ANTIBODY SPECIFICALLY BINDING TO WRS PROTEIN, AND USE THEREOF
20220252601 · 2022-08-11
Inventors
- Sumi BAE (Chungcheongbuk-do, KR)
- Sunghwa SON (Chungcheongbuk-do, KR)
- Yunsun KIM (Chungcheongbuk-do, KR)
- Jieun PARK (Chungcheongbuk-do, KR)
- Min Chul PARK (Gyeonggi-do, KR)
- Su Jin KANG (Gyeonggi-do, KR)
Cpc classification
G01N33/57484
PHYSICS
C07K2317/92
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to an antibody specifically binding to a WRS (tryptophanyl-tRNA synthetase) protein, and a use thereof. More specifically, the present invention pertains to an antibody specifically binding to a polypeptide of an amino acid sequence represented by SEQ ID NO:2 in the WRS (tryptophanyl-tRNA synthetase) protein, or a fragment of the antibody, a polynucleotide encoding the antibody, a vector comprising the polynucleotide, a cell transformed using the vector, and a use of the cell.
Claims
1. An antibody or a fragment thereof specifically binding to a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 in a WRS (tryptophanyl-tRNA synthetase) protein.
2. The antibody or the fragment thereof according to claim 1, wherein the antibody or the fragment thereof is selected from the group consisting of: (1) an antibody or a fragment thereof comprising an antibody light-chain variable region (VL) comprising a complementarity-determining region (CDR) L1 comprising an amino acid sequence represented by SEQ ID NO: 5, a complementarity-determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 6, and a complementarity-determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 7, and an antibody heavy-chain variable region (VH) comprising a complementarity-determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 8, a complementarity-determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 9, and a complementarity-determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 10; (2) an antibody or a fragment thereof comprising an antibody light-chain variable region (VL) comprising a complementarity-determining region (CDR) L1 comprising an amino acid sequence represented by SEQ ID NO: 13, a complementarity-determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 14, and a complementarity-determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 15, and an antibody heavy-chain variable region (VH) comprising a complementarity-determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 16, a complementarity-determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 17, and a complementarity-determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 18; (3) an antibody or a fragment thereof comprising an antibody light-chain variable region (VL) comprising a complementarity-determining region (CDR) L1 comprising an amino acid sequence 4240-618 represented by SEQ ID NO: 21, a complementarity-determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 22, and a complementarity-determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 23, and an antibody heavy-chain variable region (VH) comprising a complementarity-determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 24, a complementarity-determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 25, and a complementarity-determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 26; (4) an antibody or a fragment thereof comprising an antibody light-chain variable region (VL) comprising a complementarity-determining region (CDR) L1 comprising an amino acid sequence represented by SEQ ID NO: 29, a complementarity-determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 30, and a complementarity-determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 31, and an antibody heavy-chain variable region (VH) comprising a complementarity-determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 32, a complementarity-determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 33, and a complementarity-determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 34; (5) an antibody or a fragment thereof comprising an antibody light-chain variable region (VL) comprising a complementarity-determining region (CDR) L1 comprising an amino acid sequence represented by SEQ ID NO: 37, a complementarity-determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 38, and a complementarity-determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 39, and an antibody heavy-chain variable region (VH) comprising a complementarity-determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 40, a complementarity-determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 41, and a complementarity-determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 42; and (6) an antibody or a fragment thereof comprising an antibody light-chain variable region (VL) comprising a complementarity-determining region (CDR) L1 comprising an amino acid sequence represented by SEQ ID NO: 45, a complementarity-determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 46, and a complementarity-determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 47, and an antibody heavy-chain variable region (VH) comprising a complementarity-determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 48, a complementarity-determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 49, and a complementarity-determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 50.
3. The antibody or the fragment thereof according to claim 1, wherein the antibody or the fragment thereof is selected from the group consisting of: (1) an antibody or a fragment thereof comprising a light-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 3 and a heavy-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 4; (2) an antibody or a fragment thereof comprising a light-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11 and a heavy-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12; (3) an antibody or a fragment thereof comprising a light-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 19 and a heavy-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 20; (4) an antibody or a fragment thereof comprising a light-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 27 and a heavy-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 28; (5) an antibody or a fragment thereof comprising a light-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 35 and a heavy-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 36; and (6) an antibody or a fragment thereof comprising a light-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 43 and a heavy-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 44.
