KR100309072B1 - Method for sialidase assay - Google Patents

Abstract

The present invention relates to an assay for assaying the titer of sialidase using the Enzyme Linked Immunosorbent assay (ELISA) method, which provides a simple and high sensitivity as well as sialic acid already present in the sample. It can block the possibility of false-positives that occur and has excellent reproducibility with reactions on the same plate, and in particular can directly measure the titer of sialidase for the target protein.

Description

Method for measuring the titer of sialidase {Method for sialidase assay}

The present invention relates to a method for directly measuring the titer of sialidase on a target protein, and more particularly, by eliminating sialidase from a target protein using an Enzyme Linked Immunosorbent assay (ELISA) method. It relates to a method for quantifying sialic acid.

Sialic acid is a generic term for amine derivatives of N-neuraminic acid, and is widely distributed in the biological system as constituents such as mucopolysaccharide, glycoprotein, glycolipid, and oligosaccharide of human oil. In particular, they are present at the ends of glycoproteins or glycolipids of higher animals and have a profound effect on their molecular structure. These sialic acids play two roles in the interaction between cells and cell molecules. The first role is to mask the recognition determinant of the cell or molecule, and the second role is to recognize sialic acid itself. It acts as a site.

Sialidase present in Chinese Hamster Ovary (CHO) cells removes sialic acid from the sialo glycoproteins contained in the culture as follows.

The in vivo activity of glycoproteins is closely related to the presence or absence of sialic acid, and thus the glycoprotein from which sialic acid has been removed is rapidly reduced in vivo. The reason is that when sialic acid, which is present at the sugar chain terminal of the glycoprotein and is connected to the next sugar galactose and mostly α-2,3-linked, is eliminated by the sialidase present in the cell culture, the galactose residue is exposed. (Asialo) glycoproteins are recognized by galactose receptors in hepatocytes and captured and metabolized in hepatocytes.

If the cells do not produce sialidase, there will be no sialidase present in the cell culture, and it will not modify the various glycoproteins contained in the culture. It is still unclear where this sialidase comes from, taking into account the possibility that sialidase, which is present in the serum contained in the cell medium, released from the cell, or contained in the cell protoplasts, leaks as the cell breaks down. You can try A study by Munzert, E. et al. (Biotechnol. Prog. 12: 559-563, 1996) observed an increase in sialidase with an increase in lactate dehydrogenase during batch culture. There is a correlation between dead cell numbers and sialidase release from the plasma solution. Thus, sialidase in cell culture is presumed to be derived mainly from the plasma solution of dead cells.

Sialidase of CHO cells was established in 1993 by Warner T.G. Purified by Glycobiology 3: 455-463, 1993. The molecular weight of the purified sialidase reached 43,000 Daltons and the isoelectric point was found to be between pH 6.8 and 7.0. The optimal pH of sialidase was 5.9 and the titer for 2,3-bond was about four times stronger than 2,6-bond. Ferrari, J. et al. Cloned cDNA in 1994 and expected a protein consisting of 379 amino acids, which showed sequence similarity with various bacterial sialidases.

There are two methods for measuring the titer of sialidase known to date.

First, the fluorescence assay (Potier et al., Anal. Biochem. 94, 287, 1979) is a sialic acid analog to which a fluorescent substance is attached, namely 4-MU-NeuAC ((4-methylumbelliferyl-5- acetamido-3,5-dideoxy-D-glycero-aD-galacto-nonulopyanosid) onic acid) is used as a substrate.When umbelliferone is released by sialidase, the fluorescence intensity therefrom is measured. Sialidase titers will be measured. This method has the advantage of excellent sensitivity, but has various disadvantages. That is, various equipment (e.g. HPLC, fluorometer, autosampler), etc. are required, the experimenters must be highly skilled with the equipment, and the resulting fluorophore is It has the disadvantage of being unstable. In addition, the use of substrate analogs does not exclude the possibility that the actual enzyme specificity may differ from the natural substrate.

