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    • 专利摘要:
    PURPOSE: Provided is a monoclonal antibody to glutamate dehydrogenase of Sulfolobus solfataricus, which monoclonal antibody can be applied to the evolutionarily analysis of structural difference between different glutamate dehydrogenases and between microorganisms and other species, and the study of their biochemical characteristics. CONSTITUTION: The monoclonal antibody to glutamate dehydrogenase of thermophilic Sulfolobus solfataricus is separated from Sulfolobus solfataricus and produced by cell fusion method with a hybridoma cell line(KCLRF-BP-00033). It has amino acid sequence of Met-Lys-Ala-Ile-Ile and 45kDa of molecular weight.
    公开号:KR20020027855A
    申请号:KR1020000058575
    申请日:2000-10-05
    公开日:2002-04-15
    发明作者:최수영；조성우；반재훈；안지인；전성규
    申请人:최수영；조성우；
    IPC主号:
    专利说明:

    Monoclonal Antibodies to Glutamic Acid Dehydrogenase of Sulphobus solfataricus Bacterium from Sulfolobus solfataricus}
    [16] The present invention is thermophilic sulforobus solfataricus (Sulfolobus solfataricus; Below It is related to a monoclonal antibody against the glutamate dehydrogenase of the fungus (mixed with "ss") (hereinafter referred to as "GDH"). More specifically, the present invention was isolated from thermophilic sulfobus solfataricus, and the sulfobus solfataricus glutamic acid dehydrogenase of about 45 kDa whose N-terminal amino acid sequence is Met-Lys-Ala-Ile-Ile. "ss GDH") monomers.
    [17] Organisms can be divided into three primary kingdoms: eukaryotes, eubacteria, and archaebacteria (Woese, CR 1987. Bacterial evolution. Microbiol. Rev. 51: 221-271). . Archer bacteria are composed of three different phenotypes: methanogen, halophile, and extreme thermophile. While eukaryotic and eubacterial GDHs are being studied, studies of enzymes isolated from archaebacteria have been limited to the basophil phenotype. The discovery of extreme thermophilic archaebacteria was expected to show the structural requirements necessary for the enzymatic proteins in this organism to undergo thermophilic reactions. Interest among thermophilic bacteria-derived enzymes has emerged among researchers. These molecules are expected to contribute to understanding their thermophysical mechanisms and their chemical and physical properties, which are particularly suitable for many biotechnological processes.
    [18] Sulfolobus solfataricus is one of the best studied archerbacteria as a sulfur-dependent hyperthermophilic microorganism with an optimum temperature of 89 ° C and can easily obtain large amounts of biomass. Rella, R., Raia, CA, Pensa, M., Pisani, FM, Gambacorta, A., De Rosa, M., Rossi, M. 1987. A novel archaebacterial NAD + -dependent alcohol dehydrogenase.Eur.J. Biochem. 167: 475-479, Marino, G., Nitti, G., Arnone, MI, Sannia, G., Gambacorta, A., De Rosa, M. 1988. Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus. J. Biol. Chem . 263: 12305-12309). Sulphobus solfataricus grows at very high temperatures, providing a source of enzymes with abnormal physicochemical properties. In addition to growth in extreme environments, the study of sulfobus solfataricus is interesting from a phylogenetic perspective. Since the nitrogen (N) metabolism of sulfobus solfataricus is not fully known, the presumed association of GDH-rich and its amino group metabolism interests the study of this enzyme. It is already known that ss GDH is the first enzyme involved in biosynthesis of amino groups by converting 2-oxoglutarate and ammonia to glutamic acid (Consalvi, V., Chiaraluce, R., Politi, L., Gambacarta, A., De Rosa, M., Scandurra, R. 1991. Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus Eur. J. Biochem. 196: 459-467).
