CN101105491B - Dry chemical test paper for quantitatively determining human alanine aminotransferase - Google Patents

Abstract

The invention belongs to the in vitro clinical diagnosis reagent field and specifically relates to a dry chemistry test paper which quantitatively determines the activity of alanine aminotransferase in human body; the dry chemistry test paper consists of an upper and a lower supporting layers in long-belt-shapes; a loading hole, a substrate layer, a visualization layer and a test hole are provided in a test area from top to bottom; the substrate layer can adopt the materials such as glass fibre, filter paper and non-woven fabrics and is soaked with reagents such as a substrate; the visualization layer is composed of various filter papers, non-woven fabrics or synthetic film and is soaked with reagents such as chromogen. A sample penetrates into the substrate layer through the loading hole and flows to a sample surface of the visualization layer; a liquid with detected ingredients penetrates to a detecting surface; the color of chromogen is changed under chemical reaction; the liquid penetrates through the test hole on the lower supporting layer and reaches the detecting surface of the visualization layer; the photodensity of the liquid can be determined on the detecting surface. The test paper is used to rapidly test the activity of alanine aminotransferase in whole blood, serum or plasma in the human body, which is one of important indexes of the detection of liver function.

Description

The drying chemical reagent paper of quantitatively determining human alanine aminopherase

Technical field

The invention belongs to technical field of in-vitro clinical diagnostic reagent, be specifically related to a kind of drying chemical reagent paper that is applicable to biochemical indicators such as the alanine aminotransferase in mensuration human whole blood, serum or the blood plasma etc., L-aminobutanedioic acid aminopherase.

Technical background

Alanine aminotransferase claims glutamic-pyruvic transaminase again, the transfer of amino between its catalysis L-alanine and the L-glutamic acid.Alanine aminotransferase mainly is present in the hepatocellular endochylema, and the content in cardiac muscle, skeletal muscle and other tissue is lower.Alanine aminotransferase is the enzyme-specific of liver, and blood serum values raises and shows that the liver cell plasma membrane exists seepage and degeneration, and the degree of its rising is relevant with the quantity of the cell of getting involved.

The reagent of measuring alanine aminotransferase clinically can be divided into liquid reagent and drying chemical reagent paper two classes.Drying chemical reagent paper is mainly used in fast detecting.Compare with liquid reagent, drying chemical reagent paper has conveniently, flexibly, pollute advantages such as little, easy and simple to handle.

At present, detect alanine aminotransferase drying chemical reagent paper mostly be external product, mainly contain vertical and horizontal two classes.The first kind is to be the multilayer dry slides method of representative with Johson ﹠ Johnson and company of Fuji, utilize the coating technology of sensitive film that reagent such as reagent layer, auxiliary reagent layer, optical diffusion layer and distribution layer are coated on the transparent support layer, measure the variation of optical density from the reverse side of supporting layer.As patent: U.S.PAT.NO.5,508,173.Second class is the dry chemical product of Roche Holding Ag, U.S.PAT.NO.4,604,264 are coated on reagent on the fiber or fabric of multifibres, whole blood laterally filters back and last reagent layer contact reaction by glass fibre, measures the variation of optical density by a transparent membrane of top covering.

Above-mentioned these products, the complex manufacturing that has needs special technology; What have may exist some defective, as application of sample be determined at the same side and may cause interference.And the dry type Biochemical Analyzer of using at present is big-and-middle-sized equipment, and it concentrates on several biochemical projects on the instrument and measures.When measuring single alanine aminotransferase project, as occasions such as mobile blood-collecting carriages, its function and speed can be restricted.The present invention aims to provide independently minicomputer and test paper, is more suitable for the extensive rapid screening of alanine aminotransferase.

Summary of the invention

The object of the present invention is to provide a kind of easy to use, rapid quantitative to measure the drying chemical reagent paper of the alanine aminotransferase in human whole blood, serum or the blood plasma.

The drying chemical reagent paper of quantitative measurement alanine aminotransferase provided by the invention is strip, and an end is the holding area, and the other end is the test section, and its structure as depicted in figs. 1 and 2.It is made up of upper supporting layer 1, lower supporting layer 2, substrate layer 5 and color layer 6.Wherein, upper supporting layer 1 and lower supporting layer 2 are bonded together by the glue-line that carries.Have well 3 in the test section of upper supporting layer 1, the correspondence position of lower supporting layer 2 has instrument connection 4.Substrate layer 5 and color layer 6 coincide, between the upper supporting layer 1 and lower supporting layer 2 at well and instrument connection place.

