KR100412774B1 - Monoclonal antibodies to pyridoxine-5' -phosphate oxidase - Google Patents

Abstract

The present invention relates to monoclonal antibodies against pyridoxine-5'-phosphate (PNP) oxidase, more specifically isolated from sheep brain, wherein the N-terminal amino acid sequence is Met-Thr-Cys-Gly-Leu It relates to a monoclonal antibody against a Pyridoxine-5'-phosphate (PNP) oxidase monomer of about 30 kDa. The present inventors injected purified PNP oxidase into an immunogen into BALB / c mice, and prepared several monoclonal antibodies against it through cell fusion. Since these monoclonal antibodies specifically recognize PNP oxidase and can recognize PNP oxidase enzyme activity of various animal origins as well as sheep, various neuronal disorders associated with PNP oxidase, in particular brain tissue Neurological diseases that can occur due to metabolic disorders, such as epileptic seizures, convulsions, etc. The cause of the analysis and its biochemical properties can be studied. In addition, the monoclonal antibody of the present invention may be applied to the field of genetic engineering, and may be used for the isolation of PNP oxidase cDNA from cDNA library and cloning of PNP oxidase.

Description

MONOCLONAL ANTIBODIES TO PYRIDOXINE-5'-PHOSPHATE OXIDASE} Pyridoxine-5'-phosphate oxidase

The present invention relates to monoclonal antibodies against pyridoxine-5'-phosphate (hereinafter referred to as 'PNP') oxidase, and more particularly the present invention relates to sheep brains. It was isolated and relates to a monoclonal antibody against a 30 kDa PNP oxidase monomer whose N-terminal amino acid sequence is Met-Thr-Cys-Gly-Leu.

In brain tissues, pyridoxal-5'-phosphate (PLP) is a precursor of pyridoxine-5'-phosphate (PNP) oxidase and pyridoxal kinase, with pyridoxine, a vitamin B ₆ precursor. It is an important substance synthesized by action.

PLP is when not only be involved as a coenzyme of the enzyme involved in amino acids, carbohydrates, protein, fat and metabolism (metabolism) of nucleic acids from biological tissue, if Vitamin B ₆ is insufficient experience any metabolic or clinical symptoms (clinical manifestation ) Results in:

In particular, the importance of PLP in terms of brain function is critical for the synthesis and degradation of all components known as neurotransmitters, such as dopamine, serotonin, epinephrine, histamine, γ-aminobutyrate (GABA), and taurine. Is involved. Therefore, clinical symptoms of vitamin B ₆ deficiency may cause slow growth and lead to alopecia and anemia.

In addition, since the maintenance of the integrity of the physiological homeostasis of these neurotransmitter synthase enzymes is very important in the central nervous system, vitamin B ₆ deficiency in the central nervous system may cause abnormalities such as seizures and convulsions. Abnormalities of dependent enzymes directly or indirectly lead to neurological disorders such as epilepsy, Parkinsonism, Huntington's disease, and depression.

PNP oxidase is an enzyme that converts PNP, a phosphorylated form of pyridoxine, vitamin B ₆ , into pyridoxal-5-phosphate (PLP) form (Wada, H., and Snell, EE, J. Biol. Chem. , 236: 2089-2095 (1961). PNP oxidase is a flavoenzyme that requires FMN as a cofactor, since it was first purified in rabbit liver in 1975 (Kazarinoff, MN, and McCormick, DB, J. Biol. Chem., 250: 3436). Pig (Kwok, F., and Churchich, JE, J. Biol. Chem., 255: 882-887 (1980), Churchich, JE, Eur. J. Biochem., 138: 327) 332 (1984)), rats (see Cash, CD, Maitre, M., Rumigny, JF, and Mandel, P., Biochem. Biophys.Res. Commun., 96: 1715-1760 (1980)), and sheep. From brain tissue (see Choi, SY, Churchich, JE, Zaiden, E., and Kwok, F., J. Biol. Chem., 262: 12013-12017 (1987)).

Its activity is high in liver, kidney, and brain tissues, but it has some tissue specificity, low activity in heart, muscle, pancreas and bone marrow. This is related to the physiological role of PLP, in particular the importance of PLP in the central nervous system as mentioned above. Kinetics, structure, properties and regulatory phenomena of PNP oxidase have been studied for rabbit liver, pig and sheep brain tissue enzymes, but PNP oxidase is very important in mammalian central nervous system. Despite their role, there is still no research on neurophysiological and pathological significance and drug development.