4. The antibody or the fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody.
5. The antibody or the fragment thereof according to claim 1, wherein the antibody is selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.
6. The antibody or the fragment thereof according to claim 1, wherein the fragment of the antibody is selected from the group consisting of diabody, Fab, Fab′, F(ab)2, F(ab′)2, Fv, and scFv.
7. A polynucleotide encoding the antibody or the fragment thereof according to claim 1.
8. A vector comprising the polynucleotide according to claim 7.
9. A cell transformed with the vector according to claim 8.
10. A method of producing an antibody or a fragment thereof binding to WRS, comprising: producing a polypeptide comprising light-chain and heavy-chain variable regions by culturing the cell according to claim 9 under conditions in which a polynucleotide is expressed; and recovering the polypeptide from the cell or a culture medium in which the cell is cultured.
11. A composition for diagnosing cancer comprising the antibody or the fragment thereof according to claim 1.
12. A composition for diagnosing an infectious disease or infectious complications comprising the antibody or the fragment thereof according to claim 1.
13. The composition according to claim 12, wherein the infectious disease is an infectious inflammatory disease.
14. The composition according to claim 13, wherein the infectious inflammatory disease is sepsis or septic shock.
15. Use of the antibody or the fragment thereof according to claim 1 for manufacture of an agent for diagnosing cancer.
16. A method of diagnosing cancer, comprising: a) obtaining a sample from a subject; b) measuring a WRS protein expression level in the sample using the antibody or the fragment thereof according to claim 1; and c) determining that the subject has cancer when the protein expression level measured in step b) is increased.
17. Use of the antibody or the fragment thereof according to claim 1 for manufacture of an agent for diagnosing an infectious disease or infectious complications.
18. A method of diagnosing an infectious disease or infectious complications, comprising: a) obtaining a sample from a subject; b) measuring a WRS protein expression level in the sample using the antibody or the fragment thereof according to claim 1; and c) determining that the subject has an infectious disease or infectious complications when the protein expression level measured in step b) is increased.
Description
DESCRIPTION OF DRAWINGS
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[0150]
MODE FOR INVENTION
[0151] A better understanding of the present invention may be obtained through the following examples. These examples are merely set forth to illustrate the present invention, and are not to be construed as limiting the scope of the present invention.
Example 1: Production of Monoclonal Antibody
[0152] using hybridoma cell
[0153] (1) Production of hybridoma cell
[0154] 1) Animal immunization and cell fusion [0155] Preparation of immunogen: 1.5 to 2 mg of WRS protein (purity>75%, concentration>0.4 mg/ml) [0156] Animal immunization: Antibody production was induced by inoculating the immunogen into Balb/c mice. [0157] Cell fusion: At least 10,000 hybridoma cells were obtained by electro-fusion of mouse B cells and mouse myeloma cells.
[0158] 2) Selection of hybridoma cell [0159] Primary selection: Hybridoma cells producing an antigen-binding antibody were selected through indirect ELISA. [0160] Secondary selection: Three hybridoma cell lines binding to an antigen were selected through Western blotting using the positive clones obtained in the primary selection. [0161] Isotyping: Five clones having the best results in the selection process were subjected to isotyping.
[0162] 3) Subcloning, cell expansion, freezing storage, and antibody production
[0163] Subcloning, cell expansion, and freezing storage: Clones having good results were subjected to subcloning, cell expansion, and freezing storage. [0164] Antibody production: An antibody was produced in an amount of at least 2 mg from the hybridoma cell line having the best results in the selection process.
[0165] 2. Formation of ascites
[0166] 1) After adaptation of mice for at least 3 days, a pristane adjuvant was administered in an amount of 100 μl/mouse thereto. The hybridoma cell line was cultured so that it could be injected 5 to 7 days after administration of the pristane adjuvant.
