CN103320498B - Detection reagent for aspartate aminotransferase - Google Patents

Abstract

The invention discloses a detection reagent for aspartate aminotransferase. The detection reagent comprises a diluent and a reaction reagent. The diluent comprises a buffer, a surfactant, an antiseptic, a bilirubin-interference removing reagent, and an ascorbic oxidase. The reaction reagent comprises a buffer, aspartic acid, alpha-ketoglutaric acid, oxaloacetic decarboxylase, phosphoric acid, pyruvate oxidase, thiamine pyrophosphate, magnesium chloride, peroxidase, 4-aminoantipyrene, a chromogen, an antiseptic, and a stabilizer. The detection reagent has advantages of good sensitivity, accuracy, precision and linearity, and can completely meet the requirements of clinical examination.

Description

A kind of Detection reagent for aspartate aminotransferase

Technical field

The present invention relates to technical field of medical examination, be specifically related to a kind of Detection reagent for aspartate aminotransferase for POCT analyser.

Background technology

Hepatopathy is the great disease of a kind of common hazardness, its timely Treatment and diagnosis is played an important role for the prevention of disease and diagnosis and treatment, aspartic transaminase (Aspartateaminotransferase, AST) extensively exist in respectively organizing in vivo, the highest with cardiac activities, in normal human serum, content is very micro-.AST height illustrates to there is hepatocellular injury, generally sees various hepatitis B, liver cirrhosis, fatty liver, the liver and gall diseases such as alcoholic liver.Current AST generally adopts various large automatic Biochemical Analyzer to detect, but because large automatic Biochemical Analyzer equipment price is high, and complicated operation, operator need have relevant expertise and accept corresponding training, use complementary conditions to require high, need be equipped with voltage stabilized source, water purification machine etc., and floor space is large, maintenance cost is high, needs professional's time-based maintenance, and therefore basic medical unit or household person all do not have condition to buy and use.In addition, large hospital patient is many, detects loaded down with trivial details, the long flow path of formality, waiting time long, and this also brings the huge time to patient and bears, and the more important thing is that patient is affected adversely treatment because not diagnosing in time, even can lose one's life.

Real-time test (point-of-caretesting, POCT) is an emerging technical field, and principal feature is rapid results, easy and simple to handle, easily uses and miniaturization.Along with diagnosis and the progress of ancillary technique, and people are to the requirement of the understanding of disease and treatment level raising, and POCT receives publicity gradually.In fact POCT becomes the important research project of external diagnosis reagent and instrument field gradually at European & American Market, existing many products put goods on the market, but this kind of POCT instrument has a major defect at present, be exactly that analyser and reagent consumptive material are expensive especially, for China, there is huge primary care system like this, and using amount of reagent country greatly, instrument and the matched reagent consumptive material of American-European exploitation at present obviously all face the challenge.

Microfluidic chip technology is one of of paramount importance cutting edge technology in the 21 century world, it is integrated into basic operation units such as sample preparation involved in the fields such as biological and chemical, reaction, separation, detection and cell cultures, sorting, cracking on the chip of a piece tens square centimeters (even less), network is formed by microchannel, running through whole system with controlled fluid, is a kind of technology of the various functions replacing standard biologic or chemical laboratory.Microfluidic chip technology is incorporated into POCT equipment, start the new situation of POCT development, can make laboratory was complicated in the past whole blood quantitatively, the step such as blood cell serum is separated, serum-dilution and Simultaneous Determination, complete in the on-line automatic equalization of chip, reach multiple mark synchronous detection object.POCT analyser in conjunction with microfluidic chip technology has split hair caccuracy concurrently, lowly needs blood volume, simple to operate, detection reagent consumption is few, low cost and other advantages, the POCT instrument of Sheng Yi company limited as international in Bao Sheng, the Piccolo of Abaxis company, the Afinion etc. of Axis-Shield company, this type of POCT analyser all can realize a small amount of sample can analyze, easy and simple to handle, without crossed contamination, can automated job, be highly suitable for China and there is huge primary care system like this and using amount of reagent is national greatly.