Among the present invention, upper supporting layer 1 and lower supporting layer 2 can adopt macromolecule polymeric material, as tygon, Polyvinylchloride, polystyrene or dacron etc.Preferred polyester fiber wherein.The thickness of monolithic supporting layer is 100-250 μ m, and size is 6 * 50mm-12 * 80mm.Well on the last lower supporting layer and instrument connection are circular, and diameter is identical, is 4-8mm.Well 3 is used to drip sample, and instrument connection 4 is used for observing and assaying reaction district change in color.

Among the present invention, substrate layer 5 is used for accepting sample, and sample spreads in substrate layer, and with the agent dissolves that is immersed on the substrate layer, but the test section in the sample is transported to the sample face of color layer.Substrate layer 5 preferred reaction district areas can hold about 10-30 μ l blood, and about 6-10 μ l sample can be transported to the material of color layer.The material that is fit to do substrate layer comprises glass fibre, various filter paper or nonwoven fabrics etc., special preferred glass fibers, its short texture, but test section in the blood sample and reagent can rapid osmotic to color layer, shortened the reaction time.

Among the present invention, color layer 6 is for being impregnated with various filter paper, nonwoven fabrics or the synthetic film etc. of detectable and chromogen, the synthetic film of materials such as preferred polysulfones, polyethersulfone or nylon, the synthetic film that especially preferably has the hole that pore size changes in gradient, the small aperture scope on the synthetic film is 0.2-10 μ m.Synthetic film has the sample face and the detection faces that can be observed change color that are used for point sample.After blood penetration was to the macropore face of film, sample passed film gradually, and the aperture of flowing through is more and more littler.At last, material such as red blood cell arrives the just no longer down infiltration of certain position of film.The remaining sample that contains alanine aminotransferase is penetrated into the aperture face, by series reaction, the chromogen color is changed, thus the concentration of alanine aminotransferase in the indication sample.

Among the present invention, reaction reagent is distributed on substrate layer and the color layer.Quantitatively whole blood or blood serum drip in well, and sample evenly distributes and infiltrates in the reagent layer.Alanine aminotransferase in the sample (ALT) catalytic substrate L-alanine and α-ketoglutaric acid sodium produces L-glutamic acid and pyruvic acid.Pyruvic acid is produced hydrogen peroxide by pyruvate oxidase oxidation in the presence of flavin adenine dinucleotide (FAD) (FAD) and b1thiaminpyrophosphate (TPP).Under the peroxidase effect, the coloured product of the former generation of hydrogen peroxide oxidation colorless chromogenic, change color is directly proportional with alanine aminotransferase activity in the sample, can measure it with reflectance photometer.

Among the present invention, reaction reagent comprises buffer system, substrate, pyruvate oxidase, peroxidase, chromogen, activator, stabilizing agent etc.Buffer system can be phosphate buffer, Tris-HCL or Good's damping fluid etc., and concentration is 0.05M-1.0M, and the reaction optimal pH is at 6.5-8.0.Substrate adopts L-alanine and α-ketoglutaric acid sodium, and substrate produces L-glutamic acid and pyruvic acid under the catalysis of alanine aminotransferase.The concentration of L-alanine is 0.5M-1.5M, and the concentration of α-ketoglutaric acid sodium is 5mM-50mM.The pyruvic acid that generates is produced hydrogen peroxide by pyruvate oxidase oxidation in the presence of flavin adenine dinucleotide (FAD) (FAD) and b1thiaminpyrophosphate (TPP).The suitable concentration of pyruvate oxidase is 30-100U/ml, and FAD concentration is 0.1mg/ml-0.2mg/ml, and TPP concentration is 0.4mg/ml-0.8mg/ml; The activator of pyruvate oxidase adopts Mg²⁺, Mg²⁺Concentration be 1.0g/L-3.0g/L; The stabilizing agent of enzyme adopts disaccharides, polysaccharide or protide stabilizing agent etc., and as sucrose, trehalose and bovine serum albumin(BSA) etc., the weight concentration in system is 0.1-2.0%.Under the peroxidase effect, the coloured product of the former generation of hydrogen peroxide oxidation colorless chromogenic.Peroxidase concn is 30-100U/ml.Chromogen can be the chromogen substance of one-component or two kinds of components as indicator, and the preferred chromogen wavelength of absorption place by force is different from the wavelength of strong absorption place of sample, and the wavelength of preferred especially strong absorption place is the chromogen of 610-650nm, and concentration is 0.03%-0.5%.

