in Example 1-1 were administered intraperitoneally to four 8-10 week old mice. Two weeks later, the second injections with the same dose of the co-purified MRS-AIMP3 proteins were made into the abdominal cavity of mice in order to increase the immunity of the mice after the first immunization. One week later, three days before the cell fusion experiment, the co-purified MRS-AIMP3 proteins were injected into the tail vein of mice as a booster.
(19) Those immunized mice were anesthetized with ether and blood was drawn from the heart with a heparinized syringe. Collected blood was left overnight at 4° C. and centrifuged to separate serum. Separated serum was properly and stored at −80° C.
(20) 1-3. Preparation of Hybridoma Cells
(21) First, myeloma cells were prepared for cell fusion. Myeloma cells were cultured and adjusted the cell density to 2.5˜5.0×10.sup.4/ml. 24 hours prior to cell fusion, myeloma cells were diluted by ⅓. The immunized mice in Example 1-2 were anesthetized with ether and spleens were collected. B cells were isolated, washed with SF-DMEM2 (DMEM+2×AA) and eluted. Cell suspension was collected, placed in a tube and allowed to settle down. Supernatant was transferred to a new tube and centrifuged for 5 minutes at 1500 rpm. Supernatant of the centrifuged splenocytes was removed and tapped before filling with SF-DMEM2. B cells and myeloma cells were respectively centrifuged and washed and washing was repeated one more time. Supernatant of the washed myeloma cells was removed, and cells were tapped before filling with SF-DMEM2. In addition, after removing supernatant of the washed B cells and tapping, red blood cells (RBC) were added to 1 ml of LB (lysis buffer), followed by filling with SF-DMEM2. Then, B cells and myeloma cells were centrifuged, respectively, and supernatant was removed, tapped and filled with 10 ml of SF-DMEM2. Cell concentration was determined by counting the B cells and myeloma cells diluted by 100 fold in e-tubes, respectively [concentrations of B cells (1×10.sup.8, 8×10.sup.7, 5×10.sup.7), and myeloma cells (1×10.sup.7, 8×10.sup.6, 5×10.sup.6). The ratio of B cells and myeloma cells was determined to be 10:1. B cells and myeloma cells of the determined concentrations were put together in a tube and centrifuged. Supernatant of the centrifuged cells was removed and then semi-dried by placing on an alcohol pad and tapped for 30 seconds to 1 minute. Here, PEG (2 ml) was added slowly by pipetting and reacted for 1 minute, and SF-DMEM2 was added while shaking the tube and centrifuged. After centrifugation, supernatant was removed and, without tapping, HT media [HT50×(HT(sigma) 1 vial+SF-DMEM1 10 ml) 1 ml, FBS 10 ml, SF-DMEM1(DMEM+1×AA) 30 ml] was dropped with increasing speed and the volume was increased to 50 ml. This suspension was incubated again in an incubator at 37° C., 5% CO.sub.2 for 3 hours.
(22) 1-4. Selection of Hybridoma Cells Producing Monoclonal Antibodies and Cloning
(23) In order to select cells which recognize MRS well and do not interact with AIMP3 among the fused cell groups prepared in Examples 1-3, and to check whether antibodies were produced, the following experiments were performed.
(24) First, medium was exchanged 8 to 9 days after cell fusion, and cells were cultured in cDMEM2 until growing well from 96 wells to 24 wells. On day 5˜7 after replacing the medium, supernatant of the color-changed wells was withdrawn and filled with cDMEM2, and then subjected to ELISA test. After ELISA test, wells were selected and transferred to 24 wells for incubation. After incubation in 24 wells, ELISA test was repeated. Specifically, the concentration of the fusion cells in 24 wells was confirmed, and the fusion cells were diluted in 15 ml of culture medium at the concentration of 0.5 cell/well in a 96 well plate. Fusion cell dilutions were dispensed at 150 μl per well. Wells containing a single cell were checked by microscopic examination. Supernatant of wells where cells had grown to a certain degree was harvested and examined by ELISA and western blot to perform the first screening. Based on the first screening results, the selected fusion cells were transferred to 24 wells, cultured, centrifuged, and supernatant was collected and confirmed by ELISA and western blot for the second screening. Absorbance (OD value) of the fusion cells grown in 24 wells was confirmed by ELISA, and only those with absorbance value greater than 1.0 were selected and transferred to a 25T/C culture flask, incubated, and centrifuged, and supernatant was collected and examined by ELISA and western blot for the third screening. Fusion cells selected based on the third screening results were then transferred to a 75T/C culture flask, cultured, and absorbance was confirmed by ELISA to select cells that recognize only MRS well and not AIMP3. Finally, clones “1E8” and “8A12” were secured.