Along with Eleventh Five-Year Plan plan purchasing and reinforcement for three grades and second-grade hospital infrastructure device, current middle rank possesses good software and hardware facilities to go to the hospital, but particularly its full-automatic biochemical testing instruments facility of the unit such as commune hospital, clinic is generally not enough for basic medical unit, also do not have the inspection professional of enough numbers, therefore foundation operation POCT analytical system that is simple and easy, cheap, real-time report has important meaning to the effect of the vast basic hospital of performance in disease prevention and diagnosis and treatment.And provide a kind of Detection reagent for aspartate aminotransferase that can be used for the POCT analyser of above-mentioned introducing microfluidic chip technology to become problem demanding prompt solution.

Summary of the invention

The present invention is directed to the above-mentioned deficiency of prior art, a kind of Detection reagent for aspartate aminotransferase that can be used for the POCT analyser introducing microfluidic chip technology is provided.

The AST mensuration reagent that the present invention can be used for above-mentioned POCT analyser (introducing microfluidic chip technology) realizes by following technical scheme: a kind of AST that can be used for POCT analyser measures reagent, comprise diluent and reaction reagent, wherein diluent consists of the following composition:

Damping fluid (pH6.5-7.5) 0.01-1.0mol/L,

Tensio-active agent 0.1-10.0%(mass percent),

Sanitas 0.1-10.0%(mass percent),

Remove bilirubin agent interfering 1-10mol/L or 1-100KU/L,

Ascorbic acid oxidase 1-100KU/L;

Wherein said reaction reagent consists of the following composition:

Damping fluid (pH6.0-8.0): 0.1 ~ 1mol/L,

Aspartic acid 5-100mol/L,

α-ketoglutaric acid 0.15-10mol/L,

Oxaloacetic decarboxylase 10-200KU/L,

Phosphatase 11-100mol/L,

Pyruvic oxidase 10-200KU/L,

Diphosphothiamine 1-100mol/L,

Magnesium chloride 1-100mol/L,

Peroxidase 10-200KU/L,

4-AA 0.1-10mol/L,

Chromogen 0.1-10mol/L,

Sanitas 0.1-1g/L,

Stablizer 0.1-30g/L.

Wherein said damping fluid (comprising the damping fluid in diluent and the damping fluid in reaction reagent) can be the amino damping fluid of trishydroxymethyl, glycine-NaOH buffer, N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid buffer, N-tri-(methylol) methylamino--2-hydroxy-propanesulfonic acid damping fluid, N-tri-(methylol) methyl-2-amino ethanesulfonic acid buffer, two (2-hydroxyethanesulfonic acid) damping fluid of piperazine-N, N-, 3-morpholine-2-hydroxypropionate sodium damping fluid, 3-(N-morpholine) ethyl sulfonic acid sodium damping fluid, 4-(2-hydroxyethyl) piperazine-1-2-hydroxy-propanesulfonic acid damping fluid, N-(2-hydroxyethyl) piperazine-N'-4-fourth sulfonate buffer, two (2-hydroxyethyl) amino-2-hydroxy-propanesulfonic acid damping fluid of 3-, 3-(hexahydroaniline)-2-hydroxyl-1-propanesulfonic acid damping fluid, 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid damping fluid, 3-(hexahydroaniline)-1-propanesulfonic acid damping fluid, 3-morpholine propanesulfonic acid damping fluid, one or more of N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid.

Described tensio-active agent is nonionogenic tenside, as being selected from TWEEN series (as tween (TWEEN) 20, polysorbate40, polysorbate60, tween 80), SPAN series is (as class of department (SPAN) 20, class 40 of department, class 60 of department, class 80 of department), TRITON series is (as TritonX-100, TritonX-114, TritonX-405) in concrete material one or more (namely can be TWEEN series, SPAN series or TRITON serial in the concrete material of one, or more than one concrete material in often kind of series, or the concrete material of one in two kinds or more series).

The described bilirubin agent interfering that goes is one or more in yellow prussiate of potash, high-potassium ferricyanide, bilirubin oxidase.

