A kind of active dry chemistry reagent piece of quantitative determination alanine aminotransferase and its systemPreparation MethodTechnical field
The present invention relates to clinical in vitro diagnosis in vitro reagent technique field, in particular to a kind of quantitative determination phenylalanine ammonia group-transferDry chemistry reagent piece of enzymatic activity and preparation method thereof.
Background technique
Alanine aminotransferase (ALT) is a kind of enzyme for participating in human body protein metabolism, can accelerate internal third ammoniaSour amino is converted into glutamic acid.Under normal circumstances, ALT is primarily present in liver, kidney, cardiac muscle, skeletal muscle, pancreas, spleen, lung, red thinIn the histocytes such as born of the same parents, at the same exist in normal body fluid such as blood plasma, bile, celiolymph, in saliva, especially most with content in liverHeight, about the 200 of blood plasma times.But it is not present in urine, in addition to having the generation of kidney damage or disease.Work in Serum ALTProperty is very low, when certain reasons increase permeability of cell membrane or when cytoclasis, ALT can largely be discharged into blood from cell,Keep the concentration in serum significantly raised.ALT is the reflection most common sensitive indicator of liver parenchyma lesion, as long as there is 1% liver cellIt is destroyed, so that it may sero-enzyme increasing be made to double.Therefore, ALT is the most sensitive inspection of hepatic disorder by world health organisation recommendationsSurvey index.
Serum ALT single index increases, to acute hepatitis B, chronic hepatitis, HBV carrier, heavy type hepatitis and liverA series of virus hepatitis such as hardening, liver cancer cannot be made a definite diagnosis, and the raising of ALT only indicates that liver may be compromised, and need foundationOther test ratings, and carry out comprehensive descision in conjunction with medical history, symptom, sign etc..If ALT value is held more than 2-3 times of normal upper limitIt is two weeks or more continuous, show there is possibility existing for disease in the liver and gallbladder, when hepatitis B patient ALT is increased to the 2 times or more of Upper Limit of Normal Value,Antiviral therapy should be just carried out, ALT level is higher, illustrates that the immune function of patient's body is more active, the effect of antiviral therapyAlso better.In addition to hepatitis, other many diseases can cause ALT to increase.It is living in oxyhepatitis and chronic hepatitis and cirrhosisDynamic, the permeability changes of liver plasma membrane, ALT just from spilling into blood circulation into the cell, and result of taking a blood sample in this way is with regard to inclinedHeight, ALT reflect hepatocellular damage degree.But ALT lacks specificity, can cause changing for liver plasma membrane permeability there are many reasonBecome, such as fatigue drinks, catches a cold or even emotional factor.
Recent studies indicate that the raising of ALT level is not only one of important indicator of hepatic disorder, there is research groupIt was found that ALT and gamma-glutamyl based transferase are also the good sign object of chronic systemic inflammation, metabolic syndrome patient this twoIndex significantly increases, i.e., ALT is also related with metabolic syndrome, such as hyperlipidemia, hypertension, diabetes, atherosclerosis,The risk that the horizontal higher crowd of ALT suffers from metabolic syndrome is also relatively high, and Individuals at High Risk of Metabolic Syndrome need to pay close attention to ALT waterIt is flat, and correct evaluation metabolic syndrome is combined with other indexs.In conclusion Serum ALT levels and hepatopathy correlation are big, togetherWhen it is also closely related with comprehensive disease, Serum ALT levels can be used as the important indicator of clinical diagnosis hepatopathy, it is also possible to become generationOne of the index of thanking property disease risks prediction.
The method of measurement alanine aminotransferase can be divided into liquid reagent and dry chemistry reagent two by types of agents at presentKind method.The detection method of liquid reagent mainly has spectrophotometry, Optical Rotation, gas chromatography, high performance liquid chromatographyDeng.These methods carry out detection to alanine aminotransferase activity and need specific equipment, and equipment itself costly, does not haveThere is economy；It needs professional technician to operate instrument simultaneously, does not have due universality.The method measurement period is long, analysisProcess is complex, cannot achieve the real time measure of alanine aminotransferase.
