JPH0653077B2 - Standard composition for measuring bound sialic acid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a standard composition for measuring bound sialic acid, which is used as a standard composition when measuring sialic acid in a body fluid.

[Prior Art] Sialic acid (hereinafter, referred to as conjugated sialic acid) is found in many mammalian glycoproteins or in a very small amount of bacterial cell wall. Such bound sialic acid is considered to be synthesized in the liver by a complicated reaction process in mammals, for example. Bound sialic acid is also contained in human body fluids in various amounts, and varies depending on the physical condition and health condition. Therefore, measuring the amount of bound sialic acid present in body fluids provides useful information for diagnosing inflammation, observing the progress of medical conditions, etc., and is of great clinical significance.

In measuring such bound sialic acid, a standard composition thereof (standard composition for measuring bound sialic acid) is required, but the standard composition generally used conventionally is obtained from serum. It is a thing. However, the serum used as a sample is relatively difficult to obtain, its components are not constant, and it has various drawbacks such as being expensive.

Despite such drawbacks, no technical improvement has been made so far and no new standard composition has been developed so far.

[Problems to be Solved by the Invention] The present invention has been made in view of the above circumstances,
The purpose is to provide a standard composition for binding type sialic acid measurement, in which a sample as a raw material can be obtained relatively easily, the quality is stable, and the cost is low.

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to achieve the above-mentioned object, and as a result, milk protein that is relatively inexpensive and easily available, glycoprotein derived from the milk protein, or enzymatic degradation thereof. It was found that a standard composition for binding type sialic acid measurement with stable quality can be obtained by using the product as a raw material, and the present invention has been completed. That is, the present invention which has achieved the above object is a standard composition for binding sialic acid measurement, which comprises an enzymatic degradation product obtained by treating a glycoprotein derived from milk protein with a proteolytic enzyme.

[Operation] Casein or whey is most preferred as the milk protein used in the present invention. For example, skim milk, sodacasein,
Examples thereof include caseins such as acid casein and whey such as cheese whey and acid whey. Concentrated or dried products of these may be used. From the viewpoint of increasing the content of bound sialic acid per unit weight, glycoprotein can be fractionated from the above protein resource and used as a standard composition. Furthermore, it is also possible to carry out limited digestion using a proteolytic enzyme to take out the glycopeptide and use this as a standard composition.

Known glycoproteins in milk include immunoglobulins contained in whey, β-lactoglobulin, lactoferrin, and κ-casein contained in casein. A known method may be used to fractionate these glycoproteins or a mixture thereof from milk proteins. For example, “Milk P
rotein ”(edited by HA McKenzie), JP-A-58-28233, JP-A-59-91848 and the like.

The enzyme used for the limited decomposition is not particularly limited as long as it is a proteolytic enzyme, and examples thereof include trypsin, chymotrypsin, pepsin, papain, chymosin, pancreatin and the like, or a mixture thereof.

The treatment conditions of the above-mentioned enzymes are not particularly limited, and the optimum conditions for each enzyme may be selected.
For example, when β-lactoglobulin is treated with tribucin, tribucin is added to a Tris buffer having a pH of 7 to 8 so that the E / S ratio (enzyme-substrate ratio) is about 1/100 to 1/2000, and 30 The enzyme reaction may be allowed to proceed at -40 ° C for 15 minutes to 6 hours. When κ-casein is treated with chymosin, the E / S ratio is 1/100 to 1/2000 in an acetate buffer solution of pH 5.5 to 7.
Add chymosin so that
The enzymatic reaction may be allowed to proceed for 3 hours.

Examples of the method for fractionating and collecting glycopeptides include gel filtration, ion exchange chromatography, affinity chromatography, and chromatofocusing.
One or two or more of these methods may be adopted. Alternatively, trichloroacetic acid (TCA) may be added to remove the precipitate, and then the supernatant may be desalted. Further, as a method of obtaining a glycopeptide from κ-casein, a method of adding calcium (5 mmol or more) to remove a precipitate, and then collecting a supernatant thereof can be mentioned.

If necessary, a surfactant, an antiseptic, a stabilizer and the like can be added to the standard composition of the present invention. Examples of the storage form of the prepared standard composition include cold-temperature storage in a state of being melted in an aqueous solution, storage in a freeze-dried state, and the like, and any of them can be stored for a long period with stable quality.

