KR20070039704A - A MONOCLONAL ANTIBODY FOR DETECTION OF HYDROXYLATED ASPARAGINE 803 OF HIF-1alpha; AND ANTIGEN PEPTIDE - Google Patents

Abstract

The present invention relates to monoclonal antibodies and antigens that specifically recognize hypoxia inducible factor 1α (HIF-1α) in which asparagine residues are hydrated, and to produce monoclonal antibodies using peptides in which asparagine residues are hydrated. It was. The monoclonal antibody of the present invention is responsive to HIF-1α in which 803 asparagine residues are hydrated by FIH-1, but does not react to HIF-1α in which 803 asparagine residues are not hydrated. It can be usefully used for measuring activity and searching for inhibitors.

Hypoxia inducible factor 1α

Description

Monoclonal antibody for detection of hydroxylated asparagine 803 of HIF-1α and antigen peptide, where the asparagine residue 803 specifically recognizes hydrated HIF-1α

1 is the structure of (2S, 3R) -L-threo-FmocNH-β-OTBDMS-Asn-OH [Fmoc-Asn (βO-TBDMS)] for the introduction of hydrated asparagine residues,

2 shows the results of sequence and molecular weight analysis of peptides used for antibody preparation,

Figure 3 shows the result of binding the antigen peptide and BSA and OVA protein, M is a marker, 1 is EVN (OH) -BSA, EVN (OH) -OVA, GGL (OH) -BSA and GGL (OH) -OVA Indicates,

4 is a C-terminal peptide sequence and molecular weight analysis of HIF-1α used for antibody titer analysis,

5 is a result of purifying Factor inhibiting HIF-1α (FIH-1),

6 is a result of analyzing the structure and molecular weight of C-terminal peptides of HIF-1α hydrated asparagine residue by FIH-1,

7 is a result of binding a hydrated peptide and a non-hydrated peptide with a monoclonal antibody by immunoassay (ELISA), A is a reaction result according to the concentration of the hydrated peptide and the non-hydrated peptide and monoclonal antibody (SHN-HIF1α) , B is the result of the reaction of monoclonal antibody according to the concentration of the hydrated peptide and the non-hydrated peptide antigen, ○, △ and □ represents the non-hydrated peptide, ●, ▲ and ■ represents the hydrated peptide,

8 is a measure of antibody usefulness when measuring FIH-1 inhibitor activity, A is the result of confirming the reactivity by adjusting the amount of hydrated peptide, ○ is 0% hydration, ● is 20% hydration, ▲ is 40% hydration, ■ is 70% hydration, ◆ is 100% hydration, B is the result of analysis of SHN-HIF-1α production of hydrated peptides according to the concentration of NOG (N-oxyalylglycine), a inhibitor of FIH-1 activity. 0.2mM NOG, ◆ represents 0.4mM NOG, ▲ represents 1mM NOG, and ● represents 5mM NOG.

The present invention relates to a monoclonal antibody that specifically recognizes HIF-1α hydrated by FIH-1 asparagine 803, and an antigen peptide for producing the same.

Oxygen concentration in living cells is a very essential factor for cell survival, but when oxygen concentration is high, excessive oxygen is generated by oxygen stress, causing apoptosis, and in normal oxygen supply (normoxia), PHD (proline) Hydroxylase and FIH-1 (Hygroxia-inducible factor HIF inhibitor-1) inhibit HIF-1α activity, which is important for blood vessel formation and tissue energy supply. At low oxygen concentrations (hypoxia), PHD and FIH-1, which use oxygen as a substrate, are inhibited, HIF-1α is activated, binds to HIF-1β, moves to the nucleus, and binds to proteins such as 300 / CBP. Binding to the HRE promoter increases expression of proteins essential for angiogenesis and intracellular energy supply (Nathali et al . , Biochem . Pharmacol ., 68, 971-980, 2004). However, even when the oxygen supply is not smooth, the activities of PHD and FIH-1 are not inhibited, and thus HIF-1α activity is inhibited and blood vessel formation is inhibited, resulting in necrosis of brain cells, leading to stroke.

