WO2022155991A1 - 一种检测嘌呤类物质的试剂盒及其应用 - Google Patents

一种检测嘌呤类物质的试剂盒及其应用 Download PDF

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WO2022155991A1
WO2022155991A1 PCT/CN2021/074330 CN2021074330W WO2022155991A1 WO 2022155991 A1 WO2022155991 A1 WO 2022155991A1 CN 2021074330 W CN2021074330 W CN 2021074330W WO 2022155991 A1 WO2022155991 A1 WO 2022155991A1
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kit
detection
sample
tested
detecting
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PCT/CN2021/074330
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English (en)
French (fr)
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温尧林
申凯
韦苏
逄龙
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苏州凯祥生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90206—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90219—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • G01N2333/90222—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3) in general
    • G01N2333/90225—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3) in general with a definite EC number (1.10.3.-)
    • G01N2333/90235—Ascorbate oxidase (1.10.3.3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/9029—Oxidoreductases (1.) acting on -CH2- groups (1.17)

Definitions

  • the invention relates to the field of biotechnology, in particular to a kit for detecting purine substances and its application.
  • Xanthine/hypoxanthine is a purine base widely distributed in the organs and body fluids of humans and other organisms. Its normal physiological concentration is 0.5-2.5 ⁇ mol/L in serum and 40-160 ⁇ mol in urine. /L. Uric acid is usually produced under the action of xanthine oxidase (xo) and excreted in urine. Changes in the concentrations of xanthine, hypoxanthine and uric acid in the human body can directly reflect the state of human immunity and metabolism, suggesting diseases related to purine metabolism.
  • xanthine oxidase inhibitors such as allopurinol and febuxostat
  • xanthine crystals were found in the kidneys and ureters, reminding the patient to take the medicine Renal function and urinary xanthine levels need to be monitored (information sourced from the febuxostat filing); in addition, cerebrospinal fluid (CSF) xanthine/hypoxanthine levels have been used as treatment guidelines and during hydrocephalus treatment markers of disease progression.
  • CSF cerebrospinal fluid
  • the food industry has high demands on the freshness of meat. After the death of fish, the nucleotides in the body are degraded into hypoxanthine and xanthine, and their content increases with the increase of storage time. Therefore, the content of xanthine in fish meat can also be used to evaluate the freshness of fish meat. All in all, the detection of xanthine is very important in both the food field and the medical field.
  • XOD activity detection kits are mostly used for scientific research purposes. They are mainly used for the detection of xanthine content by means of microplate reader. The operation is complicated and the detection throughput is small. The biochemical analyzer detection can be directly applied to clinical detection. Simple and high throughput. In addition, the developed direct color judgment product can further simplify the operation and is not limited by time and place. Non-professionals can also operate by themselves to judge the level of xanthine content.
  • the technical problem to be solved by the present invention is to overcome the defects of complicated operation, high cost and long time in the detection of purine substances in the prior art, thereby providing a quick, intuitive and stable test kit for detecting purine substances
  • a kit for detecting or assisting the detection of purine substances comprising reagent 1, reagent 2 and reagent 3, reagent 1 is a chromogenic agent, reagent 2 is a reaction enzyme, and reagent 3 is a weak acid salt; the reaction enzyme includes peroxidation enzyme and xanthine oxidase.
  • the kit further includes vitamin C and ascorbic acid enzyme; the vitamin C and ascorbic acid enzyme are packaged separately.
  • the color developer consists of color developer A and color developer B, where color developer A is 4-AAP, and color developer B is MADB or TBHBA; Color developer B is individually packaged.
  • the reaction enzyme is packaged independently; the weak acid salt is dipotassium hydrogen phosphate or potassium dihydrogen phosphate.
  • Peroxidase:xanthine oxidase:4-AAP:MADB or TBHBA is 20-600:0.1-15:0.1-5:0.1-10 in a ratio of KU:KU:g:g.
