Reagent for total α-amylase activity test

FIELD: medicine.

SUBSTANCE: reagent for testing total α-amylase activity in serum, blood plasma and urine contains as a substratum 2-chlorine-4-nitrophenl-4-O -β-D-galactopyranosylmaltoside (GalG2CNP), sodium chloride, potassium sulphocyanate, sodium azide, EDTA, calcium acetate, Triton X-100, 2-morpholinoethanesulfonic acid (MES) and water. The pH value of said reagent is 5.5-6.5.

EFFECT: use of the reagent enables high precision and results reproducibility α-amylase test.

1 tbl, 4 ex

 

The invention relates to the field of medicine and biochemistry, namely the creation of reagents, allowing to determine the activity of the total α-amylase in serum, plasma and urine.

For determining the activity of α-amylase various techniques have been developed, based on the ability of the enzyme to hydrolyze internal α-1,4-glycosidic bonds oligo - and polysaccharides.

The most common use for determining the activity of total α-amylase is a method comprising using as the substrate 4,6-ethylidene-(G7)-p-nitrophenil(G1)-α,D-maltoheptaoside (4,6-Tilden(G7)-p-nitrophenyl-(G1)-α,D-maltoheptaoside; Et-G7PNP)approved by the International Federation of clinical chemistry (IFCC). In the reaction of α-amylase cleaves Et-G7PNP to G2PNP, G3PNP, G4PNP, Et-G3Et-G4and Et-G5. Then α-glucosidase hydrolyzes G2PNP, G3PNP and G4PNP with the formation of glucose and colored substance p-NITROPHENOL. The rate of accumulation of colored product is directly proportional to the activity of total α-amylase in the sample (Kruse-Jarres JD, Kaiser S., Hafkenscheid J... et al. Evaluation of a new α-amylase assay using 4,6-ethylidene-(G7)-l-4-nitrophenil(G1)-α-D-maltoheptaoside as substrate // J. Clin. Chem. Clin. Biochem. 1988. Vol.27. P.103-113).

The main disadvantage of this method is the two-phase process of the reaction and the use of additional enzyme for p which produce a colored product.

Closest to the claimed reagent prototype is a set of reagents for determining the activity of total α-amylase kinetic colorimetric method, which consists of two reagents, the following compounds: reagent 1 - sodium chloride 50-500 mmol/l, potassium radamisty (KSCN) 50-400 mmol/l, sodium azide 1-90 mmol/l, calcium chloride 0.5 to 10.0 mmol/l MES (2-morpholinepropanesulfonic acid) 50 mmol/l, water - the rest, the pH of the reagent 5-8; reagent 2 containing: 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside (2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosyl; GalG2CNP) 2,4-12 mmol/l MES, 50 mmol/l, water(rest)pH reagent 5-8. The basis set for determining the activity of total α-amylase laid kinetic colorimetric method, which consists in the fact that the α-amylase hydrolyzes GalG2CNP with the formation of a colored product 2-chloro-4-nitrophenol and unpainted - 4-O-β-D-galactopyranosylmaltose. The rate of accumulation of the colored product is directly proportional to the activity of α-amylase in the sample (Morishita Y., IinumaY., Nakashima N., et al. Total and pancreatic amylase measured with 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside // Clinical Chemistry. 2000. Vol 46. P.928-933).

The disadvantages of the prototype are:

- small shelf life of reagents: 3 months at 4°C;

- the complexity of using the system of the two reagents.

An object of the invention is to increase the stability of the reagent for measuring the population activity total α-amylase in serum, plasma and urine with high accuracy and reproducibility of the analysis results.

The goal of the project is achieved by the proposed reagent containing the following components: a substrate GalG2CNP 0.5 to 20 mmol/l, sodium chloride 50-500 mmol/l KSCN 50-400 mmol/l, sodium azide 1-90 mmol/l, EDTA (ethylenediaminetetraacetic acid) 0.2 to 5.0 mmol/l, Triton X-100 0,075%vol., calcium acetate 0.7 to 10.0 mmol/l MES, 50 mmol/l, (rest), pH 5,5-6,5.

This reagent is characterized by the accuracy of determining the activity of total α-amylase 100±5% reproducibility (coefficient of variation) of less than 5%. The shelf life of the reagent at 2-8°C is 2 years.

The inclusion of the new reagent components Triton X-100, which is a non-ionic surface-active agent, EDTA - forming complex compounds with most cations, as well as replacement of calcium chloride on the preservative calcium acetate can improve the stability of the reagent and extend its shelf life up to 2 years.

The determination of the activity of total α-amylase is carried out at a temperature of reagent 37°C as follows: 25 ál of sample add 1 ml of reagent and after 1 min start to read the optical density at equal intervals of time within 1-3 min at a wavelength of 405 nm, while the number of reads should be at least three. Actively the th total α-amylase (A) in u/l calculated according to the formula:

A=ΔΕ/min×3060, where ΔΕ/min - average change in optical density per minute; 3060 - the conversion factor by the equation the Bouguer-Lambert-Bera.