    [19] GDH is divided into two types depending on the coenzyme involved in this reaction. That is, NADP-dependent enzymes involved in ammonia assimilation and NAD-dependent enzymes involved in glutamic acid catabolism. A series of GDH, NADP-dependent GDHs from other origins, ie bulls, chickens, and human livers, were found to be very similar in sequence and similar hexamers of 45-56 kDa monomers (Smith, EL, Austern, BM). , Blumenthal, KM, Nyc, JF 1975. Glutamate dehydrogenase, pp. 293-367. In P. D. Boyer (ed.), The Enzymes 3rd ed. Vol. 11. Academic Press, New York. Based on this evidence, some suggest that these enzymes have a similar structure and commonality among evolutionary sources. However, NAD-dependent GDHs isolated from Saccharomyces cerevisiae and Neurospora crassa are assemblies of four 116 kDa monomers. Despite the large amount of information on some GDHs, it is uncertain about the catalytic mechanism of the enzyme and its role by various effectors. Only recently has the three-dimensional structure of GDH derived from microorganisms been known. More recently, crystallisation of bovine liver GDH has been reported. However, there is a relatively low correlation between microbial and mammalian GDH. And little has been reported about the comparison of the detailed structure and function of various GDHs.
    [20] The inventors have prepared monoclonal antibodies against sulfobus solfataricus derived purified GDH for the purpose of studying structure-function studies and evolutionary correlations between various types of GDHs. Structural differences between microbial and mammalian GDHs were studied using biosensor technology (Pharmacia BIAcore) and monoclonal antibodies against sulfobus solfataricus and cerebellar GDH. The BIAcore system enables quantitative analysis of molecular interactions in real time. Thus, the coupling and separation rate constants are calculated immediately. The results suggest the possibility of structural differences in epitopes between mammalian GDH and microbial GDH.
    [21] An object of the present invention is to provide a monoclonal antibody against glutamic acid dehydrogenase of thermophilic sulfobus solfataricus.
    [1] 1 is a photograph showing the results of SDS-PAGE purification of the finally selected five monoclonal antibodies purified by Protein A column chromatography. From lane 1 we call gdhmAb 1,2,3,4,5, respectively.
    [2] FIG. 2 (A) is a photograph showing the result of SDS-PAGE of GDH purified from S. solfataricus.
    [3] FIG. 2 (B) shows Western blot analysis of five monoclonal antibodies (gdhmAb 1,2,3,4,5) finally selected through the screening process of monoclonal antibodies and purified sulfoverse solfataricus GDH. It is a photograph showing.
    [4] 3 is a photograph showing Western blot analysis results of epitope mapping by treatment of purified sulfoverse solfataricus GDH with V-8 protease. Each lane is the result of Western blot using the anti-ss monoclonal antibody gdhmAb 1,2,3,4,5.
    [5] 4 (A) is a photograph showing the result of SDS-PAGE of the whole protein isolated from various microorganisms.
    [6] Lane 1: molecular weight marker lane 2: sulfoverse solfataricus
    [7] Lane 3: two. Coli Lane 4: Bacillus Subtilis
    [8] Lane 5: Neisseria Flava Lane 6: Proteus Bulgari
    [9] Lane 7: Pseudomonas Putita Lane 8: Staphylococcus Oreus
    [10] Lane 9: Streptococcus Salivarius
    [11] Figure 4 (B) is a photograph showing the results of Western blot analysis of the whole protein isolated from various microorganisms using a monoclonal antibody against sulfobus solfataricus GDH.
    [12] Lane 1: Sulfuroverse Solfataricus Lane 2: E. collie
    [13] Lane 3: Bacillus subtilis Lane 4: Neisseria Flava
    [14] Lane 5: Proteus Bulgari Lane 6: Pseudomonas Putita
    [15] Lane 7: Staphylococcus Oreus Lane 8: Streptococcus Salivarius
    [22] The present inventors injected a purified thermophilic sulfobus solfataricus bacterium glutamate dehydrogenase into an immunogen into BALB / c mice, and prepared several monoclonal antibodies against it by cell fusion. These monoclonal antibodies specifically recognize glutamic acid dehydrogenase of thermophilic sulfobus solfataricus and are not immunologically related to glutamic acid dehydrogenase of various microorganisms as well as other species. Therefore, if the enzyme's immunological specificity is well utilized, it may be applied to study structural differences related to glutamic acid dehydrogenase, and evolutionary analysis between microorganisms and other species and their biochemical properties.