  • peroxidase xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbate 20-1300: 0.1-18: 0.1-5: 0.1-10: 1-12: 1- 360, the proportional relationship is: KU:KU:g:g:mM:KU;
  • Dipotassium Phosphate Peroxidase: Xanthine Oxidase: 4-AAP: MADB or TBHBA: Vitamin C: Ascorbate 0.2-35: 20-1300: 0.1-18: 0.1-5: 0.1-10 : 1-12: 1-360, the proportional relationship is: g: KU: KU: g: g: mM: KU.
  • the kit further includes one or two or three of stabilizers, surfactants and preservatives.
  • the stabilizer is selected from one or more of BSA, alcohols or sugars;
  • the alcohols are glycerol, mannitol, ethylene glycol, polyethylene glycol 6000, and sorbitol.
  • the surfactant is selected from Triton X-100, Twwen-20 or alkyl glycosides Any one or more of ; the preservative is NaN 3 or Proclin300.
  • the kit further includes a urate oxidase inhibitor; the optional urate oxidase inhibitor is potassium oxonate;
  • the kit also includes bilirubin oxidase
  • the kit further includes independently packaged urate oxidase.
  • peroxidase xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid: potassium oxonate for 20-1300:0.1-18:0.1-5:0.1-10:1 -12:1-360:0.5-5, the proportional relationship is: KU:KU:g:g:mM:KU:g;
  • Dipotassium phosphate Peroxidase: Xanthine oxidase: 4-AAP: MADB or TBHBA: Vitamin C: Ascorbic acid: Potassium oxonate 0.2-45: 20-1300: 0.1-18: 0.1-5: 0.1-10: 1-12: 1-360: 0.5-5, the proportional relationship is: g: KU: KU: g: g: mM: KU: g;
  • dipotassium phosphate peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid: potassium oxonate: bilirubin oxidase 0.2-35:20- 600: 0.1-18: 0.1-5: 0.1-10: 1-12: 1-360: 1-4: 1-108, the proportional relationship is: g: KU: KU: g: g: mM: KU: g: KU;
  • dipotassium phosphate peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: uricase 0.2-45: 20-1300: 0.1-18: 0.1-5: 0.1-10 : 30, the proportional relationship is: g:KU:KU:g:g:KU.
  • the box further includes auxiliary materials selected from one or more of lactose, citric acid, sodium bicarbonate, sodium carboxymethyl starch, PVP-K30, PEG6000 and micropowder silica gel.
  • auxiliary materials selected from one or more of lactose, citric acid, sodium bicarbonate, sodium carboxymethyl starch, PVP-K30, PEG6000 and micropowder silica gel.
  • kits for detecting or assisting the detection of purine substances comprises a test paper or a cotton swab; the test paper or the cotton swab contains the reagents in any one of the kits of claims 1-7.
  • the kit further includes a standard colorimetric card and/or a xanthine standard.
  • a method for preparing a kit for detecting or assisting in the detection of purine substances comprising the steps of weighing each component according to the kit, dissolving it with water, and diluting to volume, preparing it into a solution, and sub-packing;
  • the kit includes the steps of weighing each component according to the kit and preparing it into a target dosage form.
  • a system for detecting or assisting the detection of purine substances comprising the kit or the kit prepared by the method.
  • the system further includes a biochemical analyzer, a microplate reader or a UV-Vis spectrophotometer.
  • the kit the kit prepared by the method, and the application of the system in the detection or auxiliary detection of purine substances in the sample to be tested.
  • a method for detecting or assisting the detection of purine substances in a sample to be tested comprising the steps of detecting the sample to be tested with a kit or system prepared by the kit or method; the method is a non-disease diagnosis method.
  • a method for detecting or assisting in the detection of purine substances in a sample to be tested the specific steps are:
  • the sample volume required for the detection of 1 g effervescent tablet is greater than or equal to 10 mL;
  • the required volume of the sample to be tested is 0.3-3 mL;
  • it also includes the step of adding ascorbic acid enzyme and Vc successively.
  • the step of adding potassium oxonate is also included.
  • the sample to be tested is food, blood, urine, sweat, saliva, tears or tissue; the blood, urine, sweat, saliva, tears or tissue are from humans or animals.