Second, the TBA titer (thiobabituric acid assay) (Uchida et al., J. Biochem., 82, 1425-1433, 1977) is a method of using a natural substrate, which allows sialidase to be applied to substances such as sialo oligosaccharides. It is a method of quantifying sialic acid which is directly added and liberated by thiobabiturate. This method is a classical method of detecting free sialic acid, which is cumbersome to require various pretreatments before measuring the sample. In addition, there is a risk that the sialidase titer will be higher than it is because sialic acid already present in the sample is detected together.

ELISA, on the other hand, is a high sensitivity, known immunoassay for specific antibodies or antigens. For example, to measure the activity of a particular antibody, the antiserum (or other sample) is reacted with a homogenous antigen adsorbed on the inner surface of a plastic tube or the like, followed by washing off the unbound antibody. The antibody bound to the immobilized antigen is then treated with a conjugate of anti-immunoglobulin antibodies, each covalently bound to a specific type of enzyme, followed by washing off the unbound conjugate, Incubate with a suitable substrate to determine enzyme degradation products. On the other hand, in order to measure the activity of a specific antigen, the same method as the above method can be used except that the antibody is adsorbed and the enzyme is conjugated to the anti-antigen antibody.

However, no attempt has been made to use the ELISA method to detect glycoproteins in sial as in the present invention.

Accordingly, the present inventors have conducted continuous research to develop a novel method for measuring the titer of sialidase, which can solve the problems described above. As a result, the present invention provides a simple and high sensitivity. Not only can it be obtained, it can block the possibility of false-positive caused by sialic acid already present in the sample and has excellent reproducibility by reaction on the same plate, especially for the target protein. It has been found that the titer of sialidase can be measured directly and the present invention has been completed.

Thus, the object of the present invention is to achieve a simple yet high sensitivity, to block the possibility of gastric-positive events caused by sialic acid already present in the sample and to have excellent reproducibility with reactions on the same plate, in particular the target protein. It is to provide a novel method for measuring the titer of sialidase, which can directly measure the titer of sialidase.

1 is a schematic diagram showing the principle of the titer measuring method according to the present invention:

2 is a graph showing a standard curve of sialidase titer obtained using the 4-MU-NeuAc titer method;

3 is a graph showing a standard curve of sialidase titer obtained using the titer measuring method according to the present invention; And

Figure 4 is a graph showing the results of measuring the change in sialidase titer in cell culture using the method for measuring titers according to the present invention.

In order to achieve the object as described above, the present invention provides a method for measuring the titer of sialidase, characterized by using the ELISA method. The method of the present invention is characterized by detecting a sial glycoprotein by adding a sial oligosaccharide-specific substance (antibody) to a sial glycoprotein (antigen) treated with sialidase. In the present invention, the substance that specifically recognizes the sally oligosaccharide is preferably MAA-Lectin (Maackia amurensis-Lectin). In one example of the present invention, MAA-lectin is bound to digoxigenin, and in this case, by treatment with an anti-digoxigenin antibody conjugated with an enzyme, a substrate is added to measure the enzyme degradation product. Sial can detect glycoproteins.

According to the method of the invention, the sialo glycoprotein is preferably immobilized on the plate. According to one embodiment of the present invention, the sialo glycoprotein is erythropoietin (EPO), and the sialidase may be derived from CHO cells.

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

The present invention relates to a method for directly measuring the titer of sialidase acting on sialo glycoproteins. In a preferred embodiment of the present invention, the sialo glycoprotein (antigen) is immobilized on a plate to which an antibody against sialo glycoprotein (for example, erythropoietin) is adsorbed, and then treated with sialidase for a predetermined time, Remove sialidase by washing out. To this is added MAA-lectin (antibody) which selectively recognizes only sialo oligosaccharides. MAA-lectins are lectins isolated from the legume tree called 'Maackia amurensis'. Lectin is a group of proteins widely distributed in the natural world that can aggregate red blood cells and many other types of cells, the existence of which was known since 1899 when Stillmark isolated hemagglutinin from castor fruit. The term WC First introduced by Boyd et al. (Science 119, 419, 1954). Lectins are currently used to mean 'non-immune sugar-binding proteins or glycoproteins that aggregate cells and / or precipitate glycoconjugates'. Thus, MAA-lectins have specificity for sugar conjugates containing sialic acid.