Dry chemistry reagent is mainly used in emergency treatment, on-site test.With easy, quickly, flexibly and be not necessarily to professionalThe advantages that operation.Domestic at present to be essentially qualitative or semiquantitative determination, this dry chemistry reagent piece is by filter paper, celluloseThe one kind such as piece, porous glue film multilayer material whole reagents fixed in advance, two layers and three layers by the processing such as stacking, bonding, punching pressIn conjunction with.It is little to measure the range of linearity, encountering has using meeting interference experiment result under drug condition.There are many examinations in analysis testAgent cannot be pre-mixed, and complicated test fluid needs to pre-process, and which limits dry chemistry reagent piece application fields.Quantitative inspectionThe dry chemistry reagent card of survey is all external production.Using Johson & Johnson and company, Fuji as the multilayer dry slides method of representative, sense is utilizedThe reagents such as reagent layer, auxiliary reagent layer, optical diffusion layer and distribution layer are coated in transparent support layer by the coating technology of ray film,From the variation of the reverse side of support layer measurement optical density.Though above-mentioned dry chemistry reagent piece can quantitative detection, distribution layer or diffusion layerOrganic reagent need to be used, requires material installation high, production technology complexity, and very harmful to preparation personnel health, pollutionEnvironment.
Summary of the invention
In view of this, the present invention provides a kind of active dry chemistry reagent piece of quantitative determination alanine aminotransferase andPreparation method.The dry chemistry reagent piece testing result accuracy is high, and precision is good, and stability is good, long shelf-life, operation letterJust, applied widely.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of active dry chemistry reagent pieces of quantitative determination alanine aminotransferase, folded comprising fourLayer, is followed successively by support layer, color layer, reagent layer and diffusion layer；
Reagent in reagent layer includes buffer system, hydrophilic colloid, pyruvate oxidase, peroxidase, pyrophosphoric acidThiamine, chelating agent, activator 1, activator 2, surfactant；Dosage of each component are as follows:
Reagent in color layer includes chromogen, hydrophilic colloid and surfactant.
Testing principle are as follows: liquid sample is added dropwise in analoids diffusion layer, is uniformly distributed and infiltrates into following reagent layer, expandsScattered layer contains alanine aminotransferase substrate: l-Alanine and α-ketoglutaric acid.Alanine aminotransferase is catalyzed the third ammonia of L-The transamination of acid is to α-ketoglutaric acid, to generate pyruvic acid and glutamic acid.Pyruvic acid is oxidized to acetyl by pyruvate oxidasePhosphate and hydrogen peroxide.Hydrogen peroxide make under the catalytic action of peroxidase 4-AA in color layer andN- ethyl-N- (2- hydroxyl -3- sulfopropyl) -3,5- dimethoxyaniline sodium is oxidized to blue quinonoid compound, in 590nm wavelengthUnder, the change rate through reflective spectrophotometry measurement certain time internal reflection rate calculates alanine amino by standard curve and turnsMove the activity of enzyme.By reaction temperature control under the conditions of 37 DEG C ± 1 DEG C, measurement can be completed in 5 minutes.It is added dropwise in diffusion layerSample is measured in opposite one side, and colored intensity is high, reduces the bubble that the measurement of sample-adding end generates, excess liq, turbidity and causeInterference.Simultaneously because color layer is hydrophilic colloid, there is very high colour developing evenness.
The dry chemistry reagent piece of above structure is mainly used for measuring serum, urine, whole blood equal samples, mixes the sample with before measurementSuitable isotonic solution is diluted, then is measured.
Preferably, buffer system is Tris buffer, borate buffer solution, citrate buffer solution, MOPS in reagent layerBuffer or phosphate buffer, pH value is 6.5~7.5.