Therefore, the present inventors have developed a monoclonal antibody against the PNP oxidase present in sheep brain to develop a monoclonal antibody that specifically recognizes PNP oxidase that can be effectively used in the study of PNP oxidase. The present invention has been accomplished by discovering that antibodies can widely recognize PNP oxidase of various mammals and birds.

After all, it is an object of the present invention to provide monoclonal antibodies against PNP oxidase isolated from sheep brains.

Figure 1a is a photograph showing the result of SDS-PAGE of purified PNP oxidase from sheep brain tissue.

FIG. 1B is a photograph showing Western blot analysis results of six monoclonal antibodies finally selected through the screening process of monoclonal antibodies and purified PNP oxidase.

Figure 2a is a photograph showing the result of SDS-PAGE of whole brain tissue protein.

Figure 2b is a photograph showing the results of Western blot analysis of the six monoclonal antibodies and the amount of whole brain tissue protein selected through the screening process of monoclonal antibodies.

FIG. 3A is a photograph showing the results of SDS-PAGE of six finally selected monoclonal antibodies purified by Protein A column chromatography.

3B is a graph showing that the activity of positive brain tissue PNP oxidase is inhibited by purified monoclonal antibody.

Figure 4a is a photograph showing the results of SDS-PAGE of the whole protein isolated from the brain of various mammals and birds.

Lane 1: human brain tissue crusher lane 2: bovine brain tissue crusher

Lane 3: porcine brain tissue lysate lane 4: dog brain tissue lysate

Lane 5: feline brain tissue lysate lane 6: let brain tissue lysate

Lane 7: chicken brain lysate

Figure 4b is a photograph showing the results of Western blot analysis of the total protein isolated from the brain of various mammals and birds using monoclonal antibodies against PNP oxidase (each lane is the same as Figure 4a above).

Figure 5a is a photograph showing the result of SDS-PAGE of the whole protein isolated from human brain cells.

Lane 1: human neurons SK-N-SH Lane 2: glia cells U-373-MG

Lane 3: glia cell U-373-MG diluent

Figure 5b is a photograph showing the results of Western blot analysis of the whole protein isolated from human brain cells using a monoclonal antibody against PNP oxidase (each lane is the same as Figure 5a above).

Figure 6a is a photograph showing the result of SDS-PAGE of the whole protein isolated from various tissue cells of the let.

Lane 1: brain tissue crusher lane 2: liver crusher

Lane 3: Kidney Shredding Lane 4: Heart Shredding

Lane 5: gastric crusher lane 6: lung crusher

Lane 7: testicular crusher lane 8: muscle tissue crusher

Figure 6b is a photograph showing the results of Western blot analysis of the total protein isolated from the various tissue cells of the rat using a monoclonal antibody against PNP oxidase (each lane is the same as the contents of Figure 6a above).

The present invention relates to a monoclonal antibody against pyridoxine-5'-phosphate oxidase, which is isolated from sheep brain and produced by hybridoma with accession number KCLRF-BP-00033.

The monoclonal antibody is characterized in that the monoclonal antibody against pyridoxine-5′-phosphate oxidase having an N-terminal amino acid sequence of Met-Thr-Cys-Gly-Leu and a molecular weight of about 30 kDa.

Furthermore, the monoclonal antibody is characterized in that it immunologically reacts with pyridoxine-5'-phosphate oxidase present in the brains of mammals and humans except humans.

In addition, the monoclonal antibody is characterized in that it immunologically reacts with pyridoxine-5′-phosphate oxidase present in human brain cells.

In addition, the monoclonal antibody is characterized in that it immunologically reacts with pyridoxine-5′-phosphate oxidase present in each tissue cell of the rat such as liver, kidney, heart, stomach, lungs, testes, muscles, including the brain. .

Hereinafter, the present invention will be described in more detail.

About 60 kDa of PNP oxidase, consisting of two 30 kDa monomers, was isolated purely from DEAE cellulose, P-pyridoxyl-sepharose, Sephadex G-100 column chromatography method from the whole protein of the brain tissue ( See, Choi, SY, Churchich, JE, Zaiden, E., and Kwok, F., J. Biol. Chem., 262: 12013-12017 (1987)).