In the present invention, 'MAA-lectin-digoxigenin', which binds MAA-lectin with digoxigenin, is added to sialo glycoproteins treated with sialidase, followed by washing unbound MAA-lectin-digoxigenin. Remove To this is added an 'anti-digoxigenin antibody-enzyme conjugate' in which an antibody against digoxigenin is covalently bound to a specific type of enzyme such as peroxidase, and the unbound conjugate is washed off. By adding an appropriate substrate and measuring the enzymatic degradation product using a method such as color reaction, the glycoprotein can be detected by sial after treatment with sialidase. Since sialidase removes sialic acid from sialo glycoproteins, the amount of binding of MAA-lectin will be lower than before sialidase treatment, thus resulting in a lower degree of color reaction than before sialidase treatment. From this it is possible to directly measure the titer of sialidase on sialo glycoprotein. That is, as the binding amount of MAA-lectin decreases and thus the color reaction decreases, the titer of sialidase is higher.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples, which are only intended to help the understanding of the present invention, and are not intended to limit the scope of the present invention in any way.

Preparation Example 1 Preparation of Sialidase Stock Solution

CHO cells were grown in T-flask (Nalgene) fed 5% CO ₂ at 37 ° C. Medium was DMEM / F12 with 2 mM glutamine (Gibco), 5% Fetal bovine serum (Hyclone), antibiotic (100 U / ml penicillin G, 100 μg / ml streptomycin, 25 μg / ml amphotericin B) ) Was added.

For enzyme titer determination, cells grown confluent in the medium ( ⁵ × 10 ⁵ cells / cm ² ) were treated with trypsin and passaged with 100 mm tissue culture dishes at a ratio of 1: 4. After 1-2 days, cells were obtained and 10 ml of cold PBS (Phosphate buffered saline: 8 g / l NaCl, 0.2 g / l KCl, 1.13 g / l Na ₂ HPO ₄ and 0.2 g / l KH ₂ PO ₄ , pH 7.5) Washed twice. Pour 10ml of PBS 3 times and left for 10 minutes at 4 ℃ to loosen the cells. The cells were collected and added to a centrifuge tube and some cells were counted. The remaining cells were centrifuged at 100 x g for 10 minutes. After removing PBS, the cells were added to cold water and suspended to 1 × 10 ⁷ cells / ml (3.3 mg / ml). Cells were completely disrupted by five passes through a 22 gauge needle.

Example 1 Measurement of Titer of Sialidase Using ELISA Method

To determine the effect of sialidase on sialic acid bound to glycoproteins, monoclonal antibodies that recognize the target protein were adsorbed onto a microtiter plate (Maxisorp; Nunc, Wiesbaden, Germany), and The target protein was bound to the stomach again and treated with sialidase. Since sialidase removes sialic acid bound to glycoproteins, the degree of binding of sialic acid-recognized MAA-lectins is reduced, and subsequent color development lowers the color development compared to controls not treated with sialidase. It was expected to show.

100 μl of CFC-6, a monoclonal antibody against sipo glycoprotein EPO, was added at a concentration of 2.5 μg / ml, added to a 96-well microtiter plate, and reacted at 37 ° C. for 1-2 hours. Blocking solution (0.5% gelatin hydrolyzate) was added and blocked for 1 hour at room temperature. Washed four times with PBST (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4, 0.05% Tween 20). 100 μl of the target protein EPO was added to each well and allowed to react at 37 ° C. for 1 hour (control). After washing four times with PBST again, the enzyme stock solution obtained in Preparation Example 1 was diluted and added at a predetermined rate, and then reacted at 37 ° C. for 1 hour. After washing four times with PBST, MAA-lectin-digoxigenin solution (1 mg / ml) was added to Tris buffered saline, 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, 1 mM MgCl ₂ , 1 mM MnCl ₂ , 1 mM CaCl ₂ ) Was diluted 1,000-fold and added to 100 µl, followed by reaction at room temperature for 1 hour. After washing with PBST, peroxidase conjugated anti-digoxigenin polyclonal antibody was diluted 2,000-fold with TBS, and added to 100 µl and reacted at 37 ° C for 1 hour. Dissolve 20 mg of O-Phenylene diamine dihydrochloride in 20 ml of substrate buffer (0.1 M citric acid, 0.1 M sodium phosphate, pH 5.0) and add 30 μl of 25% H ₂ O ₂ to the solution. 100 μl was added to each well and developed. Sialidase titers were determined by reading the values from OD ₄₉₂ using an ELISA autoreader and substituting them into the standard curve (FIG. 3) (FIG. 4).