[0167] 2) The cultured hybridoma cell line was collected in a 50 ml tube, washed three times with 10 ml PBS, and centrifuged.
[0168] 3) After centrifugation, the supernatant was removed by suction, after which the number of cells required per 100 μl was calculated, added with 1×PBS, mixed well, and then transferred into a 1.5 ml tube.
[0169] 4) The above solution was placed in a 1 ml syringe, and the air in the syringe was removed by turning the syringe needle upwards.
[0170] 5) 100 μl of the solution was injected intraperitoneally to each Balb/c mouse, after which the mice were placed in a cage, and whether ascites was present was observed.
[0171] 6) From 5 days after injection of the hybridoma cell line into the mice, abdominal bloating was observed every day.
[0172] 7) When abdominal bloating was noted, ascites fluid was collected from the abdominal cavity of the mice using a product with an injection needle of 23G or less (using a 3 ml or 5 ml syringe).
[0173] 8) The ascites fluid, collected and placed in a tube, was incubated at RT for 10 min to allow red blood cells to aggregate, followed by centrifugation.
[0174] 9) After centrifugation, only the supernatant was placed in a new 1.5 ml tube and stored at −70° C.
[0175] 3. Production of antibody
[0176] 1) The produced ascites fluid was taken out at −70° C. and thawed at 4° C., and the type of beads to be used was determined by confirming the subtype of the antibody to be purified. The amount of beads used was 0.5 the volume of ascites fluid.
[0177] 2) Well-mixed Protein A beads or G beads were placed in the calculated amount in a 5 ml chromatography column, and bead washing was performed by allowing 5 ml of 1×PBS to flow into the column.
[0178] 3) After completion of washing, the thawed ascites fluid was placed in the column, and the column was capped.
[0179] 4) Rotation binding was performed at 4° C. for 1 hr so that the beads and the antibody were bound to each other.
[0180] 5) After rotation binding, the entire solution was subjected to a flow-through process.
[0181] 6) Column washing was performed using 100 ml of 1×PBS.
[0182] 7) 100 μl of a neutralization buffer was added to a 1.5 ml tube, and 1 ml of an IgG elution buffer was added to the column to enable neutralization immediately after IgG elution. A total of 10 fractions were obtained under the same conditions.
[0183] 8) A portion of each fraction was loaded on a 12% SDS-PAGE gel, and the band was confirmed through gel staining. During staining, fractions were stored at 4° C.
[0184] 9) The fractions having distinct bands were collected, placed in dialysis tubing, and sealed with a clip to prevent leakage. The dialysis tubing and a stirrer bar were placed in a beaker containing 1 L of 1×PBS, and dialysis was performed at 4° C. for 1 hr using a stirrer.
[0185] 10) Dialysis was performed using 1 L of fresh 1× PBS overnight (15 hr) under the same conditions as in 9) above.
[0186] 11) The next day, the solution was collected from the dialysis tubing and immediately quantified using a BCA assay kit.
[0187] According to the above method, three hybridoma cell lines producing monoclonal antibodies specifically binding to WRS were selected. The antibodies produced from the selected hybridoma cell lines were named 3B10H5, 6A3B4 and 1D4C3.
Example 2: Selection of Monoclonal Antibody Through Phage Display
[0188] (1) ScFv phage display biopanning
[0189] 1) 1 ml of 1×PBS and 10 μg of a WRS antigen were placed in an immunotube and vortexed, followed by coating at 37° C. and 200 rpm for 1 hr in the state in which the inlet of the tube was sealed with tape.
[0190] 2) 50 μl of ER2537 E. coli cells were inoculated into 20 ml of SB media, followed by incubation at 200 rpm and 37° C. until OD600=0.5.
[0191] 3) The coating solution was discarded, followed by washing once with tap water and then blocking with 5 ml of 3% skim milk at RT for 1 hr. Here, 600 1 of 3% skim milk was placed in the phage library (400 1 per tube), followed by blocking at RT for 1 hr.
[0192] 4) The blocked phage was placed in the Ag coating tube after blocking (about 5 μl was left for input test), followed by binding in a shaking incubator at 150 rpm and 37° C. for 1 hr 30 min.