Preferably, chelating agent is EDTA.
Preferably, activator 1 is Flavin adenine dinucleotide disodium salt.
Preferably, activator 2 is magnesium sulfate.
Preferably, surfactant is polyoxyethylene sorbitan monooleate 80, sodium taurocholate, polyoxyethylene lauralEther, 3- [3- (gallbladder amido propyl) dimethylamino] propane sulfonic acid inner salt, polyethylene glycol are to one or more of isooctyl phenyl ether.
Preferably, the pH value of buffer system is 7.0~7.2.
Preferably, surfactant is polyethylene glycol to isooctyl phenyl ether.
Preferably, dosage of each component in reagent layer are as follows:
Preferably, hydrophilic colloid is selected from polyvinyl alcohol, amylopectin, cellulose esters, agarose, polyvinylpyrrolidineOne of ketone, polyacrylamide, hydroxypropyl methyl cellulose, Copolymer of Methyl Vinyl Ether/Maleic Anhydride are a variety of.
Preferably, hydrophilic colloid is the mixture of hydroxypropyl methyl cellulose and polyvinyl alcohol.
Preferably, chromogen is made of A and B in color layer, the A is 4-AA, and B is selected from phenol, to chlorinePhenol, P-hydroxybenzoic acid, N- ethyl-N- (2- hydroxyl -3- sulfopropyl) -3,5- dimethoxyaniline sodium, N- ethyl-N- (2- hydroxylBase -3- sulfopropyl) -3- methylaniline sodium, one of 3,5- dichloro sodium hydroxybenzenesulfonate or a variety of.
Preferably, dosage of each component in color layer are as follows:
10~200g/m of hydrophilic colloid2；
0.1~20g/m of surfactant2；
0.6~25mmol/m of chromogen2；
Preferably, in color layer chromogen by 4-AA and N- ethyl-N- (2- hydroxyl -3- sulfopropyl) -3,5-Dimethoxyaniline sodium composition, surfactant is polyethylene glycol to isooctyl phenyl ether in color layer, each component in color layerDosage are as follows:
It is highly preferred that dosage of each component in color layer are as follows:
Preferably, diffusion layer is by buffer system, hydrophilic high molecular polymer, l-Alanine, α-ketoglutaric acid, auxiliaryEnzyme, surfactant, particulate matter；Dosage of each component in diffusion layer are as follows:
Preferably, buffer system is Tris buffer, borate buffer solution, citrate buffer solution, MOPS in diffusion layerBuffer or phosphate buffer, pH value is 6.5~7.5.
Preferably, hydrophilic high molecular polymer be selected from polyurethane, polyamide, sodium carboxymethylcellulose, polyvinyl alcohol,One of amylopectin, xanthan gum, sodium alginate, gum arabic, peach gum, chitosan, polyvinylacetate are a variety of；ParentAqueous polymer has porous structure, can filter out the interfering substance in sample.
Preferably, hydrophilic high molecular polymer is polyurethane.
Preferably, coenzyme is P1,P5-DiAP.
Preferably, surfactant is polyoxyethylene sorbitan monooleate 80, sodium taurocholate, polyoxyethylene lauralEther, 3- [3- (gallbladder amido propyl) dimethylamino] propane sulfonic acid inner salt, polyethylene glycol are to one or more of isooctyl phenyl ether.
Preferably, surfactant is polyethylene glycol to isooctyl phenyl ether.
Preferably, particulate matter is selected from pigment, crystallite colloid, silica, resin bead, bead, diatomite, fiberOne of plain ester is a variety of.
Preferably, particulate matter is silica and cellulose esters.
Preferably, support layer is the polymer of polyethylene terephthalate, with a thickness of 50~300 microns.
Preferably, support layer is with a thickness of 100~200 microns.
Preferably, the surface of support layer is by ultraviolet irradiation, corona or coating bottom layer treatment.