  • the sample to be tested is blood, urine, sweat, saliva, tears or tissue; the blood, urine, sweat, saliva, tears or tissue are from experimental animals; it also includes adding a urate oxidase inhibitor; optionally the uric acid
  • the oxidase inhibitor is potassium oxonate.
  • the step of pre-processing the sample to be tested with a purification reagent is also included.
  • a method for detecting or assisting in the detection of purine substances in a sample to be tested the specific steps are:
  • test paper or cotton swab in the kit, and determine the concentration of xanthine and hypoxanthine in the sample to be tested according to the color change of the test paper or cotton swab.
  • the sample volume required by the test paper method is 0.5-1mL
  • Dipotassium hydrogen phosphate peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA on pre-contact test paper: 1-20: 5-100: 0.01-5: 0.1-5: 0.1-10, concentration The proportional relationship is: g/L:KU/L:KU/L:g/L:g/L.
  • Dipotassium hydrogen phosphate peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: urate oxidase on pre-contact test paper: 1-20: 5-100: 0.01-5: 0.1-5: 0.1 -10:30, the concentration ratio relationship is: g/L:KU/L:KU/L:g/L:g/L:KU/L.
  • Dipotassium Phosphate Peroxidase: Xanthine Oxidase: 4-AAP: MADB or TBHBA: Ascorbate: 10-30: 20-400: 0.1-15: 0.1-5: 0.1- 10:1-10
  • concentration ratio relationship is: g/L:KU/L:KU/L:g/L:g/L:KU/L.
  • the sample volume required for the cotton swab method is 0.5-1 mL.
  • a method for detecting or assisting in the detection of purine substances in a sample to be tested the specific steps are:
  • sample volume 0.06-0.2mL
  • the required sample volume is 0.005mL
  • the required sample volume is 0.1 mL.
  • the dosage form can be tablet, capsule or effervescent tablet
  • a single dose is a combination of all the ingredients.
  • the enzyme, stabilizer, developer and vitamin C can also be mixed together to prepare a freeze-dried preparation to ensure the activity of the enzyme;
  • the reagents in the kit are all solid reactants, specifically whole powders, ordinary tablets/capsules/granules or effervescent tablets.
  • the pretreatment steps of the purifying agent activated carbon are as follows: soak in 10% NaOH for 3 hours, filter out, bake at 105° C. for 24 hours for activation treatment, and set aside.
  • the present invention provides a kit for detecting or assisting in the detection of purine substances, including reagent 1, reagent 2 and reagent 3, reagent 1 is a chromogenic reagent, reagent 2 is a reaction enzyme, and reagent 3 is a weak acid salt; the Reactive enzymes include peroxidase and xanthine oxidase.
  • the kit also includes vitamin C and ascorbate; the vitamin C and ascorbate are packaged separately.
  • the kit adds a certain amount of vitamin C to the reagent for detecting purine substances to eliminate the background in the urine (there is a certain amount of xanthine in the normal human body, and the normal human body is colorless), and the purine in the urine is increased during the detection. Distinguishing degree of similar substances (xanthine/hypoxanthine) exceeding the standard and normal human urine;
  • the present invention provides a method for detecting or assisting the detection of purines in a sample to be tested, including the step of using a kit or a kit or system prepared by the method to detect the sample to be tested; the method is a non- Disease diagnosis methods.
  • the method detects the sample to be tested, it is fast (the reaction time is short, only 3 minutes), convenient (easy to carry, suitable for flow detection), and intuitive (can be distinguished by the naked eye); the method is used to detect purine substances (yellow purine and hypoxanthine) can obtain qualitative or semi-quantitative results without any instruments and professional training personnel; when the sample volume is large, the operation is not limited, easy to operate and enhance the user experience (such as urine samples, Can be measured by direct method, reducing exposure and sampling).