    [23] The monoclonal antibody of the present invention is isolated from S. solfataricus, and is a monoclonal antibody against glutamic dehydrogenase produced by a hybridoma cell line with an accession number of KCLRF-BP-00033. It features.
    [24] In addition, the monoclonal antibody is characterized in that the N-terminal amino acid sequence is Met-Lys-Ala-Ile-Ile, the molecular weight is about 45kDa.
    [25] In addition, the monoclonal antibody of the present invention is characterized in that it reacts immunologically with glutamic acid dehydrogenase present in the thermophilic sulfobus solfataricus .
    [26] Hereinafter, the present invention will be described in more detail.
    [27] Glutamic acid dehydrogenase was isolated from thermophilic sulfobus solfataricus (Ahn, JY., Lee, KS, Choi, SY, Cho, SW. 2000. Regulatory Properties of Glutamate Dehydrogenase from Sulfolobus solfataricus . Mol. Cells. 10 : 25-31).
    [28] To prepare monoclonal antibodies against ss GDH, a complete Freund's adjuvant mixture containing GDH was immunized by intraperitoneal injection of BALB / c mice to obtain splenocytes from these BALB / c mice and mouse myeloma cells. And fused. Hypoxanthine-aminopterin-thymidine (HAT) medium (Dulbecco's modified Eagle's medium) containing 20% BCS, antibiotics and HAT was used to grow only fusion cells. In order to screen clones that produce monoclonal antibodies after the fusion reaction, immunoblot analysis was performed using the supernatant of the fusion cell culture and the purified GDH as antigen.
    [29] Clones that consistently produce monoclonal antibodies against GDH were selected, and Western blot analysis of positive brain tissue proteins confirmed that the monoclonal antibodies produced by these clones specifically reacted with GDH.
    [30] In addition, to investigate cross-reactivity to mammalian GDH and other microbial GDH, whole proteins were prepared from various mammals and microorganisms, and analyzed according to the Western blot method using the monoclonal antibodies prepared above. As a result, it was confirmed that the PNP oxidase of mammalian, avian and human cell origin has a monomer molecular weight of about 30 kDa and is very similar immunologically.
    [31] Thus, by using the monoclonal antibody of the present invention specifically recognizes PNP oxidase of various animal origin, various neurological disorders related to PNP oxidase, in particular, can occur due to metabolic abnormalities of neurotransmitters in brain tissue. It may be applied to the analysis of the causes of neurological diseases such as seizure and convulsions and to study their biochemical properties. In addition, by applying the monoclonal antibody of the present invention in the field of genetic engineering, it can be used not only for isolation of PNP oxidase cDNA from cDNA library and cloning of PNP oxidase, but also for mass production of expression protein after cloning. Monoclonal antibodies may be used to determine the distribution of the brain tissue of this enzyme.
    [32] Hereinafter, the present invention will be described in detail by examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
    [33] Example 1: Bacterial Species and Growth Conditions
    [34] The ss MT-4 cell line was obtained from ATCC. In general, the ss MT-4 cell line grows with nutrients from 0.2% yeast extract at 87 ° C, pH 3.5 and aerobic conditions (De Rosa, M., Gambacorta, A., Nicolaus, B., Giardina, P., Poerio , E., Buonocore, V. 1984. Glucose metabolism in the extreme thermoacidophilic archaebacteriumSulfolobus solfataricus. Biochem. J. 224: 407-414). Minimal growth medium was used containing 1% casamino acid. Cells were harvested in a stationary growth phase by continuously shaking using a Lab-line orbit environ shaker.