[0193] 5) The solution was removed from the Ag coating tube after phage binding, and washing was performed twice using tap water. Thereafter, washing was performed twice using 0.05% PBST. Here, washing with 0.05% PBST was conducted in a manner in which a sample was added with about 1 ml of PBST, vortexed, and then added with excess PBST, after which the PBST was discarded (After the 2nd round, washing was performed five times).
[0194] 6) For elution of the phage bound to the antigen, 1 ml of 100 mM triethylamine (TEA solution) was added thereto, followed by incubation at RT for 8 min.
[0195] 7) During incubation, 0.5 ml of 1 M Tris-HCl (pH of 7.4) was placed in a 50 ml tube, and after incubation, the eluted phage solution was added thereto, mixed through pipetting, and neutralized.
[0196] 8) 8.5 ml of the ER2537 E. coli cells incubated in 2) above were added to the neutralized phage, followed by infection at 120 rpm and 37° C. for 1 hr (Output).
[0197] 9) The output sample was centrifuged at 3,000 rpm and 4° C. for 5 min, after which the supernatant was discarded, and the pellets were pipetted using 100 1 of SB media and spread on a 150 mm dish (LB agar plate, Amp+).
[0198] 10) Input and output titration [0199] Input titer: 2.32 μl of library stock left from step 6 in 500 μl of SB.fwdarw.2.32 μl in 500 μl of SB.fwdarw.2.32 μl in 500 μl of SB.fwdarw.1 μl in 100 μl of E. coli cell.fwdarw.100 μl spreading.fwdarw.colony counting (n) on the next day.fwdarw.Input=n*10.sup.−10 [0200] Output titer: 1 μl of output sample in 1 ml of SB (Output 1).fwdarw.10 1 in 100 μl of SB (Output 2).fwdarw.Output 1, 2 (n1, n2) all spreading by 100 μl.fwdarw.Output1=n1*10.sup.−5, Output2=n2*10.sup.−6
[0201] 11) The next day, 5 ml of SB media was placed in the 150 mm output dish on which the output sample was spread, and all colonies were scraped with a scraper.
[0202] 12) Thereafter, 3 ml of the scraped bacteria solution was placed in a 15 ml tube, 1.5 ml of 50% glycerol (autoclave) was added thereto and mixed therewith, and the resulting mixture was divided into 3 vials and stored at −70° C. as a stock.
[0203] 13) 50 μl of the panning output stock was placed in a 50 ml tube containing 20 ml of SB-ampicillin and grown at 200 rpm and 37° C. until OD600<1.
[0204] 14) 1 ml of a helper phage was added thereto, followed by infection at 120 rpm and 37° C. for 1 hr.
[0205] 15) Kanamycin was added at a final concentration of 70 μg/ml, followed by incubation at 200 rpm and 30° C. overnight (15 hr).
[0206] 16) The next day, the output phage solution cultured overnight was centrifuged at 4° C. and 12,000 rpm for 20 min. The supernatant was placed in a 50 ml tube containing 5 ml of a 5× PEG buffer, inverted, and incubated on ice for 30 min.
[0207] 17) The incubated output phage solution was centrifuged at 4° C. and 12,000 rpm for 20 min. The supernatant was discarded, and the pellets were resuspended in 400 μl of PBS, transferred into a 1.5 ml microtube, and centrifuged at 14,000 rpm and 4° C. for 2 min. The supernatant was transferred into a new tube and used as an input library for the next round of panning.
[0208] 18) A stock for each round was made and stored at −70° C. [0209] From the 2nd round, the WRS antigen was used in a decreased amount. [0210] When using the 2nd library, TG1 E. coli cells were used instead of ER2537 E. coli cells.
[0211] 2,112 candidates were selected from the phage library through biopanning according to the above method.
[0212] (2) ELISA screening
[0213] 1) The enriched round stock was taken out at −70° C. and thawed.
[0214] 2) In order to obtain a single colony, the thawed stock was diluted at 1/1,000 or 1/10,000, spread on a 90 mm LB (+Amp) plate, and cultured overnight (15 hr) at 37° C. in an incubator.