Example 2
Production of Monoclonal Antibodies Against MRS and Purification
(25) 2-1. Hybridoma Cell Culture and Production of Monoclonal Antibodies Against MRS
(26) From the finally selected fusion cells (hybridoma cells, “1E8” and “8A12”) in Example 1, following two methods can be used to obtain, respectively.
(27) 1) 500 μl of pristane was injected into the abdominal cavity of female mice at 7 to 8 weeks of age. Fusion cells cultured in a 75T/C culture flask were collected, centrifuged, and pipetted in phosphate buffer after removing supernatant. After 7-10 days of pristane administration, the fusion cells selected in Example 1-4 were injected intraperitoneally to mice at 8×10.sup.5˜4×10.sup.7, respectively. After 1-2 weeks when the mouse peritoneum is filled with ascites, the ascites was drawn using an 18G needle. The ascites was kept at 4° C. overnight and centrifuged the following day to remove the mass material including the yellow fat layer and to separate only supernatant. Isolated supernatant was aliquoted and stored at −20° C.
(28) In order to purify antibodies from the ascites fluid, a column was filled with an appropriate amount of Protein A stored in the storage solution (20% ethanol), flushed with 20% ethanol, and washed using binding buffer (20 mM sodium phosphate, pH 7.0) of 5 bed volumes. The ascites solution was diluted in phosphate buffer in an appropriate amount and loaded onto the Protein A column. After binding with 3 bed volumes of binding buffer (20 mM sodium phosphate, pH 7.0), 0.5 mL fractions were eluted with 3 bed volumes of elution buffer (0.1M glycine buffer, pH 3.0˜2.5). Each fraction was neutralized with 35 μl of neutralization buffer (1M Tris-HCl, pH 9.0). After standing overnight in 70% ethanol at refrigeration temperature, it was again stored in the storage solution (20% ethanol) until the next use. The purity of the fractions was confirmed via SDS-PAGE and desalted on Amersharm GE columns.
(29) 2) The hybridoma cells were cultured in up to 860 mL of culture media using Cellstack-5 (Corning, N.Y.). Serum-free medium (Thermo) was supplemented with 5 mM GlutaMAX (Gibco) and 1×Cholesterol lipid concentrate (Gibco) and inoculated with the cells at the initial concentration of 1.4˜2.0×10.sup.5 cell/mL. After 4˜5 days of dispensing, cells were removed by centrifugation at 2000 rpm for 10 minutes to recover the supernatant. After checking pH of the supernatant, it was adjusted to pH 7.6 using the prepared 20×binding solution (1M potassium phosphate dibasic) (pH 9.0). Then it was filtered using a 0.22 um filter to obtain a neutralized antibody culture solution.
(30) 2-2. Purification of Monoclonal Antibodies Against MRS
(31) The antibody culture solution obtained in Example 2-1 or 2-2 was purified by the following method. A column was charged with an appropriate amount of Protein A and flowed with 10 column volumes of distilled water, followed by the same amount of 1× binding solution (50 mM potassium phosphate dibasic) (pH 9.0). Then, the column was flowed with the obtained antibody culture solution to bind antibodies to Protein A, and then washed with 1× binding solution (50 mM Potassium phosphate dibasic) (pH 9.0). Next, eluates were obtained by flowing 2 column volumes of the elution solution (0.2M citric acid) (pH 3.0), neutralized using 1M Tris, and concentrations were checked by measuring absorbance at 280 nm. The GE PD-10 column was equilibrated with 25 ml of physiological saline and then centrifuged (1000 g, 2 minutes). Then, 2.5 ml of the antibody eluate obtained from the protein A column was added into the column and centrifuged (1000 g, 2 minutes) to exchange the antibody solution with saline. The antibody concentration was then measured using absorbance at 280 nm, aliquoted and stored at −80° C.
(32) 2-3. Sequence Analysis of Monoclonal Antibodies Against MRS and Cloning
(33) Sequences of 1E8 antibody and 8A12 antibody obtained in the above examples were analyzed by YBIO Inc. and AbClon Inc., Korea. RNA was extracted from the hybridoma cells obtained in Example 1 to synthesize cDNA. Next, PCR was performed using VL, CL, VH, or CH specific primers. PCR products of expected size were separated on the agarose gel and purified to check the sequence through sequencing analysis. CDR domains were identified by Kabat numbering, and Fab was synthesized from the identified sequences, and it was proven that the antibody had high binding ability to MRS by using ELISA method.