To prepare monoclonal antibodies against PNP oxidase, complete Freund's adjuvant mixtures containing PNP oxidase modified with SDS were immunized by intraperitoneal injection of BALB / c mice and then splenocytes obtained from these BALB / c mice. And fusion with mouse mialoma cells. Hypoxanthine-aminopterin-thymidine (HAT) medium (Dulbecco's modified Eagle's medium) containing 20% BCS, antibiotics and HAT was used to grow only fusion cells. After the fusion reaction, immunoblot analysis was performed using the supernatant of the fusion cell culture and the purified PNP oxidase as antigens for screening clones producing monoclonal antibodies.

Clones that consistently produce monoclonal antibodies against PNP oxidase were selected, and Western blot analysis of whole brain tissue proteins confirmed that the monoclonal antibodies produced by these clones specifically reacted with PNP oxidase.

On the other hand, in order to confirm whether the monoclonal antibody inhibits PNP oxidase enzyme activity, the purified monoclonal antibody and the purified PNP oxidase were reacted, and then the enzyme activity of PNP oxidase was measured. As a result, only two clones were found to significantly inhibit PNP oxidase.

In addition, in order to investigate cross-reactivity to animal PNP oxidase other than sheep, whole proteins were prepared from various mammals and birds and analyzed according to the Western blot method using the monoclonal antibodies prepared above. As a result, the PNP oxidases of mammalian, avian and human cell origin have a monomer molecular weight of about 30 kDa, which are all immunologically similar, as all show a band near 30 kDa, the molecular weight of PNP oxidase present in sheep's brain. It was confirmed.

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.

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.

Example 1 : Pure Separation of PNP Oxidase

The supernatant obtained by crushing and centrifuging sheep brain tissue was subjected to the N-terminal column by DEAE cellulose (Amersham Phamacia, USA), P-pyridoxyl-Sepharose, Sephadex G-100 (Amersham Phamacia, USA) column chromatography. PNP oxidase of approximately 60 kDa consisting of two 30 kDa monomers having an amino acid sequence of Met-Thr-Cys-Gly-Leu was isolated (see Choi, SY, Churchich, JE, Zaiden, E., and Kwok). , F., J. Biol. Chem., 262: 12013-12017 (1987)). 1A is a photograph showing the result of SDS-PAGE of PNP oxidase purified from sheep brain tissue as described above.

Example 2 : Preparation of Monoclonal Antibodies Against PNP Oxidase

Example 2-1 : Preparation and Injection of Immunogen

The final concentration of SDS was added to the PNP oxidase (100 μg / 200 μl) isolated in Example 1, completely denatured by heating at 100 ° C. for 1 minute, and then the same volume as the solution containing the denatured PNP oxidase. Complete Freund's adjuvant was mixed and sonicated for 15 seconds three times. This immunogen-adjuvant mixture was injected intraperitoneally into 6-8 week old female BALB / c mice. Three additional injections were made at 3-4 week intervals after the first injection, and last injection without adjuvant 3-4 days prior to preparation of fusion cells.

Example 2-2 : Preparation of Clones Producing Monoclonal Antibodies Against PNP Oxidase

Feeder cells for promoting the growth of the following fusion cells were prepared as follows one day before the preparation of the fusion cells: 12-18 week old BALB / c mice were killed by cervical dislocation After removing the skin, 5 ml of a 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.

To prepare fusion cells, splenocytes obtained from BALB / c mice immunized in Example 2-1 were prepared using SP2 / 0-Ag-14 mouse myeloma cells (Shulman H. et al., Nature, 276: 269-270 (1978)), and 1500 ml of 50% polyethylene glycol dissolved in serum-free DMEM (Gibco BRL, USA) was slowly added and fused at 37 ° C. for 90 seconds. To terminate the fusion reaction, 1 ml of DMEM was added slowly for 1 minute, followed by 2 ml for 1 minute, and 20 ml of DMEM 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 (DMEM containing 20% BCS, antibiotics and HAT) at 1 at 650 × g. Cells obtained by centrifugation for a minute were suspended again in 120 ml of HAT medium. The cell suspension was incubated in 96-well microtiter plates at 1 ml.

About two weeks after the fusion reaction, the supernatant of the cell culture solution and PNP oxidase purely separated in Example 1 were used to screen clones that produce monoclonal antibodies (hereinafter referred to as 'mAb'). As antigen, the following Immunodot blot analysis (primary screening) and Western blot analysis (secondary screening) were performed.