FIH-1 has recently been discovered and its tertiary structure is known for its activity (Lee et al . , J. Biol . Chem , 278, 7558-7563, 2003; Elkins et al . , J. Biol . Chem , 278, 1802). -1806, 2003). Used as FIH-1 is also a cofactor for Fe ^{2 +} (cofactor) as in the PHD and hydration of 2-oxoglutarate (oxoglutarate), 803 asparagine residue in the use as an oxygen (O ₂₎ substrate HIF-1α (Lando et al., Science , 295, 858-865, 2002). The enzyme has been found to be more than two times lower in Km values for 2-oxoglutarate and oxygen than PHD, so that some enzymes can function even at partial pressures of oxygen that PHD cannot act on (Koivunen et al . , J. Biol . Chem . , 279, 9899-9904, 2004). In the event of stroke, intracellular oxygen partial pressure decreases, and if FIH-1 continues to function, the formation of new blood vessels for oxygen supply is continuously suppressed, which increases the probability of necrosis of many brain cells. Therefore, inhibitors of FIH-1 are likely to be used as novel stroke treatments.

Methods for measuring FIH-1 activity include measuring the generation of carbon dioxide (CO ₂ ), an enzyme reaction product (Koivunen et al ., J. Biol . Chem ., 279, 9899-9904, 2004), or increasing mass using peptide substrates ( HIF-1α / p300 binding using the method of increasing oxygen by addition of oxygen atoms (Lando et al., Science , 295, 858-865, 2002) and the fact that p300 and HIF-1α binding are inhibited by hydration of HIF-1α. How to measure inhibition (Freedman et al ., Nature Struct . Biol., 10, 505-512, 2003) and the like can be used. However, high throughput screening (HTS) of thousands of samples using specific devices and radioisotopes is not easy to measure carbon dioxide generation, and peptides hydrated by FIH-1 measure p300 and binding inhibition. This method has a disadvantage in that the search conditions such as peptide concentration control are difficult. In addition, the method for measuring FIH-1 activity by mass spectrometry requires high HIF-1α peptide substrate concentrations, and therefore cannot be used for HTS due to high enzyme consumption and quantitative. Therefore, it is necessary to develop monoclonal antibodies with high specificity for hydrated HIF-1α peptides.

The inventors of the present invention have attempted to prepare antibodies that can be used in the HTS of FIH-1 inhibitors, and furthermore, in the field of cell biology, asparagine can be used for hydrated HIF-1α studies. As a result, residue 803 asparagine The present invention was completed by developing a monoclonal antibody that specifically recognizes hydrated HIF-1α. .

The main object of the present invention is to provide a monoclonal antibody that specifically recognizes HIF-1α hydrated asparagine residue 803.

In order to achieve the above object, the present invention provides a monoclonal antibody that specifically recognizes HIF-1α hydrated 803 asparagine residues.

It also provides a cell line producing the monoclonal antibody.

In another aspect, the present invention provides an antigen peptide hydrated asparagine residue for the antibody production.

The present invention also provides a method for screening a FIH inhibitor using the antibody.

Hereinafter, the present invention will be described in detail.

The monoclonal antibody of the present invention specifically recognizes HIF-1α hydrated with the asparagine residue No. 803, and is produced by a preparation method comprising the following steps.

1) preparing a peptide comprising asparagine in which residue 803 of HIF-1α is hydrated as a fragment derived from an active portion of HIF-1α;

2) immunizing mice using said peptide;

3) fusing mouse splenocytes with myeloma cell line and selecting the hybridoma cell line producing monoclonal antibody of step 1); And

4) separating and purifying the monoclonal antibody from the cell line of step 3).

In the present invention, the peptide used in step 1) is derived from an active moiety in which the asparagine residue of HIF-1α is hydrated, and includes the amino acid sequence described in SEQ ID NO: 5 in which the asparagine residue is hydrated. The antigen peptide is most preferably one having an amino acid sequence represented by SEQ ID NO: 1 or 2, but is not limited thereto. The peptide sequence may be combined with other amino acids and chemicals in order to increase the antibody production capacity or to bind the carrier protein to the amino acid terminal and the carboxy terminus, in the present invention, but BSA or OVA is used as a carrier protein, but is not limited thereto. As for the antigen peptide, it is preferable that the peptide which has the amino acid sequence described by SEQ ID NO: 1 is hydrated asparagine, and the peptide which has the amino acid sequence described by SEQ ID NO: 2 is preferably hydrated asparagine 11. The peptide can be synthesized by solid phase synthesis using a 9-fluorenylmethoxycarbonyl (Fmoc) group as a N-amino protecting group. For the introduction of hydrated asparagine residues we used Fmoc-Asn (βO-TBDMS) (FIG. 1) in peptide antigen synthesis. The peptide synthesis was synthesized by using decyclohexylcarbodiimide (DCC) and N-hydroxyboenzotriazole (HOBt), and each synthetic peptide was purified by HPLC and confirmed by MALDI-TOF analysis, respectively, by EVN (OH) and GGL (OH). Named (see Figure 2). The synthesized peptide antigens were linked to the carrier protein BSA or OVA. EVN (OH) -BSA and EVN (OH)-OVA are bound using GMBS (N-γ-maleimidobutyryloxylsuccinimide ester), and GGL (OH) -BSA and GGL (OH) -OVA using GA (glutaraldehyde) It is preferable to combine.