Determine significant differences of the proposed reagent, compared with the prototype are:

the reagent further comprises Triton X-100 and EDTA in experimentally selected, the optimal quantities, which improves the stability of the latter during storage;

in the reagent calcium chloride is replaced with a more suitable component is calcium acetate, which improves the stability of the reagent during storage;

liquid reagent ready to use, which can significantly simplify the procedure of analysis;

- all components in the reagent used in the optimal experimentally selected concentrations that can increase functionality and to extend the scope of the reagent.

The proposed reagent is clinically tested and approved for production, sale and use on the territory of the Russian Federation. The proposed reagent available for any clinical diagnostic, outpatient and biochemical laboratories and allows you to get accurate, reliable results of the analysis, the corresponding foreign standards.

Example 1

To prepare the reagent doing weighed on an analytical balance for each component. The Perrin is placing them in glass beakers and dissolved in a small amount of distilled water. The solutions are thoroughly mixed on a magnetic stirrer. Solution components except GalG2CNP, transferred into a volumetric flask with a capacity of 1 liter and adjusted the pH under control of a pH meter to 5,73. Then add a portion GalG2CNP. The volume of solution was adjusted to the mark with distilled water and mix thoroughly. The result is a reagent for the determination of total α-amylase in serum, plasma and urine, containing the following components:

Sodium chloride, mmol/l to 50.0

KSCN, mmol/l - 400,0

Sodium azide, mmol/l to 90.0

EDTA, mmol/l 0,2

Calcium acetate, mmol/l 0,7

Triton X-100, vol.% - 0,075

GalG2CNP, mmol/l 0,5

MES, mmol/l to 50.0

Water - the rest.

When the pH value of the reagent is 6.0.

Example 2

The reagent carried out analogously to example 1. Get reagent for the determination of total α-amylase in serum, plasma and urine, containing the following components:

Sodium chloride, mmol/l - 300,0

KSCN, mmol/l to 100.0

Sodium azide, mmol/l to 20.0

EDTA, mmol/l to 2.5

Calcium acetate, mmol/l 5,0

Triton X-100, vol.% - 0,075

GalG2CNP, mmol/l 10,0

MES, mmol/l to 50.0

Water - the rest.

When the pH value of the reagent is 5,5.

Example 3

The reagent carried out analogously to example 1.

Get reagent for the determination of total α-amylase in serum, plasma, crowie urine, containing the following components:

Sodium chloride, mmol/l - 500,0

KSCN, mmol/l to 50.0

Sodium azide, mmol/l - 1,0

EDTA, mmol/l 5,0

Calcium acetate, mmol/l 10,0

Triton X-100, vol.% - 0,075

GalG2CNP, mmol/l to 20.0

MES, mmol/l to 50.0

Water - the rest.

When the pH value of the reagent is 6.5.

Example 4

The determination of the activity of pancreatic α-amylase in the control sera.

To 1 ml of reagent, heated to a temperature of 37°C, was added 25 ál control sera "Precinorm U". After 1 min started to read the optical density at equal intervals of time within 1-3 min at a wavelength of 405 nm. The same procedure was repeated for the control sera "Precipath U", "c.f.a.s.", "Contornorm", "Contropath", "BioCal E". The results of the analysis are given in table 1.

Table 1
Control serumCertified activity value of pancreatic α-amylase (IFCC), u/lThe result, u/l
"Precinorm U" (Roche, Germany)40,8 (33,6-48,0)40
"Precipath U" (Roche, Germany)102 (84-120)102
"c.f.a.s." (Roche, Germany)162164
"Contornorm" ("Biocon", Germany)40,1 (32,9-47,3)41
"Contropath" ("Biocon", Germany)102 (84-120)101
"BioCal E" ("Biocon", Germany)171177

From table 1 it is evident that the proposed reagent provides high accuracy for determining the activity of total α-amylase in serum, plasma and urine.

The use of the reagent will allow for a comparison with prototype:

to increase the shelf life of the reagent up to 2 years by ensuring its stability;

- to expand the scope and improve the functionality of the reagent by providing opportunities to work with him in terms of clinical-diagnostic, outpatient and biochemical laboratories.

Reagent for determining the activity of total α-amylase kinetic colorimetric method containing the substrate 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosyl (GalG2CNP), sodium chloride, potassium radamisty, sodium azide, a water-soluble salt of calcium, 2-morpholinepropanesulfonic acid (MES) and water, characterized in that it complements the flax contains EDTA and Triton X-100, and as the calcium salt is calcium acetate, in the following ratio of components:

Sodium chloride, mmol/l50,0-500,0
Potassium radamisty, mmol/l50,0-400,0
Sodium azide, mmol/lof 1.0 to 90.0
EDTA, mmol/l0,2-5,0
Calcium acetate, mmol/l0,7-10,0
Triton X-100, vol.%0,075
GalG2CNP, mmol/lof 0.5 to 20.0
MES, mmol/l50,0
WaterThe rest,

if the pH of the reagent is 5,5-6,5.



 

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