Described chromogen comprises phenols, as 2-chlorophenol, 2, 4-chlorophenesic acid, 4-chlorophenol, 2, one in 6-chlorophenesic acid, and/or aniline analogue, as N-ethyl-N-(2-hydroxyl-3-third sulfo group) meta-aminotoluene, N-ethyl-N-(3-sulfopropyl)-3-monomethylaniline sodium salt, N-ethyl-N-(2-hydroxyl-3-sulfopropyl l)-3-anisidine sodium salt (dihydrate), N-ethyl-N-(3-sulfopropyl) aniline sodium salt, N-ethyl-N-(3-sulfopropyl)-3-anisidine sodium salt, N-ethyl-N-(3-sulfopropyl) aniline sodium salt, N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3, 5-dimethoxyaniline sodium salt, N-(2-hydroxyl-3-sulfopropyl)-35-dimethoxyaniline sodium salt, N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3, 5-xylidine sodium salt), 3-hydroxyl-2, 4, one in 6-Triiodobenzoic acid, preferred 3-hydroxyl-2, 4, 6-Triiodobenzoic acid.

Described sanitas (comprising the sanitas in diluent and the sanitas in reaction reagent) is selected from the concrete material of one in potassium sorbate, Sodium Benzoate, Sodium Nitrite, proclin series sanitas (as Proclin300) or the concrete material of one in nipagin esters (as methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propylparaben, butyl p-hydroxybenzoate, p-Hydroxybenzoic acid isopropyl ester, p-Hydroxybenzoic acid isobutyl ester).

Described stablizer select in trehalose, sucrose, bovine serum albumin, tween 80, triton x-100, fatty alcohol-polyoxyethylene ether (Brij-35) one or several.

It is liquid state that described AST measures diluent in reagent, and reaction reagent is dry powder.

The preparation method that described AST measures the diluent of reagent is as follows: mix after described agent formulations is added distilled water and stir evenly.

The preparation method that described AST measures the reaction reagent of reagent is as follows: mix after described agent formulations is added distilled water and stir evenly, 0.5-10 μ l reaction reagent is joined reaction detection groove, volatilize 24-72h through freeze-drying (industry routine techniques) or 2-8 DEG C.

The test condition that described AST measures reagent is as follows: temperature: 37 DEG C; Detect predominant wavelength 510nm, commplementary wave length 750nm.

Microfluidic chip technology of the present invention is integrated into by basic operation unit on the chip of a piece tens square centimeters (even less), forms network by microchannel, runs through a kind of technology of whole system with controlled fluid.It is two-layer up and down that its feature is that chip is generally divided into, there is the through hole for application of sample on upper strata, the difform fluid channel etc. that lower floor comprises sample cell, dilution liquid bath, sample amounts groove, diluent quantitative slot, reservoir, multiple reaction detection groove, one group of self-inspection groove for system compensation, one group of overflow groove, many groups being preinstalled with reaction reagent control fluid flowing.Its detection method generally comprises following steps: sample solution and diluent are injected in described sample cell and dilution liquid bath through respective through hole by (1); (2) chip described in starter motor rotation; (3) sample solution realizes solid-liquid separation with quantitative under centrifugal action, and diluent enters diluent quantitative slot simultaneously; (4) quantitative sample and diluent flow into tempering tank and mix; (5) mixed liquid enters reaction detection groove and reaction reagent reacts; (6) in reaction detection groove, in situ detection is carried out by the test set supporting with chip.

The measuring method that described AST measures reagent is as follows: 5-20 μ l sample is joined sample cell, 20-100 μ l diluent is joined dilution liquid bath, starter motor, records absorbance A 1, record absorbance A 2 after continuing reaction 5-9min after 37 DEG C of reaction 1min.

The reaction principle that described AST measures reagent is: sample first carries out mixing hatching with diluent, to remove the interfering substances such as bilirubin, vitamins C and blood fat in sample, after mixed solution enters reaction detection groove, under the existence of the aspartic acid in reaction reagent and α-ketoglutaric acid AST in the sample, L-glutamic acid and oxaloacetic acid is generated by reaction, and oxaloacetic acid generates pyruvic acid under the existence of oxaloacetic decarboxylase, pyruvic acid and phosphoric acid at pyruvic oxidase, Hydrogen Peroxide under the existence of thiaminpyrophosphate and magnesium ion.Under Catalases catalyze, hydrogen peroxide and 4-AA and chromogen are reacted can colour generation, and the generating rate of monitoring absorbancy, can calculate the unit of activity of AST.