[0215] 3) The next day, 200 μl of SB (+Amp.) was added to each well of a 96-well cell culture plate (experimental plate), and 150 μl of SB (+Amp.) was added to each well of a copy 96-well plate to be used for stock.
[0216] 4) The copy plate was stored at 4° C. for a while and a single colony on the 90 mm LB plate was picked using a sterile toothpick and then added to each well of the experimental plate.
[0217] 5) The experimental plate was placed in a plate shaker, followed by shaking incubation at speed 2.5 until at least 80% confluence was reached.
[0218] 6) After the copy plate was taken out, the grown cells on the experimental plate were replicated on the copy plate using a 96-pin replicator.
[0219] 7) IPTG was added to the experimental plate so that the final concentration thereof was 1 mM (11 μl of 1 M IPTG for 1 ml of SB (+Amp.)), followed by shaking incubation together with the copy plate at 30° C. overnight (15 hr) using a plate shaker at speed 2.
[0220] 8) The next day, 75 μl of 50% glycerol was added to each well of the copy plate incubated overnight and stored at −70° C. The experimental plate subjected to induction overnight was taken out and centrifuged at 4° C. and 3,500 rpm for 20 min.
[0221] 9) During centrifugation, a 96-well half plate was coated at 37° C. for 1 hr with 4 μg/ml of the antigen required for screening. As a negative control, a plate not coated with the antigen was used.
[0222] 10) From the centrifuged experimental plate, the supernatant was discarded, and the plate was lightly wiped with a tissue, after which 60 μl of a 1× TES buffer was added to each well, followed by shaking at 37° C. for 20 min using a plate shaker at speed 4.
[0223] 11) Additional 90 μl of a 0.2× TES buffer was added to each well of the experimental plate, followed by shaking at 37° C. for 5 min using a plate shaker at speed 2.5.
[0224] 12) The experimental plate was incubated on ice for 30 min or more.
[0225] 13) The solution was discarded from the antigen-coated plate, and the plate was washed once with tap water and wiped with a tissue.
[0226] 14) 130 μl of 3% skim milk was added to each well of the antigen-coated plate and blocked for 1 hr at RT.
[0227] 15) About 20 min before completion of blocking, the experimental plate incubated on ice was centrifuged at 3,500 rpm and 4° C. for 20 min.
[0228] 16) The blocking solution was removed from the antigen-coated plate, and the plate was washed once with tap water and wiped with a tissue.
[0229] 17) In the experimental plate, 25 μl of the supernatant was added to each well of the antigen-coated plate and the negative control plate. Here, addition was performed at the same well position in the two plates, followed by a binding process at RT for 1 hr.
[0230] 18) During the binding process, a secondary antibody (anti-HA HRP, 1:2,000) was prepared in advance and stored at 4° C.
[0231] 19) The binding solution was discarded from the antigen-coated plate, and 130 μl of 1×PBS-T was added to each well and then discarded (washing process). This process was repeated 2 more times (a total of 3 times). Lastly, light wiping was performed using a dry tissue.
[0232] 20) 100 μl of the secondary antibody was added to each well, followed by the binding process at RT for 1 hr.
[0233] 21) The washing process of 19) above was repeated.
[0234] 22) 50 μl of a TMB solution was added to each well, followed by incubation at RT for a maximum of 15 min, after which 50 μl of a stop solution (H2504) was added to each well, whereby the reaction was terminated, and the value was measured using an ELISA reader at OD 450 nm.
[0235] Through the above method, 209 scFv candidates were selected again.
[0236] (3) Purification of scFv clone
[0237] 1) 5 μl of the selected monoclonal phage was taken out from the stock stored at −70° C. and then placed in a 14 ml tube containing 3 ml of SB.
[0238] 2) Incubation was carried out at 200 rpm and 37° C. for 2 hr. Thereafter, all of the cells were placed in a flask containing 100 ml of SB and grown at 200 rpm and 37° C. until OD600<1.
[0239] 3) 100 μl of 1 M IPTG was added so that the final concentration thereof was 1 mM, followed by induction at 200 rpm and 30° C. overnight (15 hr).
[0240] 4) The next day, the cells were collected into two 50 ml tubes and centrifuged at 3,500 rpm and 4° C. for min, after which the supernatant was discarded and only the pellets were used.