On the other hand, as a method for measuring bound sialic acid using the standard composition for measuring bound sialic acid according to the present invention, (a) neuraminidase, N-acetylneuraminic acid aldolase,
A method for measuring the amount of bound sialic acid by reducing NADH using lactate dehydrogenase and NADH, (b) a method for measuring using neuraminidase, N-acetylneuraminic acid aldolase, pyruvate oxidase, (c) There is a method of measurement using N-acetylneuraminic acid aldolase, acylglucosamine epimerase, N-acetylhexosamine oxidase, etc., which may be appropriately selected.

Example 1 First, 1 g of β-lactoglobulin (manufactured by Sigma) was dissolved in 100 ml of a Tris buffer solution having a pH of 8. Separately, a solution prepared by dissolving 1 mg of tribucin (manufactured by Sigma) in a very small amount of deionized water was prepared. Next, both solutions were incubated at 35 ° C and then mixed, and reacted at 35 ° C for 3 hours.
After adding 12% TCA to the reaction solution and leaving it for 10 minutes,
Centrifugation was performed at 3000 rpm for 10 minutes to obtain a supernatant. Next, this supernatant was dialyzed against deionized water and freeze-dried to obtain about 0.2 g of powder. It was confirmed that the bound sialic acid contained in this powder was 1.5% by weight.

Example 2 Sodacasein (New Zealand) was dissolved in an imidazole buffer solution having a pH of 7 so as to be 10% and cooled at 2 ° C. for 1 hour. Prepare a column (diameter: 2 cm, height: 90 cm) of Seraphadex G-100 (Pharmacia Co., Ltd.), which has been leveled with the same buffer and cooled to 2 ° C.,
5 ml of the above casein solution was added to this column and eluted with the same buffer. Next, the fraction eluted in the void volume was collected, dialyzed against deionized water, and freeze-dried to obtain 80 mg of powder. It was confirmed that this powder contained κ-casein as a main component, and the content of bound sialic acid was about 2% by weight.

The powder obtained by the above-mentioned method was dissolved in 50 mg deionized water, the pH was adjusted to 6.4, and 0.04 mg rennet (manufactured by Hansen) was added. Next, after reacting at 40 ° C. for 30 minutes, CaCl ₂ was added so that the concentration of the mixture was 20 mmol, paracasein was precipitated, and a supernatant was collected. This supernatant was mixed with M. W. Diafiltration was carried out with an ultrafiltration device (manufactured by Amicon) equipped with an ultrafiltration membrane of cut-off 1000, and the retentate was freeze-dried to obtain 20 mg of powder. It was confirmed that this powder contained 1.1 mg of bound sialic acid.

Example 3 The standard composition for measuring the bound sialic acid obtained in Example 2 and the standard composition obtained from blood (conventional example) were used as test liquids, and the respective reagents were reacted using the following reagents. investigated. The concentration of bound sialic acid contained in each test liquid was 100 mg / dl for both.

<Reagent> Tris buffer (0.1 M) pH 7.0 neuraminidase 0.2 U / ml N-acetylneuraminic acid aldolase 0.2 U / ml NADH 0.2 mmol mol The measuring method is as follows. That is, after adding 3 ml of the above reagent to 50 μl of each test liquid and reacting at 37 ° C., the absorbance is measured at a wavelength of 340 nm, and ΔOD / min (change in optical density) from 2 minutes to 4 minutes based on the reagent blank is measured. It was measured.

The measurement results are shown in Table 1 below.

As is clear from the results shown in Table 1 above, the standard composition for measuring bound sialic acid according to the present invention is comparable in reactivity to the conventionally used standard composition derived from serum. It is understood that the quality is stable.

[Effects of the Invention] As described above, according to the present invention, a standard composition for measuring bound sialic acid is obtained from milk protein which is relatively inexpensive and easily available and has stable quality. It can be resolved.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Eiji Eike 2-23-5-102 Irumagawa, Sayama City, Saitama Prefecture (56) References J Fac Agric Hokkai do Univ, 62 (3), P. 236-255, 1985 Lait, 57 (568), p. 492-500, 1977 J Dairy Sci, 59 (10), P.P. 1718-1726, 1976 J Dairy Sci, 66 (3), P. 390-395, 1983

Claims (1)

[Claims] 1. A standard composition for measuring bound sialic acid, which comprises an enzymatic degradation product obtained by treating a milk protein-derived glycoprotein with a proteolytic enzyme.