    [35] Example 2: ss GDH Purification and Assay
    [36] ss GDH was purified by methods developed in this laboratory (Ahn, JY., Lee, KS, Choi, SY, Cho, SW. 2000. Regulatory Properties of Glutamate Dehydrogenase from Sulfolobus solfataricus . Mol. Cells. 10: 25-31 ). Enzyme activity was measured spectrophotometrically to the reduction of absorbance at 340 nm the reduction amination of 2-oxoglutarate. All assays were performed twice and the initial rate data correlated with a standard assay mixture containing 50 mM triethanolamine, pH8.0, 100 mM ammonium acetate, 0.1 mM NADH, 2.6 mM EDTA at 60 ° C. GDH concentration was adjusted to less than 0.04 absorbance unit / min. The reaction is started by adding 2-oxoglutarate to a final concentration of 10 mM. One unit of enzyme was defined as the amount of enzyme required to oxidize 1 umole of NADH at 60 ° C.
    [37] Example 3: Preparation of Anti-GDH Monoclonal Antibodies
    [38] Example 3-1 : Preparation and Injection of Immunogen
    [39] GDH (50 μg in 150 μl) purified in Example 2 was mixed with the same volume of CFA (Complete Freund's Adjuvant) and sonicated three times for 15 seconds. This immunogen-adjuvant mixture was intraperitoneally injected in 8-10 weeks BALB / c magnetic mice. Three additional injections were made at 3-4 week intervals after the first injection. The final injection was performed 3 days before cell fusion without an adjuvant.
    [40] Example 3-2 : Preparation of Clones Producing Monoclonal Antibodies Against PNP Oxidase
    [41] Feeder cells for promoting the growth of the following fusion cells were prepared as follows one day before the preparation of the fusion cells: 16 to 18 weeks old BALB / c mice were killed by cervical dislocation. After removing the skin, 5 ml of cold 11.6% sucrose solution (0 ° C.) was injected intraperitoneally, and about 3 ml of the sucrose solution was removed and centrifuged at 650 × g for 5 minutes to obtain peritoneal cells.
    [42] To prepare fusion cells, splenocytes obtained from BALB / c mice immunized in Example 3-1 were collected in 15 ml centrifuge tubes. These splenocytes are mixed with SP2 / 0-Ag-14 mouse myeloma cells (Shulman H. et al., Nature, 276: 269-270 (1978)) and serum-free DME (Gibco BRL, USA) 1 ml of 50% polyethylene glycol 1500 dissolved in was slowly added and fused at 37 ° C. for 90 seconds. To terminate the fusion reaction, 1 ml of DME was added slowly for 1 minute, then 2 ml for 1 minute, and 20 ml of DME was added for a total of 10 minutes. Cells were then obtained by centrifuging at 650 × g for 1 minute and suspended in 20 ml of HAT (Gibco BRL, USA) medium (DME containing 20% fetal calf serum, antibiotics and HAT) at 650 × g. Centrifuged for 1 minute. The obtained cells were again suspended in 120 ml of HAT medium. This cell suspension was transferred in 1 ml 24-well microtiter plates.
    [43] Approximately two weeks after the fusion reaction, the supernatant of the cell culture solution and the pure ss GDH isolated in Example 1 were screened for screening clones that produce a monoclonal antibody (mixed with 'mAb'). As an antigen, the following immunodot blot analysis (first screening) and Western blot analysis (secondary screening) were performed.
    [44] (i) Immunoda blot analysis : ss GDH (0.5) purified on each 1 × 1 cm nitrocellulose paper, serially numbered 1 × 1 cm nitrocellulose paper, placed on a piece of nitrocellulose paper (10 × 10 cm). Mg / ml) was adsorbed and dried by air. This blot was blocked for 1 hour with Blotto (5% skim milk powder dissolved in Tris-buffered saline (TBS), washed with TBS and dried, and then placed into each 96-hole microplate hole containing each fusion cell culture. The immune response was measured as published in the Western blot analysis method below.