Be quantitative measurement alanine aminotransferase activity, the inventor has also developed reflectance spectrophotomete (separate case is applied for a patent), with the supporting use of test strips of the present invention.6-30 μ l blood sample drips on test strips, and temperature of reaction is controlled at 37 ± 0.3 ℃, and with the variation of this photometric determination test paper reaction back reflection density under 630 ± 20nm wavelength, test was finished in 3.5-5 minute.Automatically report and the storage experimental result, and print.

The present invention uses the pyruvate oxidation enzyme process to measure the alanine aminotransferase activity, the structure that adopts substrate layer and color layer to coincide, reagent stability is good, the colored intensity height, good evenness, reduced the interference that the bubble that application of sample end mensuration produces, excess liq, red blood cell etc. cause, can use whole blood and blood serum as sample.Manufacturing process of the present invention is simple, and is supporting with custom-designed compact reflective photometer, uses more convenient quick.

Description of drawings

Fig. 1 is a drying chemical reagent paper diagram of the present invention.

Fig. 2 is a drying chemical reagent paper cross-section illustration of the present invention.

Number in the figure: 1 is upper supporting layer, and 2 is lower supporting layer, and 3 is well, and 4 is instrument connection, and 5 is substrate layer, and 6 is color layer.

Fig. 3 is the regression beeline equation of the measurement result of embodiment 1.

Embodiment

Embodiment 1: upper and lower supporting layer adopts the PVC sheet, gets well 3 and instrument connection 4 with tapping and plugging machine, and bore dia is 4mm, and pitch-row is 8mm.Substrate layer 5 adopts glass fibre, is pasted on upper supporting layer.Color layer 6 adopts nylon membrane, is pasted on lower supporting layer.Use BIO-DOT specking instrument difference point sample in substrate layer 5 and color layer 6 substrate solution and the colour developing liquid that prepare, every hole 6 μ l.Test paper behind the specking is put into 30-50 ℃ vacuum drying chamber, and take out dry back.With involutory sticking being posted on together of upper and lower supporting layer, well 3 aligns with instrument connection 4.With the hobboing cutter machine test paper is cut into 8mm wide test strips.

Reagent is composed as follows:

Phosphate buffer: 50mM

L-alanine: 500mM

α-ketoglutaric acid sodium: 10mM

MgCl₂*6H₂O： 2.4g/L

Peroxidase: 30U/ml

Pyruvate oxidase: 30U/ml

Flavin adenine dinucleotide (FAD) (FAD): 0.1mg/ml

B1thiaminpyrophosphate (TPP): 0.4mg/ml

4-amino-antipyrine: 1.2g/L

DAOS (chromogen): 0.85g/L

(N-ethyl-N-(2-hydroxyl-3-sulfonic group propyl group)-3,5-dimethylamino-aniline)

Bovine serum albumin(BSA): 0.2%

The alanine aminotransferase calibration object of 6 μ l7% bovine serum albumin(BSA)s preparation is dripped in well, measure the variation of the reflection strength when reading 2.5-4 minutes with baffled photometer.The results are shown in Table 1:

Setting up linear regression equation with this is Y (reflection strength variation)=0.0004X (ALT concentration)+0.0245, r²=0.9987.As shown in Figure 3.

Embodiment 2: upper and lower supporting layer adopts the PET sheet, gets well 3 and instrument connection 4 with tapping and plugging machine, and bore dia is 4mm, and pitch-row is 8mm.Substrate layer 5 adopts glass fibre, and color layer 6 adopts the PES film, and width is 8mm.Substrate layer 5 and color layer 6 are soaked in respectively in substrate solution and the colour developing liquid, soak the back fully and take out, wipe off.The test paper that will soak is put into 30-50 ℃ vacuum drying chamber, and take out dry back.Substrate layer 5 is pasted on upper supporting layer, and color layer 6 is pasted on lower supporting layer.With involutory sticking being posted on together of upper and lower supporting layer, well 3 aligns with instrument connection 4.With the hobboing cutter machine test paper is cut into 8mm wide test strips.