In the present invention, in step 2), each peptide antigen is administered to a mouse to induce antibodies to the peptide. Methods of administering and using the peptides are well known in the art and are not particularly limited. The peptide can be administered in combination with an adjuvant. In the present invention, peptides were injected into the abdominal cavity of Balb / c mice to induce antibody formation.

In the present invention, in step 3), splenocytes of mice immunized with peptide antigens were extracted, fused with osteoblastic cell lines, and cell lines producing antibodies with high specificity to the hydrated peptide were selected. The monoclonal antibody produced in the selected cell line was named SHN-HIF1α, and isotyping confirmed that the monoclonal antibody was IgG2b and was a κ light chain.

In the present invention, in step 4), the prepared monoclonal antibody was isolated and purified by a method commonly known to those skilled in the art.

Hybridoma cell line producing the monoclonal antibody of the present invention as a cell line prepared and selected during the process of step 3 of the manufacturing method as the accession number KCTC10850BP to the Korea Institute of Bioscience and Biotechnology as of September 26, 2005 Deposited.

The present invention provides a method for screening a FIH-1 inhibitor using the monoclonal antibody. Since FIH-1 is an enzyme that targets oxygen and nutrients to cells such as stroke, FIH-1 activity inhibitors are candidates for stroke treatment. Therefore, by administering a candidate inhibitor of FIH-1 activity and measuring the activity of HIF-1α using the monoclonal antibody of the present invention, it is possible to indirectly determine whether the administered candidate substance has an effect of inhibiting FIH-1 activity. .

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

< Example  1> sign language Asparagine  Construction of Peptide Antigen and Antibody Analysis Peptides

Synthesis of peptide antigen for immunization (see FIG. 2) and antibody assay peptide (see FIG. 4) was performed by solid phase synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) as a N-amino protecting group (Merrifield, Science , 232, 341, 1986). Resin with Fmoc-amino acids and Fmoc-amino acids except for Fmoc-Asn (βO-TBDMS) (see FIG. 1) synthesized by the inventors for the introduction of hydrated asparagine residues were purchased from Novabiochem (Swiss). . Synthesis was performed using dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt). After the final synthesis, the Fmoc protecting group was removed with piperidine, and trifluoroacetic acid: water: ethandithiol: triisopropylsilane was 95: 2.5: Crude peptides were obtained by treatment with a solution containing 2.5: 1 for 2 hours, purified by HPLC and then confirmed by MALDI-TOF analysis and the peptide antigens were labeled with EVN, GGL and biotin. Peptides for antibody analysis were named bDES35 and bSMD, respectively, and had amino acid sequences set forth in SEQ ID NOs: 1,2,3, and 4 (see FIGS. 2 and 4).

< Example  2> Peptide-protein conjugate preparation

Peptide-protein conjugates were prepared to immunize the peptide antigens synthesized in Example 1 with mice. At this time, BSA (bovine serum albuimin) or OVA (ovalbumin) was used as a carrier protein.

GMBS (N- [γ-maleimidobutyryloxy] succinimide ester) was used as the sample used to bind the EVA (OH) peptide (see FIG. 2) to the protein. First, 2 mg BSA or OVA protein was dissolved in 200 μl binding buffer (0.083 M sodium phosphate, 0.9 M sodium chloride and 0.1 M ethylenediaminedihydrochloride, pH 7.2), and GMBS 20 dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mg / ml in the reaction. Μl was added and reacted at room temperature for 1 hour. The reaction solution was passed through Sephadex G50 culumn equilibrated with binding buffer to obtain GMBS-activated protein, and dissolved by adding 1 mg of peptide powder and reacted for 2 hours. The final reaction solution was dialyzed with PBS using a dialysis membrane permeating a molecular weight of 10,000 or less, and stored frozen after quantitating proteins. Peptide-protein conjugates were named EVN (OH) -BSA or EVN (OH) -OVA.