    [45] (ii) Western blot analysis : The purified protein of Example 1, appearing as a single band on SDS-PAGE, was transferred to a nitrocellulose membrane, and the membrane was washed with distilled water and dried. Then, the cells were blocked with Blotto for 1 hour and washed with TBS, reacted with the positive clone culture supernatant in the immunodot blot analysis for 1 hour, and then washed three times with TBS including Tween 20 at 5 minute intervals. Then, Goat anti-mouse IgG (AP) conjugated with alkaline phosphatase (AP) was added and reacted for 1 hour with three times TBS containing Tween 20 at 5-minute intervals. After washing, the final wash was performed for 5 minutes with AP buffer (100 mM Tris buffer containing 5 mM MgCl 2 , pH 9.5). 66 μl solution consisting of 70% dimethylformamide and 50 mg / ml nitroblue tetrazolium and solution consisting of 100% dimethylformamide and 50 mg / ml bromochloroindolyl phosphate 30 Color development was induced with 10 ml of AP buffer containing the final volume. When the color reaction reached an appropriate intensity, the reaction was stopped by washing the membrane several times with distilled water.
    [46] Purification of Monoclonal Antibodies
    [47] For purification of monoclonal antibody, 100 ml of the culture supernatant was centrifuged at 15,000 X g for 30 minutes to separate cells and insoluble aggregates, and applied to 1 ml of Protein A-Agarose column (Sigma). The column was washed with phosphate-buffered saline (PBS) until the absorbance of unbound protein dropped to background levels. The antibody was eluted with 0.1 M glycine-HCl (pH 2.5). Eluted antibodies were neutralized by addition of 1M Tris and diluted with PBS.
    [48] GDH was purified as above and showed a single band on the SDS-PAGE. Purified enzyme was denatured in the presence of SDS and injected into the animal. 94 positive clones from the two fusion experiments were first screened by immunodot-blot analysis. Goat anti-mouse IgG antibody was used as the second antibody. All monoclonal antibodies screened by this method were of IgG class. Among the hybridomas, some clones continued to lose their ability to form antibodies, or they produced monoclonal antibodies that weakly reacted with proteins in the western blot and excluded them. Of the 94 clones, 25 hybridomas were finally selected. Five representative monoclonal antibodies were purified using a protein-A affinity column, which is shown in FIG. 1. To check the specificity of the anti-GDH monoclonal antibody, the entire protein of ss was extracted and separated by SDS-PAGE. These antibodies specifically recognized the protein band corresponding to the purified GDH position on SDS-PAGE (FIG. 2).
    [49] Epitope mapping
    [50] One-dimensional epitope mapping was performed. 10 μg of purified ss GDH in SDS sample buffer was mixed with the same volume of 0.5 μg Staphyloccus aureus V-8 protease solution dissolved in SDS sample buffer. The mixture was SDS-PAGE and the isolated peptides were transferred for immunoblotting analysis as above.
    [51] Other immunoreactivity of anti-GDH monoclonal antibodies with GDH proteins was further studied by epitope mapping analysis using V-8 protease. GDH was digested by V-8 protease and immunoblotted by anti-ss GDH monoclonal antibody. 3 shows that only one subgroup that recognizes the same peptide fragment of GDH was identified among the antibodies. These monoclonal antibodies (gdhIImAb1 to gdhIImAb5) showed two bands at 50kDa and 16kDa. This indicates that the epitopes recognized by the five monoclonal antibodies are adjacent or at the same point.