[0241] 5) The pellets were suspended in 3 ml of 1× TES, added with 4.5 ml of 0.2× TES, and incubated on ice for 30 min or more.
[0242] 6) Centrifugation was performed at 12,000 rpm and 4° C. for 30 min, and the supernatant (crude extract) was filtered using a 0.45 μm filter.
[0243] 7) 200 1 of Ni-NTA beads was placed in the column, and bead washing was performed using 5 ml of 1×PBS.
[0244] 8) Thereafter, the filtered extract was further filtered twice, followed by binding to the Ni-NTA beads.
[0245] 9) After binding, washing was performed using 30 ml of a washing buffer (5 mM imidazole in PBS).
[0246] 10) 5 ml of an elution buffer (200 mM imidazole in PBS) was added, after which 1 ml of an eluent was collected in one 1.5 ml tube and 0.5 ml of an eluent was collected in each of four 1.5 ml tubes.
[0247] 11) 4 μl of a 5× sample buffer was added to each of five new 1.5 ml tubes, 16 μl of the eluent was added thereto, and the tubes were boiled at 100° C. for 7 min.
[0248] 12) 10% SDS-PAGE gel was assembled in a cassette, the cassette was placed in a tank, and the gel and the tank were filled with a 1× running buffer.
[0249] 13) 5 μl of a protein marker and 20 μl of 5 samples were sequentially loaded.
[0250] 14) Electrophoresis was carried out.
[0251] 15) After loading, the gel was separated from the cassette, and staining was performed using an instant blue staining solution until the gel was submerged.
[0252] 16) After staining, the thickness of the band was measured. A solution of the fraction in which the band appeared was placed in a dialysis tube, and dialysis was performed using 1× PBS at 4° C. for 1 hr.
[0253] 17) After 1 hr, replacement with 1 L of fresh 1× PBS was performed, followed by dialysis at 4° C. overnight (15 hr).
[0254] 18) The next day, the sample was taken out, the upper inlet of the dialysis tube was opened, and the sample was harvested.
[0255] 19) Protein quantification was carried out using the BCA test.
[0256] 20) The purified scFv after quantification was stored at −70° C.
[0257] After the purification process, the human cell lysate was used, and based thereon, three scFv clones were finally selected through Western blotting, immunoprecipitation, and IgG engineering.
Example 3: Sequencing
[0258] Total RNA was isolated from the hybridoma cells selected in Example 1 according to the technical manual of a TRIzol reagent. Total RNA was reverse-transcribed into cDNA using universal primers according to the technical manual for a PrimeScript 1st Strand cDNA Synthesis Kit. The antibody fragments of a heavy-chain variable region (VH) and a light-chain variable region (VL) were amplified through RACD (rapid amplification of cDNA ends). The amplified antibody fragment was cloned separately into a standard cloning vector. Colony PCR was performed to screen clones having inserts of the correct size. At least 5 colonies having inserts of the correct size were sequenced for each fragment. The sequences of the different clones were aligned, and consensus sequences of these clones were provided.
[0259] Each scFv monoclonal phage selected in Example 2 was sequenced after extracting plasmid DNA using the HiYield Plasmid Mini kit (Real Biotech Corporation, YPD100) according to the manufacturer's instructions.
[0260] The light-chain and heavy-chain variable region amino acid sequences of a total of six monoclonal antibodies selected in Examples 1 and 2 according to the above method and the sequences of polynucleotides encoding the same were identified, and are illustrated in
Example 4: Identification of Polypeptide in WRS to which Monoclonal Antibody Specifically Binds
[0261] In order to identify the polypeptide region recognized by the monoclonal antibodies produced in Examples 1 and 2, the WRS protein (1-471) of SEQ ID NO: consisting of 471 amino acids and the protein fragments (1-104, 1-154, 48-154, and 48-471) were prepared as follows.
[0262] 1) In order to purify the WRS fragment protein, competent cells for protein expression were transformed with the plasmid in which the WRS fragment gene was cloned into a pET28a vector.