Protein binding with GGL (OH) peptide (see FIG. 2) was performed using glutaraldehyde (GA). First, 1 mg BSA or OVA protein and 0.5 mg of peptide are dissolved in PBS, and 2 µl of 25% glutaraldehyde is added to the solution for 1 hour, and 2 µl of 25% glutaraldehyde is further added for 1 hour. I was. Unreacted GA and peptides were removed by dialysis and stored in the same manner as in Example 1. Peptide-protein conjugates were named GGL (OH) -BSA or GGL (OH) -OVA.

The formed EVN (OH) -BSA, EVN (OH) -OVA, GGL (OH) -BSA or GGL (OH) -OVA conjugates were identified by the following SDS-PAGE method. First, 3-5 μg of protein-peptide conjugate was mixed with 5 μl of sample buffer (50 mM Tris-HCl, 2% SDS, 15% glycerol, 5% 2-mercaptoethanol, 0.024% bromophenol blue) and heated at 95 ° C. for 2 minutes. Then, electrophoresis was performed for 1 hour and 30 minutes at 20 mA per gel after loading on 10% SDS-PAGE, and the gel was subjected to Coomassie stain solution (0.1% Coomassie brilliant blue R-250, 50% methanol). , 10% acetic acid) was decolorized in a solution containing 30% methanol, 10% acetic acid to observe the stained protein, it was confirmed that the protein is well coupled to the peptide (see Figure 3).

< Example  3> Anti-peptide antibody production and monoclonal antibody production

Each peptide-protein conjugate obtained by the Example 2 method was diluted in PBS at a concentration of 30 µg / 100 µl, emulsified by mixing 100 µl of the dilution with 100 µl of complete Freund's adjuvant, and injected into the abdominal cavity of Balb / c mice. . Thereafter, 100 μl of the peptide-protein conjugate diluted in PBS at a concentration of 30 μg / 100 μl at 2 weeks intervals was emulsified by mixing with 100 μl of incomplete Freund's adjuvant, and injected into the abdominal cavity three times, followed by final injection. One week serum activity was analyzed for peptides and then well immunized mice were injected with 100 μl of peptide-protein conjugate at 30 μg / 100 μl into the tail vein. Three days later, after spleen was removed from the mice, splenocytes were isolated, washed with serum-free RPMI1640 medium, and washed with the same medium, and P3 / NS1 / 1-Ag4-1 myeloma cells with an average molecular weight of 1450. 1 ml of polyethylene glycol was added for 1 minute, and 10 ml of serum-free RPMI1640 medium was added slowly, followed by fusion for 5 minutes. The fused cells were washed with the same medium, and then suspended by adding 30-40 ml of RPMI1640 medium containing 10% fetal bovine serum per spleen. Thereafter, 100 μl was dispensed into a 96 well plate. After incubation for 3 hours, 100 μl of RPMI1640 medium containing 2 × hypoxanthine-aminmlopterine-thymidine was added thereto, followed by incubation for 7-10 days in a cell incubator at 37 ° C. with 5% carbon dioxide. HIF-1α peptide specific activity hydrated in the medium was investigated in the same manner as in FIG. Cells producing high bispecific antibodies were diluted to contain 2/3 cells per well, and finally cell lines producing antibodies with very high specificity for the hydrated peptide were selected. The cell line obtained monoclonal antibody from mice immunized with EVA (OH) -OVA and named SHN-HIF1α. As a result of isotype determination, the heavy chain constant region was found to be IgG2b, and the light chain constant region was kappa (κ) iso It was found to have a type.