[Comparative Example 1] Fluorescence titer measuring method (4-MU-NeuAc titer measuring method)

To determine the titer of sialidase, 4-MU-NeuAc titer was used (Potier et al., Anal. Biochem. 94, 287, 1979). At this time, the composition of the measurement solution was as follows.

100 μl final 0.1 M sodium acetate (pH 3-6) or potassium phosphate (pH 6-8) buffer (10 μl 1 M stock), 1 mM 4MU NeuAc (25 μl from 4 mM aliquot), 25 μl enzyme sample Made with. 100 μl of sample was added to a 1.5 ml test tube and allowed to react for 30 minutes while shaking in a 37 ° C. water bath. The reaction was stopped by adding 0.9 ml of 0.2 M glycine (pH 10.4). Samples were centrifuged at 12,000 × g for 15 minutes to remove precipitate and diluted 10-fold in HPLC sample bottles (50 μl sample + 450 μl glycine buffer).

Measurements were made using an autosample injector on HPLC with fluorescence detector. At this time, the extinction wavelength was 365nm, the diverging wavelength was used 448nm.

Comparative Example 2 Method for Measuring TBA Titer

To a sample solution and a standard solution (containing 0.5-30 ml of sialic acid in 0.5 ml of water), 0.25 ml of periodate solution (0.025 M periodic acid in 0.125NH ₂ SO ₄ ) was added and left at 37 ° C for 30 minutes for oxidation. I was. Excess periodic acid was reduced using 0.2 ml of sodium arsenite solution. As soon as the yellow color of the free iodine disappeared, 2 ml of thiobarbiturate solution (0.1M, pH 9.0) was added and the sample was capped and then bathed in boiling water for 7.5 minutes. The colored solution was cooled in ice water, and 4 ml of acid-butanol solution (5% (v / v) 12N HCl containing n-butanol) was added thereto and shaken. After the butanol layer and the aqueous layer were separated, the butanol layer was taken and the intensity of the color was measured at 549 nm. The molar absorbance of sialic acid was 70,700.

The method for measuring the titer of sialidase according to the present invention is not only easy to obtain high sensitivity, but also blocks the possibility of gastric-positivity caused by sialic acid already present in the sample and is excellent in reaction on the same plate. It is reproducible and in particular can directly detect the titer of sialidase for the target protein.

Claims (7)

A method for measuring the titer of sialidase, comprising detecting a sialo glycoprotein by adding a sialo oligosaccharide-specific substance (antibody) to a sialid glycoprotein (antigen) treated with sialidase. The method for measuring the titer of sialidase according to claim 2, wherein the substance that specifically recognizes the oligosaccharide of sial is MAA-lectin (Maackia amurensis-Lectin). 4. The method of claim 3, wherein the MAA-lectin is combined with digoxigenin. The method for measuring the titer of sialidase according to claim 4, wherein the sial glycoprotein is detected by treating with an anti-digoxigenin antibody conjugated with an enzyme and then measuring the enzyme degradation product by adding a substrate. 3. The method of claim 2, wherein the sial glycoprotein is immobilized on a plate. The method for measuring the titer of sialidase according to claim 2, wherein the sialo glycoprotein is erythropoietin. The method for measuring the titer of sialidase according to claim 2, wherein the sialidase is derived from CHO cells.