(i) Immunodot blot analysis : PNP oxidase (1 mg) purified on each 1 × 1 cm nitrocellulose paper, placed on a sheet of nitrocellulose paper (10 × 10 cm), serially numbered 1 × 1 cm nitrocellulose paper. / Ml) was adsorbed and dried in air. The blot was blocked for 1 hour with Blotto (5% skim milk powder dissolved in PBS (phosphate buffered saline)), washed with PBS, dried, and placed in 96-hole microplate holes containing respective fusion cell cultures. Immune responses were measured as published in the western blot analysis method below.

(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. Subsequently, the cells were blocked with Blotto for 1 hour and washed with PBS, reacted with the positive clone culture supernatant in Immunodot blot analysis for 1 hour, and then washed three times with PBS including Tween 20 at 5 minute intervals. Then, Goat anti-mouse IgG (AP) coupled with alkaline phosphatase (AP) was added and reacted for 1 hour with 3 minutes of PBS containing Tween 20 at 5 minute intervals. After one wash, a final wash for 5 minutes with AP buffer (100 mM Tris buffer containing 5 mM MgCl ₂ , pH 9.5). 60 μl of a solution consisting of 70% dimethylformamide and 50 mg / ml nitroblue tetrazolium and a solution of 100 mg 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.

Through the cell fusion experiments, 21 clones positive in the primary screening and 12 clones in the secondary screening were selected. After diluting the culture of the clones identified as positive in the 1st and 2nd screening with HT medium to 15 cells / ml, dispense 70 μl into 96-hole plates, and add 140 μl of fresh HT medium to each of them. The supernatant was analyzed by Western blot to finally select six single specific antibody-secreting cells that produce antibodies against PNP oxidase. Figure 1b is a photograph showing the results of Western blot analysis of the finally selected six mAbs and purified PNP oxidase in Example 1.

As shown in FIG. 1B, each mAb showed a band of immune response results at the same position as the 30 kDa purified PNP oxidase, indicating that each of the six mAbs was a monoclonal antibody of PNP oxidase.

Example 3 : Confirmation of specificity of monoclonal antibody

In order to determine the specificity of the monoclonal antibody prepared in Example 2, Western blot analysis was performed on the amount of whole brain tissue protein (see Figs. 2a and 2b). Figure 2a is a photograph showing the result of SDS-PAGE of the whole brain tissue protein, the first lane shows the molecular weight marker, the second lane shows the positive whole brain tissue protein. Figure 2b is a photograph showing the results of the immune response between the six mAb and the whole protein of the brain tissue. As shown in Figures 2a and 2b, each mAb exhibits a band of immune response results at the same position as the 30 kDa PNP oxidase, it can be seen that each mAb is a mAb that specifically reacts with PNP oxidase.

Example 4 : Inhibition of PNP Oxidase Enzyme Activity of Monoclonal Antibodies

To investigate whether mAb inhibited PNP oxidase enzyme activity, 10 μg of 6 monoclonal antibodies purified by Protein A column chromatography and 1 μg of purified PNP oxidase were reacted at room temperature for 1 hour, followed by PNP oxidase. The enzyme activity of was measured. Figure 3a is a picture showing the results of purification by protein A column chromatography of the six monoclonal antibodies, SDS-PAGE, Figure 3b is the activity of the positive brain tissue PNP oxidase is inhibited by the purified monoclonal antibody It is a graph. As shown in Figure 3b, only two mAbs were found to inhibit the activity of PNP oxidase.

Example 5 : Reactivity of monoclonal antibodies with PNP oxidases of various mammalian and avian origins

Cross-reactivity of PNP oxidase and the monoclonal antibodies of mammals and birds including humans was examined by Western blot. First, brain tissues were isolated from humans, cows, pigs, dogs, cats, rats and chickens, and then crushed by mixing 10 mM potassium phosphate buffer containing 0.1 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM PMSF for each. It was. 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, Western blot analysis by the method of Example 2 (see Figs. 4a and 4b).

4A is a photograph showing the results of SDS-PAGE of brain tissue lysate of humans, cows, pigs, dogs, cats, rats, and chickens. Figure 4b is a photograph showing the results of Western blot analysis of the entire protein of Figure 4a using one of six mAbs for PNP oxidase. As shown in Figure 4b, it can be seen that mAb recognizes PNP oxidase derived from the brains of humans, cows, pigs, dogs, cats, rats and chickens. Therefore, it was confirmed that the PNP oxidase of mammalian and avian origin has a monomer molecular weight of 30 kDa and has a common specific antigenic epitope and is very similar immunologically.