< Example  4> HIF To hydrate the -1α peptide FIH -1 separation and purification

The recombinant pET-FIH-1 gene inserted into the pET-28a vector was inserted into E. coli BL21 (DE3), and then in an LB (Luria-Bertani) agar plate with antibiotic (50 μg / ml kanamycine). Colonies were selected and cultured at 37 ° C. until 600 nm absorbance was 0.6-0.8 in LB medium. Thereafter, 0.5mM IPTG (isopropyl-β-thiogalactoside) was added to incubate at 18 ° C. for 16-20 hours to induce the expression of 6 × His tagged FIH-1, followed by centrifugation at 6,000 RPM for 5 minutes. E. coli expressing 1 was harvested. E. coli was then suspended in buffer A (20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM PMSF and 5% glycerol) at a concentration of 2 g / ml and the cells were disrupted by sonication and then at 16,500 rpm. The supernatant was collected by centrifugation for 30 minutes. 20 ml of the supernatant was added to 5 ml Ni-NTA-agarose (Invitrogen, USA) equilibrated with glycerol-free buffer A, then the gel was washed with 100 ml of the same buffer and buffer B (20 mM Tris-HCl). FIH-1 was eluted by flowing a pH 7.5, 300 mM imidazole, 1 mM PMSF) at a 10-90% concentration gradient. 50 units of trombin were added to the eluted FIH1-1 and 6 × His tags were removed while equilibrating with buffer C (50 mM Tris-HCl pH7.5, 1 mM DTT, 1 mM PMSF) using dialysis. Place on equilibrated 7 ml gradient Q-Sepharose (Pharmacia amersham, Sweden), wash with 50 ml of buffer, buffer D (50 mM Tris-HCl pH7.5, 500 mM NaCl, 0.1 mM DTT, 1 mM PMSF) FIH-1 was purified by flowing) at 0-100% concentration gradient. 5 is a diagram stained by SDS-PAGE using 5 μg purified FIH-1 using 10% gel.

< Example  5> monoclonal antibody SHI - HIF1 hydration of α HIF Specific recognition verification for -1α peptide

To mimic the actual in vivo environment, the peptides bSMD41 and bDES35 prepared in Example 1 in which the biotin group was bound to the N-terminus were hydrated with FIH-1 at 37 ° C. for 1 hour and the hydrated peptides were bDES35 (OH) and bSMD41. It was named (OH). At 45 μl reaction solution (50 mM Tris-HCl, 4 mM ascorbic acid, 2 mM dithiothreitol, 0.6 mM 2-oxoglutarate (2OG) and 0.1 mM ammonium ferrous sulfate), 15 μg FIH-1, 15 μg bDES35 or bSMD41 peptide, 20 μg catalase is included. In order to obtain an unhydrated peptide, the reaction was carried out with 2OG removed from the reaction solution. The reaction solution was denatured at 95 ° C. for 2 minutes, and then centrifuged at 10,000 rpm for 5 minutes. Subsequently, the supernatant was analyzed for hydration of the peptide by MALDI molecular weight analysis. As a result, in the presence of 20G, the molecular weight of the peptide was increased by about 16, but in the absence of 20G, there was no increase in molecular weight (see FIG. 6). This confirmed that the bSMD41 and bDES35 peptides are hydrated as in vivo.

Hydration peptide specific recognition of SHN-HIF1α was examined by ELISA method. First, streptoavidin was adsorbed onto an ELISA plate at a concentration of 100 ng / well, and then 100% to 50 ng (see FIG. 7A) or various concentrations (see FIG. 7B, sequential dilution from 100 ng to 1/5) to Peptides reacted with 0% FIH-1 were combined with TBSS (20 mM Tris-HCl, pH7.5, 150 nM sodium chloride, 1% skim milk) and reacted for 1 hour before TBST (20 mM Tris-HCl, pH7.5, 150 mM). 3 times with sodium chloride, 0.1% tween 20). SHN-HIF1α antibody diluted with TBSS was then added at various concentrations (see Figure 7A, indicating SHN-HIF1α cell culture dilution grown 60-70% saturated) or 1/20 dilution culture (see Figure 7B) for 1 hour. The reaction was then washed three times with TBST. Thereafter, an anti-mouse IgG goat antibody conjugated with horseradish peroxidase diluted 1 / 10,000 was added to TBSS, which was then reacted for 1 hour, washed three times with TBST, and the bound antibody was developed with a substrate of o-phenylene diamine and hydrogen peroxide. Absorbance at 490 nm was analyzed. As shown in FIG. 7, SHN-HIF1α was found to have very high specificity for hydrated HIF-1α peptide.