Reagent is composed as follows:

Good's damping fluid: 200mM

L-alanine: 1000mM

α-ketoglutaric acid sodium: 20mM

MgCl₂*6H₂O： 2.0g/L

Peroxidase: 100U/ml

Pyruvate oxidase: 100U/ml

Flavin adenine dinucleotide (FAD) (FAD): 0.2mg/ml

B1thiaminpyrophosphate (TPP): 0.8mg/ml

KH₂PO₄： 20mM

3 ', 3 ', 5 ', 5 ,-tetramethyl benzidine (chromogen): 0.1%

Trehalose: 0.5%

Alanine aminotransferase serum dilution red blood cell with high concentration, obtain packed cell volume and be 20%, 30%, 40%, 50% and 60% whole blood sample, the application of sample amount is 20 μ l, drip respectively in well, measure the variation of the reflection strength when reading 3-4 minutes with baffled photometer.The results are shown in Table 2:

The presentation of results packed cell volume is subjected to erythrocytic interference hardly at 30%-50% whole blood sample when measuring.

Claims (2)

1. the drying chemical reagent paper of a quantitative measurement alanine aminotransferase is characterized in that being shaped as strip, and an end is the holding area, and the other end is the test section, is made up of upper supporting layer (1), lower supporting layer (2), substrate layer (5) and color layer (6); Wherein, upper supporting layer (1) and lower supporting layer (2) are bonded together by the adhesive layer that carries; Have well (3) in the test section of upper supporting layer (1), the correspondence position of lower supporting layer (2) has instrument connection (4); Substrate layer (5) and color layer (6) coincide, and are positioned between the upper supporting layer (1) and lower supporting layer (2) that well (3) and instrument connection (4) locate; Be impregnated with reaction reagent on substrate layer (5) and the color layer (6), and through 39-50 ℃ of vacuum drying chamber drying;

The material of described substrate layer (5) adopts glass fibre;

The material of described color layer (6) is synthetic film; Described synthetic film is polysulfones, polyethersulfone or nylon; Described synthetic film has the hole that pore size changes in gradient, and wherein the pore diameter range of aperture is 0.2-10 μ m;

Described reaction reagent comprises buffer system, substrate, pyruvate oxidase, peroxidase, chromogen, activator and stabilizing agent, wherein, buffer system is phosphate buffer, Tris-HCL or Good ' s damping fluid, and concentration is 0.05M-1.0M, and pH value in reaction is at 6.5-8.0;

Substrate is L-alanine and sodium alpha-ketoglutarate, and the concentration of L-alanine is 0.5M-1.5M; The concentration of sodium alpha-ketoglutarate is 5mM-50mM;

The concentration of pyruvate oxidase is 30-100U/ml; Peroxidase concn is 30-100U/ml;

Chromogen is DAOS or 3 ', 3 ', 5 ', and 5 '-tetramethyl benzidine, the chromogen absorbing wavelength is 610nm-650nm, concentration is 0.03%-0.5%;

The coenzyme of pyruvate oxidase adopts flavin adenine dinucleotide (FAD) and b1thiaminpyrophosphate, and flavin adenine dinucleotide (FAD) concentration is 0.1mg/ml-0.2mg/ml, and b1thiaminpyrophosphate concentration is 0.4mg/ml-0.8mg/ml;

The activator of pyruvate oxidase adopts Mg²⁺, concentration is 1.0g/L-3.0g/L;

The stabilizing agent of enzyme is sucrose, trehalose or bovine serum albumin(BSA), and the weight concentration in system is 0.1-2.0%.

2. the drying chemical reagent paper of quantitative measurement alanine aminotransferase according to claim 1, the material that it is characterized in that described upper supporting layer and lower supporting layer is tygon, Polyvinylchloride, polystyrene or dacron, the thickness of monolithic supporting layer is 100-250 μ m, size is 6 * 50-12 * 80mm, the specification of upper supporting layer and lower supporting layer is identical, well on it (3) and instrument connection (4) are circular, and diameter is 4-8mm.

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