JPS6098998A - Determination of alpha-amylase activity - Google Patents

Abstract

PURPOSE:To determine the activity of alpha-amylase easily and r apidly, by contacting a specific water-insoluble polysaccharide complex with alpha-amylase, and hydrolyzing the complex to produce a water-soluble pigment oligosaccharide complex. CONSTITUTION:A small-sized tube 1 having an inner diameter of 1-2mm. is filled with (A) a powdery mixture 2 of (a) water-insoluble pigment polysaccharide complex obtained by the chemical bonding of a water-soluble reactive pigment with a polysaccharide hydrolyzable with alpha-amylase and (b) microcrystalline cellulose, (B) a hydrophilic water-insoluble carrier substance (e.g. diethylaminoethylated celluose) 3 and (C) glass wool 4. The tube 1 is dipped in an aqueous solution 5 containing alpha-amylase to effect the hydrolysis of the complex 2. The produced water-soluble pigment oligosaccharide complex is made to ascend together with the aqueous solution 5 in the tube, and adsorbed to the carrier substance 3 to effect the color development. The activity of alpha-amylase can be determined from the length of the colored zone.

Description

[Detailed description of the invention] The present invention relates to a method for measuring α-amylase activity.

The activity of α-amylase, an enzyme that hydrolyzes starch, glycogen, etc., is an important indicator of the function of the pancreas and salivary glands, so its measurement is extremely important in clinical medicine. In particular, it is urgent to measure its activity in the sensitive areas of acute pancreatitis.

For this purpose, Japanese Patent Publication No. 51-14915 discloses water-soluble reactive dyes for amylose or amylose-containing substances, such as Cibacron Blue 3G, which is a type of triazine dye.
An aqueous suspension of a water-insoluble pigment polysaccharide complex to which A is covalently bonded is prepared, a sample such as serum or urine containing α-amylase is added to this, and the above complex is prepared under predetermined conditions. The body is subjected to the enzymatic action of α-amylase to produce a water-soluble pigment oligosaccharide complex, then the supernatant containing this is separated by centrifugation, etc., and the degree of coloration is analyzed by absorbance. proposed a method for measuring the activity of α-amylase.

Also, in Japanese Patent Publication No. 51-34317, starch,
A granular hydrophilic water-insoluble dye polysaccharide complex is described in which amylose or the like is cross-linked with a bifunctional cross-linking agent to form a water-insoluble gel, to which a water-soluble reactive dye is bound as described above. A method for measuring α-amylase activity in a sample is disclosed.

However, all of these methods usually require about 30 minutes to separate the supernatant after reacting the sample with the pigment polysaccharide complex and to measure the absorbance with a spectrophotometer needle as described above. It lacks urgency and is complicated to operate.

The present invention was made in order to solve the above-mentioned problems in α-amylase activity measurement using a water-insoluble dye polysaccharide complex, and includes fractionation of the supernatant after the reaction and absorbance measurement using a spectrophotometric needle. An object of the present invention is to provide a simple and rapid method for measuring α-amylase activity that does not require such complicated operations.

The method for measuring α-amylase activity according to the present invention comprises a first layer of a water-insoluble pigment polysaccharide complex formed by chemically bonding a water-soluble reactive pigment to a polysaccharide that can be hydrolyzed by α-amylase; A small-diameter tube filled with a second layer consisting of a water-insoluble carrier substance having a water content is impregnated with an aqueous sample containing α-amylase from the outside of the first layer to form the pigment polysaccharide complex. The water-soluble dye oligosaccharide complex produced by hydrolysis is transferred to the carrier layer together with the sample, adsorbed to the carrier layer and colored, and α-amylase activity is determined by the length of the colored region in this carrier layer. It is characterized by

The water-insoluble pigment polysaccharide complex used in the method of the present invention is a water-insoluble complex formed by chemically bonding a water-soluble pigment to a polysaccharide such as starch or amylose that can be hydrolyzed by α-amylase, as described above. α−
The polysaccharide may be crosslinked with a polyfunctional crosslinking agent as described above, as long as it undergoes hydrolysis by amylase to produce a water-soluble dye oligosaccharide complex having an ionic group. In addition to the above-mentioned Cibacron Blue 30-A, various water-soluble dyes can be used. A water-insoluble pigment polysaccharide complex in which a water-soluble pigment is bound to a polysaccharide in this manner is commercially available from, for example, Pharmacia.

As the carrier material, common carriers for chromatography can be used, and for example, colorless, microcrystalline diethylaminoethylated (DEAE) cellulose having ionic groups is preferably used.

Further, as the small-diameter tube, a transparent glass tube or synthetic resin tube with an inner diameter of about 100 yen is usually used.

The present invention will be explained below based on the drawings.

FIG. 1 shows a measuring tube used in the method of the invention. This measuring tube consists of a transparent small-diameter tube l with an inner diameter of approximately 1 to 2 mm, with N2 filled with the mixed powder of the water-insoluble pigment polysaccharide complex and microcrystalline cellulose on the lower side, and microcrystalline cellulose as a carrier. A packed layer 3 of crystalline DEAE cellulose is filled on the top side,
In order to fix these filling layers in the tube, the outer ends of the filling layers are filled with a suitable filling material 4, such as glass wool or cellulose cotton. Further, usually, both ends of the small-diameter tube are melt-sealed to prevent bacteria from entering the measuring tube, and the melt-sealed portions are removed before use.