Preferably, support layer is handled by ultraviolet irradiation.
The present invention also provides the preparation methods of the dry chemistry reagent piece, include the following steps:
Developing solution reagent, reaction solution reagent are sequentially coated on support layer, diffusion liquid reagent is then sprayed on supportIt is dry on layer, obtain successively include support layer, color layer, reagent layer and diffusion layer dry chemistry reagent piece；
Reagent in reagent layer includes buffer system, hydrophilic colloid, pyruvate oxidase, peroxidase, pyrophosphoric acidThiamine, chelating agent, activator 1, activator 2, surfactant；Dosage of each component are as follows:
Reagent in color layer includes chromogen, hydrophilic colloid and surfactant.
Preferably, the surface of support layer is by ultraviolet irradiation, corona or coating bottom layer treatment.
Preferably, the coating method of color layer and reagent layer includes prepared by the modes such as levelling, extension stream, the viscous, coating of rolling, workTo be preferred, preparation method is coating.
The present invention provides a kind of active dry chemistry reagent piece of quantitative determination alanine aminotransferase and its preparation sidesMethod.The dry chemistry reagent piece includes four laminations, is followed successively by support layer, color layer, reagent layer and diffusion layer；Examination in reagent layerAgent includes buffer system, hydrophilic colloid, pyruvate oxidase, peroxidase, diphosphothiamine, chelating agent, activator1, activator 2, surfactant；Reagent in color layer includes chromogen, hydrophilic colloid and surfactant.In diffusion layerReagent includes buffer system, hydrophilic high molecular polymer, l-Alanine, α-ketoglutaric acid, coenzyme, surfactant, particleSubstance.Compared with prior art, the invention has the following advantages that
(1) operating process is simple, and pattern detection is directly added dropwise without preparing in reagent.
(2) the dry no moisture of reagent layer, analoids have good stability.
(3) diffusion layer has filtering function, and haemolysis, bilirubin and ascorbic acid sample obviously do not do measurement resultIt disturbs.
(4) testing result is reproducible, accuracy is high, can carry out quantitative detection, and the range of linearity is wide.
(5) preparation process is easy, and easy to operate, harmfulness, pollution are small.
Experiment shows the active dry chemistry reagent of quantitatively determining human blood alanine aminotransferase of the present inventionPiece, testing result accuracy is high, and precision is good, and specificity is good, and stability is good, and long shelf-life is easy to operate, applied widely,The advantages that without professional supplement well the shortcoming of wet chemistry method, accuracy of measurement and precision already close toWet chemical determination method is provided a great convenience in clinical field of fast detection.
Specific embodiment
The invention discloses a kind of active dry chemistry reagent piece of quantitative determination alanine aminotransferase and its preparation sidesMethod, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that allSimilar replacement and change is apparent to those skilled in the art, they are considered as being included in the present invention.ThisThe method of invention and application are described by preferred embodiment, and related personnel can obviously not depart from the present inventionHold, in spirit and scope to method described herein and application is modified or appropriate changes and combinations, carrys out implementation and application sheetInventive technique.
In active dry chemistry reagent piece of quantitative determination alanine aminotransferase provided by the invention and preparation method thereofAgents useful for same or instrument are available on the market.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1: the preparation method of alanine aminotransferase dry chemistry reagent piece of the present invention
Transparent support layer material uses polyethylene terephthalate material, with a thickness of 0.188mm, length 30cm,Width is 30cm.Surface is handled by ultraviolet irradiation, irradiation distance 5cm, and irradiation time 60 minutes.
The formula of developing solution reagent is as follows:
The formula of reaction solution reagent is as follows:
Color layer is coated on the polyethylene terephthalate support layer Jing Guo ultraviolet processing, and reagent layer is continuously appliedIt overlays on color layer, is put into drying box, 30 DEG C dry 20 minutes.