(34) In addition, it was confirmed that the sequences of the antibodies were in agreement with the results of mass spectrometry analysis of the protein sequences of the antibodies obtained through the ascites purification after injecting the hybridoma cells into the mouse peritoneum.
(35) The obtained 1E8 Fab sequences and 8A12 Fab sequences were cloned into mouse IgG heavy chain vector (pFUSE-mIgG2a-Fc, InvivoGen) and mouse light chain sequence vector (pFUSE2-CLIg-mK, InvivoGen). Next, those vectors were subjected to co-transfection into freestyle 293F cells using PEI (Polysciences, 23966-2) to have the light and heavy chains of each antibody express simultaneously. Co-transfected 293F cells were incubated at 37° C., 8% CO.sub.2 for 7 days. Then, cells were centrifuged to obtain supernatant, and pH of the supernatant was adjusted to 7.6 using a prepared 20× binding solution (1M potassium phosphate dibasic, pH 9.0). Then, the supernatant was filtered with a 0.22 μm filter to obtain a neutralized antibody culture solution. Antibodies were collected from the antibody culture solution by the method described in Example 2-2. It was confirmed that thus obtained whole antibody of 1E8 IgG consists of a light chain having the amino acid sequence of SEQ ID NO:35 and a heavy chain having the amino acid sequence of SEQ ID NO:36, and the whole antibody of 8A12 IgG consists of a light chain having the amino acid sequence of SEQ ID NO:37 and a heavy chain having the amino acid sequence of SEQ ID NO:38.
Example 3
Binding Specificity of Antibodies Against MRS
(36) 3-1. Western Blot Experiment Using Anti-MRS Antibody
(37) In order to confirm MRS binding ability of 1E8 and 8A12 antibody obtained in the above Examples, experiments were performed as follows.
(38) H460 cells were cultured according to the method described in the above examples and treated with si-MRS for 72 hours. H460 cells were then harvested, lysed, and subjected to western blot. 1E8 antibody and 8A12 antibody were diluted 1:5000 (0.2 μg/ml) and used as primary antibodies. Commercially available anti-MRS antibody (Abcam, Ab50793) was used as well. Tubulin was used as a control.
(39) As a result, as shown in
(40) 3-2. ELISA Experiments Using Anti-MRS Antibody
(41) In order to examine the cross-reactivity of 1E8 and 8A12 antibodies obtained in the above examples with other aminoacyl-tRNA synthetase (ARS) proteins, experiments were performed as follows.
(42) Each well of 96 well plates (Corning 3690 flat bottom, 96-well half-area plates) was coated with different kinds of ARS proteins (His-MRS, MRS full, DX2 tag free, 34S-DX2, 34S-AIMP2, His-CRS, His-AIMP1, His-GRS, His-WRS, His-KRS) at the concentration of 1 μg/ml. 1E8 and 8A12 antibodies at a concentration of 500 ng/ml were added onto the 96 well plates coated with ARS proteins and allowed to react for 1 hour. Thereafter, HRP-conjugated anti-mouse IgG secondary antibody was added and reacted for 1 hour. ELISA was performed and absorbance was measured at 450 nm. TMB (3,3′,5,5′-tetramethylbenzidine) was used as substrate.
(43) As a result, as shown in
(44) 3-3. Examination of Antibody Affinity Using Surface Plasmon Resonance
(45) In order to confirm the affinity of the antibodies purified in Example 2, experiments were carried out as follows.
(46) Surface plasmon resonance (SPR) experiments were carried out using 1E8 and 8A12 antibodies and MRS+AIMP3 protein obtained in Example 1.
(47) CM5 chips were coated with MRS+AIMP3 or AIMP3 proteins and 1E8 or 8A12 antibodies were flowed at various concentrations to measure the degree of binding reaction with the proteins. Samples or buffers were injected for 8 minutes at a flow rate of 30 μl/min and washed for 20 minutes.
(48) As a result, as shown in
Example 4
Measurement of Antibody Reactivity Against MRS
(49) In order to check the immune activity of 1E8 antibody and 8A12 antibody obtained in Example 2, experiments were carried out as follows.
(50) Cultured Panc-1 cells were treated with 20 mM EDTA to detach cells, and then centrifuged. Then, the cover glass was put in a 6 well plate, 1 ml of culture medium was added, and cells at 1.0×10.sup.6 cells/ml were added and cultured at 37° C. Then, the culture medium was removed, and cells were fixed with methanol, followed by 0.2% PBST (PBS+tween 20) treatment and blocking with 2% goat serum (Abchem) for 1 hour. Subsequently, 1E8 and 8A12 antibodies were diluted at a ratio of 1:200 and reacted overnight at 4° C. Mouse IgG alexa 488 (Abchem) was diluted 1:200 with as the secondary antibody and reacted for 1 hour at room temperature. After washing with 0.2% PBST, cells were stained with DAPI (molecular probes) and observed with a fluorescence microscope (Nikon).