When using this measuring tube, as shown in FIG. 2, the measuring tube is immersed in the sample aqueous solution so that the sample aqueous solution 5 containing α-amylase can penetrate into the packed layer 2 of the pigment polysaccharide complex. Then, the aqueous sample solution rises in the small diameter tube by capillary action. As the sample aqueous solution rises in the small diameter tube in this way, the dye polysaccharide complex is hydrolyzed by α-amylase contained in the sample aqueous solution to produce a water-soluble dye oligosaccharide complex, and this dye oligosaccharide complex is hydrolyzed by α-amylase contained in the sample aqueous solution. The complex is
As shown in FIG. 3, the aqueous sample solution further rises in the small diameter tube and reaches a packed bed 3 of colorless carriers, where it is adsorbed by the carriers having ionic groups and is colored.

Here, the amount of the dye oligosaccharide complex produced is proportional to the amount of α-amylase contained in the sample aqueous solution, and therefore, the carrier layer has a colored region 6 with a length proportional to the amount of α-amylase. is formed. Therefore, by filling a small diameter tube with a predetermined diameter with a predetermined amount of the pigment polysaccharide complex and the carrier in two layers, and preparing a calibration curve for this in advance, it is possible to α− in sample aqueous solution
Amylase activity can be measured.

Fig. 4 shows a measurement tube for injecting and measuring a sample aqueous solution from above the small diameter tube.The small diameter tube 1 is connected downward from the sample tank 7, and the dye polysaccharide complex is formed in the upper part of the small diameter tube. Layer 2 is filled and below is filled with carrier WI3. According to this measuring tube, an aqueous sample solution is injected into the sample tank, allowed to flow down to the dye polysaccharide complex layer, reacted in the same manner as above to generate a water-soluble dye oligosaccharide complex, and further When allowed to flow down onto the carrier layer, a colored region with a length proportional to the amount of α-amylase is formed, and the activity of α-amylase in the aqueous sample solution can be measured based on this length.

As described above, according to the method of the present invention, a water-insoluble pigment polysaccharide complex that is hydrolyzed by α-amylase to produce a water-soluble pigment oligosaccharide complex in a small diameter tube;
A carrier layer that adsorbs the above dye-oligosaccharide complex is packed in two layers, an aqueous sample solution containing α-amylase is impregnated into the dye-polysaccharide complex packed layer, and the resulting dye-oligosaccharide complex is transferred to the carrier layer. The α-amylase activity in the sample is measured based on the length of the colored region caused by the dye-oligosaccharide complex, so unlike conventional methods, there is no need to separate the supernatant after the reaction or analyze its absorbance. Therefore, α-amylase activity can be determined very easily and quickly.

Examples of the present invention are listed below.

Example 1 Five tablets of a commercially available pigment polysaccharide complex (manufactured by Pharmacia) and 200 mg of microcrystalline cellulose were ground in a mortar and mixed uniformly.

Fill a transparent glass tube with an inner diameter of 21 cm at a position approximately 10 mm from the bottom end, and add the above mixed powder on top of the glass wool by 10 mm.
This was further filled with microcrystalline DEAE cellulose to a thickness of 40II11, and this was covered with glass wool to form a measuring tube.

Pigment starch method (edited by Kiyoshi Okuda, “Clinical Chemistry Test Manual”)
The amount of α-amylase activity measured according to page 132 (published by Ishiyaku Publishing Co., Ltd.) was 100.200.40, respectively.
Prepare 30ml of an aqueous solution of 0.800.1000 and 1500 U/L as a standard sample, immerse the lower part of the measurement tube in this sample aqueous solution, and let the α-amylase aqueous solution rise inside the glass tube by capillary action. I let it happen.

The length of the carrier cellulose layer colored by the water-soluble dye oligosaccharide complex produced by the enzymatic action of α-amylase is 2.5, 12.25, respectively, corresponding to the above active units.
．． It was the 27th and 30th sazen.

Further, the time required for each sample aqueous solution to permeate the entire measuring tube was approximately 7 minutes, although it depended to some extent on the viscosity of the sample aqueous solution and the packing density in the glass tube.

[Brief explanation of drawings]

Fig. 1 is a sectional view showing an example of the measuring tube used in the method of the present invention, Fig. 2 is a sectional view showing how to use the same, and Fig. 3 is a sectional view of the measuring tube showing the colored area in the carrier layer. , 4th ′
The figure is a sectional view showing another example of the measurement tube. 1-small diameter tube, 2- packed layer of dye polysaccharide complex, 3- packed layer of carrier substance, 5- aqueous sample solution, 6- colored area, 7-.
・Sample tank. Patent Applicant Sekisui Chemical Co., Ltd. Representative Mototoshi Fujinuma Figure 1 Figure 4 Whistle 3 Figure Procedure Nefit Ordinary Seal Button 1, Case Indication 1988 Special Application No. 205628 2, Title of the Invention α-amylase activity measurement method 3, relationship with the case of the person making the amendment Patent applicant postal code: 530 Address: 2-4-4 Nishitenma, Kita-ku, Osaka, Tokyo Patent Department TIEL (03) 434-95525, Contents of the amendment (1) In the 4th line of page 2 of the specification, the phrase “Japanese Patent Publication No. 5114915” should be replaced with “Japanese Patent Publication No. 5114915”.
1-14916 Publication”. that's all

Claims (1)

[Claims] +1) polyt that can be hydrolyzed by α-amylase
A small-diameter film filled with a first layer of a water-insoluble dye polysaccharide complex made by chemically bonding a water-soluble reactive dye to class m, and a second layer made of a hydrophilic water-insoluble carrier substance. The tube is impregnated with an aqueous sample containing α-amylase from the outside of the first layer to hydrolyze the dye polysaccharide complex, and the resulting water-soluble dye oligosaccharide complex is added to the carrier together with the sample. A method for measuring α-amylase activity, which comprises transferring the α-amylase activity to a carrier layer, adsorbing it onto a carrier layer, and coloring the α-amylase activity, and measuring the α-amylase activity based on the length of the colored region in the carrier layer.