    [52] Example 5 : Cross Reactivity of ss GDH Monoclonal Antibodies with GDH from Various Mammalian and Microbial Origins
    [53] Cross-reactivity of GDH with the ss GDH monoclonal antibodies of mammals and microorganisms including humans was examined by Western blot. Cells were isolated from dogs, cats, cattle, pigs, letts and microorganisms and then disrupted by mixing 10 mM potassium phosphate buffer containing 0.1 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM PMSF for each. Centrifuge each 25% (w / v) lysate at 10,000 x g for 1 hour, take 5 μl from each supernatant, mix with an equal volume of 2 x SDS sample buffer, boil for 3 minutes, and cool to SDS -PAGE and transfer to nitrocellulose membrane and Western blot analysis by the above method.
    [54] The immunological relationship between ss GDH and microbial derived enzymes has been trained by immunoblotting on various microbial homogenates. Escherichia. coli , Bacillus subtilis , Neisseria flava , Total proteins of Proteus vulgaris , Pseudomonas putita , Staphylococcus aureus , and Streptococcus salivarius were isolated. Total proteins isolated on SDS-PAGE were transferred and probed with five monoclonal antibodies. The immune response bands on the Western blot did not find protein bands near 45-60 kDa equivalent molecular weight in all the microorganisms tested. And all five monoclonal antibodies showed the same result. These monoclonal antibodies only reacted with ss derived GDH. These results indicate that the pattern of recognition sites of the microorganism under test and the monoclonal antibody are completely different, and that 25-27% of ss GDH and other Saccharomyces cerevisiae, neurospora cerevisiae and GDH derived from E. coli Suggests sequence similarity. Since structural information on GDH species is not yet available, it is interesting to compare the immunocross-reactivity between anti-ss GDH monoclonal antibodies and other GDH species. To investigate cross-reactivity of anti-ss GDH monoclonal antibodies with GDH species in other mammals and birds, homogenates from dogs, cats, cows, pigs, and rats are removed, whole proteins are isolated and transferred, and five Probed as monoclonal antibody. All five monoclonal antibodies could not recognize GDH in all animal species tested. Cross-reaction results with GDH derived from these various species suggest that ss GDH is immunologically unrelated to GDH in mammals.
    [55] Protein Immobilization and Analysis on BIAcore
    [56] Protein-protein interactions with ss GDH were performed with anti-ssGDH and anti-cerebellar GDH antibodies with the Pharmacia Biosensor BIAcore instrument. CM5 research grade sensor chips (Pharmacia Biosensor) were used. Indirect oriented immobilization of antibodies on the CM5 sensor chip was performed as follows. First, rabbit anti-mouse IgG Fc (ramfc) was coupled to the chip by injection of 100 ng ramfc dissolved in 10 mM sodium acetate (pH 4.5) solution at a rate of 5 μl / min at 20 ° C. The carboxy-methyl dextran matrix of the sensor chip is a 30 µl mixture of 0.05 M N-hydroxysuccinimide and 0.2 M 1-ethyl-3-[(3-dimethylamino) propyl] carbodiimide in water as a solvent. It was activated by injecting (6 minutes) to convert the carboxyl group of the sensor chip matrix into N-hydroxysuccinimide ester. The ester is subjected to a nucleophilic attack by the amino group of the protein, resulting in an amide linkage between the sensor chip and the protein. Under these conditions, ramfc's typical 3700 resonance units are immobilized on a CM5 chip. The interaction of ss and cerebellar GDH monoclonal antibodies with ss GDH is measured by the following two series of injections; Monoclonal antibodies are captured by ramfc. Then two different GDH antibody species are injected. Protein-protein interactions were performed in HBS buffer (10 mM Hepes / KOH, pH 7.5, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20). Kinetic rate constants (k on and k off ) and equilibrium separation constants (k D = k off / k on ) were obtained using the BIAlogue Kinetic Evaluation software.