The formula of diffusion liquid reagent is as follows:
Diffusion liquid is uniformly sprayed on reagent layer, is put into drying box and dries.10mm is cut to strip cutting machineThe test-paper of × 10mm size, is sealed with aluminium foil bag.
Detection method: 6 μ L test samples are added dropwise and are incubated for 5min under analoids, 37 DEG C of constant temperatures, use reflecting lightThe change rate of reflectivity, the work of alanine aminotransferase in sample is calculated according to standard curve when degree meter measurement 3min to 5minProperty.
Embodiment 2: the accuracy analysis of the method for the invention
Test sample: 20 examinee's blood samples；
Contrasting detection method: it is tried with commercially available Detection reagent for alanine aminotransferase box (alanine substrate method) 2:1 liquidAgent detects on 7080 automatic clinical chemistry analyzer of Hitachi, and 2.5 μ L samples, one 160 μ of reagent is added by automatic clinical chemistry analyzerL, 2 80 μ L of reagent, the absorbance change rate of 6.5min to 8min measurement 340nm is reacted at 37 DEG C, calculates sample according to standard curveThe activity of alanine aminotransferase in product.
20 samples are measured respectively with 1 detection method of embodiment and contrasting detection method, and testing result is shown in Table 1.
Table 1 analyzes result
The results show that calculated R according to testing result2Value is 0.9999, is greater than 0.95, shows the method for the inventionTesting result and contrast agent box testing result no significant difference have high accuracy (degree of conformity).
Embodiment 3: the Precision Analyze of the method for the invention
Test sample: any one blood serum sample；
Detection 10 times is repeated to same sample to be tested with 1 detection method of embodiment, testing result is shown in Table 2.2 test sample of tableAlanine aminotransferase activity (10 times) and standard rate (coefficient of variation)
The results show that standard rate is 1.89%, less than the 10% of standard requirements, show that the method for the invention has heightPrecision.
Embodiment 4: the linear analysis of the method for the invention
Test sample: high activity alanine aminotransferase sample (1000U/L)；
Alanine aminotransferase sample is diluted to 6 different activity, be followed successively by 0U/L, 200U/L, 400U/L,600U/L, 800U/L, 1000U/L carry out 2 detections, meter to each of above-mentioned sample activity using the detection method of embodiment 1Coefficient R value is calculated, testing result is shown in Table 3.
3 linear test result of table
The results show that be 0.9996 according to the calculated R value of testing result that 1 detection method of embodiment obtains, close to1, show that the method for the invention has the good linearity in the range of 0U/L-1000U/L.
Embodiment 5: the preparation method of alanine aminotransferase dry chemistry reagent piece of the present invention
Transparent support layer material uses ethylene glycol terephthalate material, and with a thickness of 0.188mm, length 20cm is wideDegree is 20cm.Surface pass through sided corona treatment, corona intensity 1000W, the corona time 20 seconds.
The formula of developing solution reagent is as follows:
The formula of reaction solution reagent is as follows:
Color layer is coated on the polyethylene terephthalate support layer Jing Guo ultraviolet processing, and reagent layer is continuously appliedIt overlays on color layer, is put into drying box and dries.
The formula of diffusion liquid reagent is as follows:
Diffusion liquid is uniformly sprayed on reagent layer, is put into drying box and dries.10mm is cut to strip cutting machineThe test-paper of × 10mm size, is sealed with aluminium foil bag.
Detection method: 6 μ L test samples are added dropwise and are incubated for 5min under analoids, 37 DEG C of constant temperatures, use reflecting lightThe change rate of reflectivity, the work of alanine aminotransferase in sample is calculated according to standard curve when degree meter measurement 3min to 5minProperty.
Embodiment 6: the stability analysis of the method for the invention
Condition of storage: sealing is placed in 2-8 DEG C of refrigerator.
Round of visits: when 2-8 DEG C 0, March, September, 15 months, 18 months.