< Example  6> using monoclonal antibody FIH -1 inhibitor detection

The present inventors used a mixture of hydrated bSMD41 (OH) and unhydrated bSMD41 as shown in Figure 8A to determine the reactivity of the monoclonal antibody according to the bSMD41 hydration ratio. First, 50 ng of streptovidin was coated on an ELISA plate, and peptides prepared so that bSMD41 (OH) was 0, 20, 40, 70 and 100% were added to TBSS by sequential dilution of 5 ng / well from peptide concentration of 5 ng / well. . Next, the culture medium used in Example 5 containing the monoclonal antibody SHN-HIF1α was added by diluting 1/20 to TBSS, and then the same method as in Example 5 was performed. As shown in FIG. 8A, it was confirmed that the monoclonal antibody SHN-HIF1α binds asparagine residue of the peptide according to the degree of hydration.

To find out whether the present invention is useful in the search for FIH-1 inhibitors, the inventors have found that N-oxalylglycine (NOG) (Koivunen et al ., J. Biol . Chem ., 279, known to compete with 2OG to inhibit the activity of FIH-1). 9899-9904, 2004) was used to confirm the inhibitory search utility of SHN-HIF1α. 1 μl of a NOG inhibitor dissolved in various concentrations dissolved in DMSO in 9 μl FIH-1 reaction solution (reaction conditions are the same as in Example 2 but FIH-1 concentration was 1/20 and bSMD41 concentration was used as 1/40). After the reaction for 30 minutes, 2 μl of the reaction solution was added to 50 ng of streptoavidine-coated plates in 100 μl of TBSS, and serially diluted 1/5, and analyzed in the same manner as in Example 5. As shown in FIG. 8B, inhibition of antibody-peptide binding was shown according to the inhibitor NOG concentration. At 0.2 mM, the activity was partially inhibited, 1 mM NOG strongly inhibited hydration of the peptide, and 5 mM NOG completely inhibited the activity.

This demonstrates that the monoclonal antibodies of the present invention are very useful for the search for FIH-1 inhibitors.

As described above, the antibody-producing peptides of the present invention (EVN (OH) and GGL (OH)) produce antibodies with high hydration peptide specificity, and the monoclonal antibody (SHN-HIF1α) obtained by the antigen is FIH-. The monoclonal antibody is very useful for the detection of HIF-1α hydrated by FIH-1, because it is highly reactive with the HIF-1α peptide hydrated by 1, but has little reactivity with the non-hydrated peptide. This can be useful for navigation.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A monoclonal antibody for detection of hydroxylated asparagine          803 of HIF-1a and antigen peptide <130> 5p-08-33 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 16 <212> PRT <213> EVN <220> <221> VARIANT <222> (3) <223> hydroxylated asparagine <400> 1 Glu Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Cys   1 5 10 15 <210> 2 <211> 16 <212> PRT <213> GGL <220> <221> VARIANT <222> (11) <223> hydroxylated asparagine <400> 2 Gly Gly Leu Thr Ser Tyr Asp Cys Glu Val Asn Ala Pro Ile Gln Gly   1 5 10 15 <210> 3 <211> 35 <212> PRT <213> bDES35 <400> 3 Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys Glu Val Asn   1 5 10 15 Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu Glu Leu Leu              20 25 30 Arg Ala Leu          35 <210> 4 <211> 41 <212> PRT <213> bSMD41 <400> 4 Ser Met Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys Glu   1 5 10 15 Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu Glu              20 25 30 Leu Leu Arg Ala Leu Asp Gln Val Asn          35 40 <210> 5 <211> 8 <212> PRT <213> antigen motif <220> <221> VARIANT <222> (3) <223> hydroxylated asparagine <400> 5 Glu Val Asn Ala Pro Ile Gln Gly   1 5

Claims (6)

803 A monoclonal antibody that specifically recognizes hydrated HIF-1α with an asparagine residue. A hybridoma cell line producing the monoclonal antibody of claim 1 (Accession No .: KCTC10850BP). An antigen peptide for producing the monoclonal antibody of claim 1, wherein the asparagine residue is hydrated and comprises the following amino acid sequence: EVN (OH) APIQG. The antigenic peptide of claim 3, wherein the antigenic peptide is represented by SEQ ID NO: 1. The antigenic peptide of claim 3, wherein the antigenic peptide is represented by SEQ ID NO: 2. Method for screening inhibitors of FIH-1 activity by measuring the degree of HIF activity using the monoclonal antibody of claim 1.

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