[0263] 2) The transformed cells were spread on an LB (+Kanamycin) plate, followed by culture at 37° C. for 15 hr.
[0264] 3) The next day, a single colony was inoculated into 3 ml of LB (+Kan), followed by culture at 200 rpm and 37° C. for 3 hr.
[0265] 4) All of the small cultured cells were placed in 500 ml of LB (+Kan), followed by culture at 37° C. and 200 rpm for 4 hr.
[0266] 5) When 0.8<OD value<1 was measured, 250 μl of a 1 M IPTG stock was added thereto (final 0.5 mM IPTG), followed by induction at 18° C. and 200 rpm overnight (15 hr).
[0267] 6) The next day, the induction-treated cells were centrifuged at 4,000 rpm for 10 min.
[0268] 7) The supernatant was removed, and the pellets were suspended in 10 ml of washing buffer 1.
[0269] 8) The cells were lysed using a sonicator. Treatment with 35% AMPL for 2 sec and storage on ice for min were performed. This process was repeated 14 times (a total of 15 sonications).
[0270] 9) Centrifugation was performed at 15,000 rpm and 4° C. for 30 min to separate pellets and the supernatant from each other.
[0271] 10) 200 μl of Ni-NTA beads were placed in poly-prep chromatography columns, and 5 ml of washing buffer 1 was added to reach equilibrium.
[0272] 11) After centrifugation, the supernatant was filtered using a 0.45 μm filter in a 50 ml tube and was allowed to flow into the column containing the beads. This procedure was performed once more.
[0273] 12) Washing was performed using washing buffer 1.
[0274] 13) Washing was performed using washing buffer 2.
[0275] 14) Washing was performed using washing buffer 3.
[0276] 15) Washing was performed using washing buffer 4.
[0277] 16) The washed column was placed on a 1.5 ml tube and an elution buffer was then passed therethrough, and thus an eluate was collected.
[0278] 17) A 5× sample buffer and DW were placed in a 5 ml tube and subjected to a flow-through process, and a washing buffer and the eluate were added thereto and then boiled in a heat block for 5 min.
[0279] 18) A premade 15-well comb and 15% SDS-PAGE gel were assembled in a cassette, the cassette was placed in the tank, and the gel and the tank were filled with a 1× running buffer.
[0280] 19) The protein marker and sample were sequentially loaded.
[0281] 20) During gel loading, dialysis tubing was heated in a DW bath at 100° C. for 10 min. The DW was replaced with fresh DW and the heating process in a DW bath was repeated twice more, followed by cooling using 200 ml of cold 1× PBS.
[0282] 21) After loading, the gel was separated from the cassette, and staining was performed by pouring instant blue until the gel was submerged (
[0283] Western blotting was performed according to a typical method using the WRS protein produced through the above method, fragments thereof, and the six monoclonal antibodies produced in Examples 1 and 2 as primary antibodies.
[0284] As a result, as shown in
Example 5: Analysis of Binding Affinity of Antibody
[0285] In order to evaluate binding affinity of the six monoclonal antibodies produced in Examples 1 and 2 and two commercial antibodies (Abnova, anti-WRS antibody (Cat #H00007453-M02) and Novus biological, anti-WRS antibody (Cat #NBP2-32186)), indirect ELISA assay was performed on the full-length WRS protein of SEQ ID NO: 1.
[0286] Briefly, the binding affinity of the antibodies was evaluated according to the following method.
[0287] 1) The WRS protein was diluted to 1 μg/ml in PBS, loaded in an amount of 100 μl/well into a 96-well plate, and reacted at room temperature for 1 hr, whereby the wells were coated therewith.
[0288] 2) After completion of coating, washing was performed once with a PBST (0.05% Tween-20) buffer, and 3% BSA and PBST (0.1% tween-20) were dispensed, followed by a blocking reaction at room temperature for 1 hr.
[0289] 3) The biotin-attached antibody was diluted with a blocking buffer according to each concentration and then reacted at room temperature for 1 hr.
[0290] 4) Washing was performed with PBST (0.05% Tween-20).
[0291] 5) Streptavidin-HRP was diluted with a blocking buffer, followed by reaction at room temperature for 1 hr.