    [57] To further compare the structural differences between ss GDH and mammalian GDH, the immunoreactivity between ss GDH and anti ss GDH monoclonal antibodies was quantitatively tested using Pharmacia Biacore. As described above, kon and koff values were measured for ss gdhmAb5 monoclonal antibody and cerebellar GDH monoclonal antibody. Each measurement was performed on different surfaces at least two to four times. The results of the kinetic experiments are summarized in Table 1. The binding affinity between ss GDH and anti-ss GDH monoclonal antibody (Kd = 11nM) was much stronger than that for anti-cerebellar GDH monoclonal antibody (Kd = 450nM). The difference in binding affinity for ss GDH between anti-ssGDH monoclonal and anti-cerebellar GDH monoclonal antibodies was mainly determined by the binding rate constants for anti ss GDH monoclonal and anti-cerebellar GDH monoclonal antibodies (kon), 2.1 x 10 3 M -1 s -1 and 1.6 x 10 2 M -1 s -1 . These results indicate that ss GDH differs in molecular recognition process from antibodies to ss GDH enzyme and antibodies to cerebellar enzyme. Thus, this suggests that the molecular structure of the ss GDH protein epitope surface is different from that of cerebellar GDH.
    [58] Sulfur bus Sol Fataricus GDHAssociation rate constant (kon) (M -1 s -1 )Dissociation rate costant (koff) (s -1 )Equilibrium constant (Kd) (nM) Sulforverse Solfataricus mAb2.1 x 10 3 23.5 x 10 -6 11.2 ± 0.3 Cerebellum mAb1.6 x 10 2 72.1 x 10 -6 450.6 ± 0.5
    [59] In the present invention a library of monoclonal antibodies against ss GDH was prepared, which were used to test the structural relationship between the extreme thermophilic Archiebacteria ss GDH and other sources of GDH. Immunoblot analysis, a cross-reaction test between anti-GDH monoclonal antibodies and other GDH species, showed that some microbial and vertebrate GDHs were immunologically different from SS GDH. Epitope mapping analysis predicted that the various GDH species tested had different amino acid sequences or protein structures than ss GDH.
    [60] Structural differences between microbial and mammalian GDH have been further studied through monoclonal antibodies and biosensor technologies for ss and cerebellar GDH. The binding affinity for anti-ss GDH monoclonal antibody to ss GDH was 40 times higher than that for bovine GDH. These results indicate that the molecular recognition process of ss GDH against anti ss GDH antibodies is different from that for anti-cerebellar GDH antibodies.
    [61] Accordingly, the monoclonal antibodies of the present invention are useful for studying structural differences between glutamic acid dehydrogenases of different origin, evolutionary analysis between microorganisms and other species, and their biochemical properties.
    权利要求:
    Claims (3)
    [1" claim-type="Currently amended] A monoclonal antibody against glutamic acid dehydrogenase of sulfobus solfataricus, isolated from Sulfolobus solfataricus, produced by a hybridoma cell line with accession number KCLRF-BP-00033.
    [2" claim-type="Currently amended] According to claim 1, wherein the monoclonal antibody is N- terminal amino acid sequence of Met-Lys-Ala-Ile-Ile, the molecular weight of about 45kDa, characterized in that the glutamic acid dehydrogenase of Sulfurobus solfataricus bacteria Against monoclonal antibodies.
    [3" claim-type="Currently amended] According to claim 1 or 2, wherein the monoclonal antibody sulfobus solfattaricus, characterized in that the immunological reaction with glutamic acid dehydrogenase present in Sulfolobus solfataricus bacteria. Monoclonal antibody against bacterial glutamate dehydrogenase.
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    同族专利:
    公开号 | 公开日
    KR100412773B1|2003-12-31|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2000-10-05|Application filed by 최수영, 조성우
    2000-10-05|Priority to KR10-2000-0058575A
    2002-04-15|Publication of KR20020027855A
    2003-12-31|Application granted
    2003-12-31|Publication of KR100412773B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR10-2000-0058575A|KR100412773B1|2000-10-05|2000-10-05|Monoclonal antibodies to glutamate dehydrogenase from Sulfolobus solfataricus|
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