It is detected with the detection method of embodiment 5 in above-mentioned round of visits, accuracy in computation, precision, linear modelIt encloses, testing result is shown in Table 4.
4 stability test result of table
The results show that the calculated R of testing result obtained according to 5 detection method of embodiment2Value is all larger than 0.95, standardRate is respectively less than 10%, the R value of standard requirements all close to 1, shows that the method for the invention stores 18 under the conditions of 2-8 DEG CMonth, analoids still have good accuracy, precision, the range of linearity.
Embodiment 7: the anti-Interference Analysis of the method for the invention
Test sample: bilirubin (20mg/dL) is added in check sample serum, check sample serum in check sample serumMiddle addition ascorbic acid (3mg/dL)；
3 detections are carried out to above-mentioned sample using the detection method of embodiment 5, experiment with computing sample is relative to check sampleBias produced by measurement result, testing result are shown in Table 5.
5 anti-interference test result of table
The results show that the calculated D of testing result obtained according to 5 detection method of embodimentobsRespectively less than Dc, do not observeIt is influenced to clinical significance, shows the method for the invention in the active bilirubin of 20mg/dL and the active Vitamin C of 3mg/dLAcid does not significantly interfere with measurement result.
Comparative example 1: the preparation method of alanine aminotransferase dry chemistry reagent piece described in other methods
Transparent support layer material uses polyethylene terephthalate material, with a thickness of 0.20mm, color layer and reactionLayer uses nitrocellulose filter, and diffusion layer uses polyester screen cloth.Color layer, conversion zone and diffusion layer are separately immersed in colour developingIn liquid, reaction solution and diffusion liquid, takes out, strike off after soaking completely.It is put into 40 DEG C of drying box, takes out, will develop the color after dryLayer is pasted on support layer, then is successively compacted conversion zone and diffusion layer.
The formula of developing solution reagent is as follows:
Polyethylene glycol is to isooctyl phenyl ether 2.0g/L；
4-AA 1.0mmol/L；
N- ethyl-N- (2- hydroxyl -3- sulfopropyl) -3,5- dimethoxyaniline sodium 5.0mmol/L；
The formula of reaction solution reagent is as follows:
The formula of diffusion liquid reagent is as follows:
It is cut to the test-paper of 7mm × 7mm size with strip cutting machine, is sealed with aluminium foil bag.
Detection method: 9 μ L test samples are added dropwise and are incubated for 5min under analoids, 37 DEG C of constant temperatures, use reflecting lightThe change rate of reflectivity, the work of alanine aminotransferase in sample is calculated according to standard curve when degree meter measurement 3min to 5minProperty.
The Precision Analyze of 2 comparative example of comparative example, 1 method the method
Test sample: any one blood serum sample；
Detection 10 times is repeated to same sample to be tested with 1 detection method of comparative example, testing result is shown in Table 6.
6 test sample alanine aminotransferase active (10 times) of table and standard rate (coefficient of variation)
The results show that standard rate is 5.21%, hence it is evident that be greater than 1 standard rate 1.89% of embodiment, show institute of the present inventionMethod is stated with high precision.
The linear analysis of 3 comparative example of comparative example, 1 method the method
Test sample: high activity alanine aminotransferase sample (1000U/L)；
Alanine aminotransferase sample is diluted to 6 different activity, be followed successively by 0U/L, 200U/L, 400U/L,600U/L, 800U/L, 1000U/L carry out 2 detections, meter to each of above-mentioned sample activity using the detection method of comparative example 1Coefficient R value is calculated, testing result is shown in Table 7.
7 linear test result of table
The results show that being 0.9942 according to the calculated R value of testing result that 1 detection method of comparative example obtains, and implementIt is good linear to show that the method for the invention has in the range of 0U/L-1000U/L for example R value 0.9996 closer to 1Degree.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the artFor member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answeredIt is considered as protection scope of the present invention.