(51) As a result, as shown in
Example 5
Identification of Binding Site of Anti-MRS Antibody
(52) In order to identify the domains of 1E8 antibody and 8A12 antibody obtained in Example 2, the following experiments were performed.
(53) Six fragments of different lengths and positions were prepared based on the GST, catalytic domain, and tRNA binging domains of the MRS protein, and MRS protein and each of the MRS fragments were cloned into pcDNA3 vector (EV). The position of each MRS fragment is shown in Table 1 below. At this time, since Myc protein is linked to the N-terminus of MRS protein, Myc protein was used as a control.
(54) Then, 2 μg of the cloned vector DNA were transfected into H460 cells using Turbofect (Thermo) according to the manufacturer's instructions. After 24 hours, cells were harvested and subjected to western blot. 1E8 antibody and 8A12 antibody were diluted 1:5000 (0.2 μg/mL) as the primary antibody.
(55) As a result, as shown in
(56) Through this observation, it was confirmed that antibodies bind to 598˜900 aa.
(57) Based on this results, four fragments of different lengths and positions were prepared from the 598˜900 aa portion of MRS protein, and each fragment was cloned into pcDNA3 vector (EV). Then, western blot was performed in the same manner as described above.
(58) As a result, as shown in
(59) From this finding, it was confirmed that both antibodies bind to the 861˜900 aa position of the MRS protein.
(60) TABLE-US-00001 TABLE 1 MRS Sequence fragment Position Sequence number 1 1~266 aa MRLFVSDGVPGCLPVLAAAGRARGRAEVLISTVGP 40 EDCVVPFLTRPKVPVLQLDSGNYLFSTSAICRYFF LLSGWEQDDLTNQWLEWEATELQPALSAALYYLVV QGKKGEDVLGSVRRALTHIDHSLSRQNCPFLAGET ESLADIVLWGALYPLLQDPAYLPEELSALHSWFQT LSTQEPCQRAAETVLKQQGVLALRPYLQKQPQPSP AEGRAVTNEPEEEELATLSEEEIAMAVTAWEKGLE SLPPLRPQQNPVLPVAGERNV 2 267~417 aa LITSALPYVNNVPHLGNIIGCLVSADVFARYSRLR 41 QWNTLYLCGTDEYGTATETKALEEGLTPQEICDKY HIIHADIYRWFNISFDIFGRTTTPQQTKITQDIFQ QLLKRGFVLQDTVEQLRCEHCARFLADRFVEGVCP FCGYEEARGDQ 3 267~597 aa LITSALPYVNNVPHLGNIIGCVLSADVFARYSRLR 42 QWNTLYLCGTDEYGTATETKALEEGLTPQEICDKY HIIHADIYRWFNISFDIFGRTTTPQQTKITQDIFQ QLLKRGFVLQDTVEQLRCEHCARFLADRFVEGVCP FCGYEEARGDQCDKCGLKINAVELKKPQCKVCRSC PVVQSSQHLFLDLPKLEKRLEEWLGRTLPGSDWTP NAQFITRSWLRDGLKPRCITRDLKWGTPVPLEGFE DKVFYVWFDATIGYLSITANYTDQWERWWKNPEQV DLYQFMAKDNVPFHSLVFPCSALGAEDNYTLVSHL IATEYLNYEDGKFSKS 4 1~597 aa MRLFVSDGVPGCLPVLAAAGRARGRAEVLISTVGP 43 EDCVVPFLTRPKVPVLQLDSGNYLFSTSAICRYFF