Advantage of the present invention and beneficial effect: reagent of the present invention has good sensitivity, accuracy, precision and linear, can meet Clinical Laboratory requirement completely.Reagent of the present invention can be used for the POCT analyser introducing microfluidic chip technology, thus realization operation is simple and easy, cheap, the foundation of the POCT analytical system of real-time report.

Accompanying drawing explanation

The analyte that Fig. 1 is diluted to different concns with the high density serum specimen come from hospital's collection detects, the linear result figure of gained AST.

The AST end value that Fig. 2 and automatic clinical chemistry analyzer measure compares result figure, and wherein X-axis represents determination data in automatic clinical chemistry analyzer Hitachi 7180, and Y-axis represents determination data on POCT analyser.

Embodiment

To further illustrate the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many amendments to the present invention, such amendment also falls into scope of the present invention.

Following experimental technique if no special instructions, is ordinary method, and the experiment material used if no special instructions, all can easily obtain from commercial company.

Embodiment 1

Diluent:

Amino damping fluid (pH7.0) 0.1mol/L of trishydroxymethyl

TWEEN80 5％

Proclin300 0.1%

Bilirubin oxidase 1KU/L

Ascorbic acid oxidase 1KU/L

Reaction reagent:

Amino damping fluid (pH6.0) 0.1mol/L of trishydroxymethyl

Aspartic acid 5mol/L

α-ketoglutaric acid 0.5mol/L

Oxaloacetic decarboxylase 20KU/L

Phosphatase 11 0mol/L

Pyruvic oxidase 30KU/L

Diphosphothiamine 40mol/L

Magnesium chloride 20mol/L

Peroxidase 20KU/L

4-AA 1mol/L

N-ethyl-N-(3-sulfopropyl)-3-anisidine sodium 2mol/L

Potassium sorbate 0.1g/L

Bovine serum albumin 5g/L

Embodiment 2

Diluent:

Amino damping fluid (pH7.5) 0.5mol/L of trishydroxymethyl

TRITONX-100 5%

Methyl p-hydroxybenzoate 1%

Yellow prussiate of potash 1mol/L

Ascorbic acid oxidase 2KU/L

Reaction reagent:

3-(N-morpholine) ethyl sulfonic acid sodium damping fluid (pH7.2) 0.5mol/L

Aspartic acid 20mol/L

α-ketoglutaric acid 2mol/L

Oxaloacetic decarboxylase 50KU/L

Phosphoric acid 20mol/L

Pyruvic oxidase 100KU/L

Diphosphothiamine 20mol/L

Magnesium chloride 20mol/L

Peroxidase 10KU/L

4-AA 0.5mol/L

N-ethyl-N-(2-hydroxyl-3-third sulfo group) meta-aminotoluene 2mol/L

Proclin300 1g/L

Trehalose 10g/L

Embodiment 3

Diluent:

3-morpholine propanesulfonic acid damping fluid (pH6.5) 1.0mol/L

SPAN20 10%

Sodium Benzoate 1%

High-potassium ferricyanide 10mol/L

Ascorbic acid oxidase 10KU/L

Reaction reagent:

Two (2-hydroxyethanesulfonic acid) damping fluid (pH8.0) 1mol/L of piperazine-N, N-

Aspartic acid 100mol/L

α-ketoglutaric acid 10mol/L

Oxaloacetic decarboxylase 200KU/L

Phosphoric acid 50mol/L

Pyruvic oxidase 200KU/L

Diphosphothiamine 80mol/L

Magnesium chloride 30mol/L

Peroxidase 100KU/L

4-AA 5mol/L

N-ethyl-N-(3-sulfopropyl)-3-anisidine sodium salt 5mol/L

Proclin300 1g/L

Fatty alcohol-polyoxyethylene ether 10g/L

Be described below in conjunction with the performance of form to the embodiment of the present invention 1 gained reagent.