[0292] 6) Washing was performed five times with PBST (0.05% Tween-20) to remove all unattached residue.
[0293] 7) 50 μl/well of TMB was added thereto, followed by reaction at room temperature for 5 min, after which the same amount of 2 M H.sub.2SO.sub.4 was added to terminate the reaction.
[0294] 8) Absorbance was measured using a spectrophotometer (Sunrise, Tecan) (450 nm).
[0295] 9) The EC.sub.50 values were calculated from the results of 8) above.
[0296] The results thereof are shown in Table 1 below.
TABLE-US-00001 TABLE 1 3B6 4G4 4H9 6A3B4 1D4C3 3B10H5 Abnova Novus EC.sub.50 508.1 95.3 55.4 78.4 59.8 64.7 1655.6 532.8
[0297] As is apparent from Table 1, it was confirmed that the six antibodies according to the present invention exhibited very high affinity to the WRS protein compared to the two commercial antibodies.
Example 6: Analysis of Binding Specificity of Antibody
[0298] In order to evaluate the binding specificity of the six monoclonal antibodies produced in Examples 1 and and two commercial antibodies (Abnova, anti-WRS antibody (Cat #H00007453-M02) and Novus biological, anti-WRS antibody (Cat #NBP2-32186)), 20 μg of an HCT116 cell lysate was treated with each of a primary antibody and a secondary antibody under the following conditions, and Western blotting was performed according to a typical method.
[0299] * Primary antibody (room temperature, 1 hr)
[0300] 3B6, 4G4, 4H9, 1D4C4, 3B10H5, 6A3B4: 1 μg/ml
[0301] Abnova Ab: 1:5,000 dilution Novus Ab: 1:10,000 dilution
[0302] * Secondary antibody (room temperature, 1 hr)
[0303] Anti-human HRP (GenScript, A00166): 1:5,000 dilution: 4H9, 3B6, 4G4
[0304] Anti-mouse HRP (Millipore, AP181P): 1:10,000 dilution: Abnova, 1D4C3, 3B10H5, 6A3B4
[0305] Anti-rabbit HRP (Millipore, AP187P): 1:10,000 dilution: Novus
[0306] The results thereof are shown in
[0307] As shown in
[0308] Therefore, it was confirmed that the antibodies according to the present invention exhibited very high binding specificity compared to the commercial antibodies.
Example 7: Validation of Cross-Reactivity
[0309] In order to evaluate whether the six monoclonal antibodies produced in Examples 1 and 2 exhibit cross-reactivity with CRS (cysteinyl-tRNA synthetase), AIMP1 (aminoacyl tRNA synthase complex-interacting multifunctional protein 1), GRS (glycyl tRNA synthetase), and KRS (lysyl tRNA synthetase), which are other ARS (aminoacyl-tRNA synthetase) proteins secreted from the cells, in addition to WRS, indirect ELISA assay was performed according to the following method.
[0310] 1) Antigen coating: 1 μg/ml in PBS, 100 μl/well, 4° C., overnight coating
[0311] 2) Washing: 0.05% PBST (0.05% Tween 20), 200 μl/well, 3 times
[0312] 3) Blocking: 0.5% BSA in 0.05% PBST, 200 μl/well, RT, 1 hr
[0313] 4) Primary antibody binding: 500 ng/ml in 0.05% PBST, 100 μl/well, RT, 1 hr
[0314] 5) Secondary antibody binding: anti-mouse HRP (AP160P) 1:10,000 in 0.05% PBST, 100 μl/well, RT, 1 hr
[0315] 6) TMB detection
[0316] 7) Reaction termination (2 M H.sub.2SO.sub.4)
[0317] 8) Absorbance measurement: 450 nm
[0318] The results thereof are shown in
[0319] As shown in
INDUSTRIAL APPLICABILITY
[0320] The antibody or the fragment thereof according to the present invention specifically binds to WRS and has no cross-reactivity with other proteins included in the same ARS family, making it possible to detect and inhibit WRS, and can thus be effectively used for detecting WRS and diagnosing WRS-related diseases such as cancer, inflammatory diseases, or infectious diseases, thereby exhibiting high industrial applicability.