LLSGWEQDDLTNQWLEWEATELQPALSAALYYLVV QGKKGEDVLGSVRRALTHIDHSLSRQNCPFLAGET ESLADIVLWGALYPLLQDPAYLPEELSALHSWFQT LSTQEPCQRAAETVLKQQGVLALRPYLQKQPQPSP AEGRAVTNEPEEEELATLSEEEIAMAVTAWEKGLE SLPPLRPQQNPVLPVAGERNVLITSALPYVNNVPH LGNIIGCVLSADVFARYSRLRQWNTLYLCGTDEYG TATETKALEEGLTPQEICDKYHIIHADIYWRFNIS FDIFGRTTTPQQTKITQDIFQQLLKRGFVLQDTVE QLRCEHCARFLADRFVEGVCPFCGYEEARGDQCDK CGKLINAVELKKPQCKVCRSCPVVQSSQHLFLDLP KLEKRLEEWLGRTLPGSDWTPNAQFITRSWLRDGL KPRCITRDLKWGTPVPLEGFEDKVFYVWFDATIGY LSITANYTDQWERWWKNPEQVDLYQFMAKDNVPFH SLVFPCSALGAEDNYTLVSHLIATEYLNYEDGKFS KS 5 598~900 aa RGVGVFGDMAQDTGIPADIWRFYLLYIRPEGQDSA 44 FSWTDLLLKNNSELLNNLGNFINRAGMFVSKFFGG YVPEMVLTPDDQRLLAHVTLELQHYHQLLEKVRIR DALRSILTISRHGNQYIQVNEPWKRIKGSEADRQR AGTVTGLAVNIAALLSVMLQPYMPTVSATIQAQLQ LPPPACSILLTNFLCTLPAGHQIGTVSPLFQKLEN DQIESLRQRFGGGQAKTSPKPAVVETVTTAKPQQI QALMDEVTKQGNIVRELKAQKADKNEVAAEVAKLL DLKKQLAVAEGKPPEAPKGKKKK 6 298~900 aa SRLRQWNTLYLCGTDEYGTATETKALEEGLTPQEI 45 CDKYHIIHADIYRWFNISFDIFGRTTTPQQTKITQ DIFQQLLKRGFVLQDTVEQLRCEHCARFLADRFVE GVCPFCGYEEARGDQCDKCGLKINAVELKKPQCKV CRSCPVVQSSQHLFLDLPKLEKRLEEWLGRTLPGS DWTPNAQFITRSWLRDGLKPRCITRDLKWGTPVPL EGFEDKVFYVWFDATIGYLSITANYTDQWERWWKN PEQVDLYQFMAKDNVPFHSLVFPCSALGAEDNYTL VSHLIATEYLNYEDGKFSKSRGVGVFGDMAQDTGI PADIWRFYLLYIRPEGQDSAFSWTDLLLKNNSELL NNLGNFINRAGMFVSKFFGGYVPEMVLTPDDQRLL AHVTLELQHYHQLLEKVRIRDALRSILTISRHGNQ YIQVNEPWKRIKGSEADRQRAGTVTGLAVNIAALL SVMLQPYMPTVSATIQAQLQLPPPACSILLTNFLC TLPAGHQIGTVSPLFQKLENDQIESLRQRFGGGQA KTSPKPAVVETVTTAKPQQIQALMDEVTKQGNIVR ELKAQKADKNEVAAEVAKLLDLKKQLAVAEGKPPE ARKGKKKK 7 660~860 aa FVSKFFGGYVPEMVLTPDDQRLLAHVTLELQHYHQ 46 LLEKVRIRDALRSILTISRHGNQYIQVNEPWKRIK GSEADRQRAGTVTGLAVNIAALLSVMLQPYMPTVS ATIQAQLQLPPPACSILLTNFLCTLPAGHQIGTVS PLFQKLENDQIESLRQRFGGGQAKTSPKPAVVETV TTAKPQQIQALMDEVTKQGNIVRELK 8 660~900 aa FVSKFFGGYVPEMVLTPDDQRLLAHVTLELQHYHQ 47 LLEKVRIRDALRSILTISRHGNQYIQVNEPWKRIK GSEADRQRAGTVTGLAVNIAALLSVMLQPYMPTVS ATIQAQLQLPPPACSILLTNFLCTLPAGHQIGTVS PLFQKLENDQIESLRQRFGGGQAKTSPKPAVVETV TTAKPQQIQALMDEVTKQGNIVRELKAQKADKNEV AAEVAKLLDLKKQLAVAEGKPPEAPKGKKKK 9 730~900 aa GSEADRQRAGTVTGLAVNIAALLSVMLQPYMPTVS 48 ATIQAQLQLPPPACSILLTNFLCTLPAGHQIGTVS PLFQKLENDQIESLRQRFGGGQAKTSPKPAVVETV TTAKPQQIQALMDEVTKQGNIVRELKAQKADKNEV AAEVAKLLDLKKQLAVAEGKPPEAPKGKKKK
INDUSTRIAL APPLICABILITY
(61) As described above, the antibody or fragment thereof of the present invention specifically binds to human-derived MRS, and has no cross-reactivity with other proteins including the same ARS family, thus making it possible to detect MRS. Can be used.