1, precision

Table 1, precision assessment result

2, linear

The analyte being diluted to different concns with the high density serum specimen come from hospital's collection detects, and AST linearly the results are shown in Fig. 1.

3, methodology Comparability test

Compare with the AST end value that automatic clinical chemistry analyzer measures, result is as Fig. 2, wherein X-axis represents determination data in automatic clinical chemistry analyzer Hitachi 7180, POCT analyser (the Taiwan Bao Sheng world of micro flow chip technology is introduced in Y-axis representative, Amishield, TMO-100) upper determination data.

4, detection sensitivity

The appraisal procedure of the detection sensitivity standard deviation of 10-20 dummy signal strength, and the standard deviation of 3-15 least significant non-zero sample signal strength, calculate gained with statistical software EPEvaluatorrelease6.Experimental result shows reagent of the present invention good sensitivity, can meet the improvement of U.S. clinical laboratory completely and amend legislation.

Table 2 reagent detection sensitivity

From above-mentioned detected result, reagent of the present invention has good sensitivity, accuracy, precision and linear, can meet Clinical Laboratory requirement completely.

Claims (1)

1. a Detection reagent for aspartate aminotransferase, is characterized in that: this reagent comprises diluent and reaction reagent,

Diluent:

Amino damping fluid: pH 7.0 0.1 mol/L of trishydroxymethyl

TWEEN 80 5%

Proclin300 0.1%

Bilirubin oxidase 1 KU/L

Ascorbic acid oxidase 1 KU/L

Reaction reagent:

Amino damping fluid: pH 6.0 0.1 mol/L of trishydroxymethyl

Aspartic acid 5 mol/L

α-ketoglutaric acid 0.5 mol/L

Oxaloacetic decarboxylase 20 KU/L

Phosphatase 11 0 mol/L

Pyruvic oxidase 30 KU/L

Diphosphothiamine 40 mol/L

Magnesium chloride 20 mol/L

Peroxidase 20 KU/L

4-AA 1 mol/L

N-ethyl-N-(3-sulfopropyl)-3-anisidine sodium 2 mol/L

Potassium sorbate 0.1 g/L

Bovine serum albumin 5 g/L;

Or

Diluent:

Amino damping fluid: pH 7.5 0.5 mol/L of trishydroxymethyl

TRITON X-100 5%

Methyl p-hydroxybenzoate 1%

Yellow prussiate of potash 1 mol/L

Ascorbic acid oxidase 2 KU/L

Reaction reagent:

3-(N-morpholine) ethyl sulfonic acid sodium damping fluid: pH 7.2 0.5 mol/L

Aspartic acid 20 mol/L

α-ketoglutaric acid 2 mol/L

Oxaloacetic decarboxylase 50 KU/L

Phosphoric acid 20 mol/L

Pyruvic oxidase 100 KU/L

Diphosphothiamine 20 mol/L

Magnesium chloride 20 mol/L

Peroxidase 10 KU/L

4-AA 0.5 mol/L

N-ethyl-N-(2-hydroxyl-3-third sulfo group) meta-aminotoluene 2 mol/L

Proclin300 1 g/L

Trehalose 10 g/L;

Or

Diluent:

3-morpholine propanesulfonic acid damping fluid: pH 6.5 1.0 mol/L

SPAN 20 10%

Sodium Benzoate 1%

High-potassium ferricyanide 10 mol/L

Ascorbic acid oxidase 10 KU/L

Reaction reagent:

Two (2-hydroxyethanesulfonic acid) damping fluid: pH 8.0 1 mol/L of piperazine-N, N-

Aspartic acid 100 mol/L

α-ketoglutaric acid 10 mol/L

Oxaloacetic decarboxylase 200 KU/L

Phosphoric acid 50 mol/L

Pyruvic oxidase 200 KU/L

Diphosphothiamine 80 mol/L

Magnesium chloride 30 mol/L

Peroxidase 100 KU/L

4-AA 5 mol/L

N-ethyl-N-(3-sulfopropyl)-3-anisidine sodium salt 5 mol/L

Proclin300 1 g/L

Fatty alcohol-polyoxyethylene ether 10 g/L.