Example 6 : Reactivity of monoclonal antibody with PNP oxidase of several human brain cells

The cross-reactivity of PNP oxidase and monoclonal antibodies of human neurons was examined by Western blot. First, human neurons (neural cells SK-N-SH (ATCC HTB 11), glia cell U-373-MG (ATCC HTB 17)) were added to 10 ml of HT (Gibco BRL, USA) medium (10% FBS, Incubated in DMEM containing antibiotics and centrifuged at 650 × g for 1 min to obtain cells, each containing 10 mM potassium phosphate buffer containing 0.1 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM PMSF By crushing. Centrifuge each 25% (w / v) lysate at 10,000 × g for 1 hour, take 5 μl from each supernatant, mix with an equal volume of 2 × SDS sample buffer, boil for 3 minutes, and cool to SDS -PAGE and transfer to nitrocellulose membrane, Western blot analysis by the method of Example 2 (see Figs. 5a and 5b).

Figure 5a is a photograph showing the results of SDS-PAGE of human neurons and glia cell lysate. Figure 5b is a photograph showing the results of Western blot analysis of the entire protein of Figure 5a using one of the six mAbs for PNP oxidase. As shown in Figure 5b, it can be seen that mAb recognizes PNP oxidase derived from human neurons. Therefore, it was confirmed that the PNP oxidase of human neuronal origin has a monomer molecular weight of 30 kDa and has a common specific antigen determination site (epitope), which is very similar immunologically.

Example 7 : Reactivity of monoclonal antibodies with PNP oxidases of various tissue origin

Cross-reactivity of several tissue PNP oxidases of the rat with the monoclonal antibody was examined by Western blot. First, liver, kidney, heart, stomach, lung, testis and muscle tissue, including brain tissue, were isolated from the rats, and then 10 mM potassium phosphate buffer containing 0.1 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM PMSF for each. Was mixed and crushed. 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, Western blot analysis by the method of Example 2 (see Figs. 6a and 6b).

Figure 6a is a photograph showing the results of SDS-PAGE of liver, kidney, heart, stomach, lungs, testes, muscle tissue crushing fluid including the brain tissue of the let. Figure 6b is a photograph showing the results of Western blot analysis of the entire protein of Figure 6a using one of the six mAbs for PNP oxidase. As shown in Figure 6b, it can be seen that mAb recognizes PNP oxidase derived from liver, kidney, heart, stomach, lungs, testes, muscle tissue, including the brain tissue of the rat. Therefore, it was confirmed that the PNP oxidase of various tissue origins of the let has a monomer molecular weight of 30 kDa and has a common specific antigenic epitope, which is very similar immunologically.

As described and demonstrated in detail above, the present invention provides a monoclonal antibody against PNP oxidase isolated from sheep brain. These monoclonal antibodies specifically recognize PNP oxidase and can recognize PNP oxidase enzymatic activity of various animal and tissue origins as well as sheep, leading to neuropathy associated with PNP oxidase, especially in brain tissue. It can be applied to the analysis of the causes and the biochemical properties of neurological diseases such as seizure, convulsions, etc. that can occur due to metabolic abnormalities. 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.

Claims (5)

Monoclonal antibody against pyridoxine-5′-phosphate oxidase isolated from sheep brain and produced by hybridoma cell line with accession number KCLRF-BP-00032. The monoclonal antibody against pyridoxine-5′-phosphate oxidase of claim 1, wherein the N-terminal amino acid sequence is Met-Thr-Cys-Gly-Leu and has a molecular weight of about 30 kDa. The pyridoxine-5'-phosphate oxidase according to claim 1 or 2, characterized in that it is immunologically reacted with pyridoxine-5'-phosphate oxidase present in the brains of mammals and humans except humans. Monoclonal antibodies. 3. The method of claim 1 or 2, wherein said method reacts immunologically with pyridoxine-5'-phosphate oxidase present in brain neurons of a human selected from neuronal cells SK-N-SH or glia cells U-373-MG. Characterized in a monoclonal antibody against pyridoxine-5'-phosphate oxidase. The method according to claim 1 or 2, wherein the immunological reaction with pyridoxine-5'-phosphate oxidase is present in each tissue cell of the rat, including the brain, liver, kidney, heart, stomach, lungs, testes and muscle. Characterized in a monoclonal antibody against pyridoxine-5'-phosphate oxidase.