  • the reagents in the kit of the present invention can be divided into two doses, three doses or four doses, which improves the stability and accuracy of the product; the enzyme can also be made into a freeze-dried preparation, which is prepared and used immediately;
  • the present invention uses the detection of purine substances for auxiliary screening and diagnosis of related diseases for the first time; and can also play a certain role in monitoring the patient's medication process;
  • the present invention provides a system for detecting or assisting the detection of purine substances, the system includes the kit or the kit prepared by the method; the system further includes a biochemical analyzer, a microplate reader or an ultraviolet Visible spectrophotometer. There is no product of the same type on the market that can be used for high-throughput detection;
  • kit, system and method of the present invention provided by the present invention have a wide detection range, such as related biological samples/tissues: blood, urine, sweat, saliva, tears, organs, and other body fluids;
  • kits, system and method of the present invention can be used. More intuitive detection and high accuracy;
  • the kit, system and method of the present invention can be applied to the detection of food such as fish, beer, vegetables and other foods containing purine.
  • Figure 1 (a) is the result of the experimental group in Example 1 of the present invention; (b) is the result of the control group in Example 1 of the present invention;
  • Fig. 2 is the test result among the embodiment 2;
  • Fig. 3 is the test result in embodiment 3;
  • Fig. 4 is the standard colorimetric card of the cotton swab method among the embodiment 4;
  • Figure 5 is the result of the cotton swab method in Example 4.
  • Fig. 6 is the standard colorimetric card of the test paper method among the embodiment 5;
  • Fig. 7 is the result of the test paper method among the embodiment 5;
  • Fig. 8 is the standard curve in embodiment 6;
  • Fig. 9 is the standard curve of the microplate reader method in embodiment 7.
  • Fig. 10 is the standard curve of the ultraviolet-visible spectrophotometer method among the embodiment 8;
  • Figure 11 is the result of the test paper method for total content of xanthine and hypoxanthine in kidney samples in Example 9;
  • Figure 12 is the result of total content of xanthine and hypoxanthine in urine sample
  • Figure 13 is the stability comparison test result of the kit (quantitative microplate reader).
  • Figure 14 is (a) single standard solution of xanthine standard product; (b) single standard solution of hypoxanthine standard product; (c) single standard solution of adenine standard product; (d) single standard solution of guanine standard product.
  • Reagent 1-1 Reagent 1-2 Reagents 1-3 25g/L dipotassium hydrogen phosphate 15g/L dipotassium hydrogen phosphate 15g/L dipotassium hydrogen phosphate 15mL/L Glycerol 8g/L MADB 400KU/L POD 20g/L polyethylene glycol 6000 10KU/L Ascorbicase 2g/L 4-AAP 3mL/L ethylene glycol 10KU/L XOD 25g/L Mannitol 10mM/L Vc 5g/L trehalose 4g/LBSA 5g/L alkyl glycoside (APG) 2g/L preservative NaN 3 4g/L potassium oxonate (OA)
  • Sample Take normal urine as blank control, add 4 different concentrations of xanthine standard solutions to the urine, so that the final concentrations are: 200 ⁇ M, 400 ⁇ M, 700 ⁇ M, 1000 ⁇ M.
  • Reagent R1-A 1L of Reagent 1-1 to Reagent 1-2 in powder state to dissolve and mix
  • Reagent R1-A add 1L of Reagent 1-1 to Reagent 1-3 in powder state to dissolve and mix, denoted as Reagent R1-A
  • Reagent R1-B spare.
  • Control group The vitamin C in the above steps was removed, and the results of other steps remained unchanged, as shown in Figure 1(b).
  • adding vitamin C can eliminate the background in urine (the content of xanthine in normal human body is below 160 ⁇ M), and after adding VC, normal human urine is colorless, and the detection of xanthine/xanthine in urine increases.
  • each kilogram of R2-A contains 10KU of ascorbate
  • each kilogram of R2-B contains 500KU of POD and 18KU of XOD.
  • each tablet is 1g, namely effervescent tablets R2-A and R2-B;
  • Sample take normal urine as blank control, take 5 normal urine, add xanthine standard solution to the urine respectively, so that the final concentration of xanthine in urine is: 150 ⁇ M, 300 ⁇ M, 500 ⁇ M, 700 ⁇ M and 900 ⁇ M;
  • Purifying agent activated carbon (type JH303, powder) pretreatment: soaked in 10g/ml NaOH for 3 hours, filtered out, baked at 105°C for 24 hours for activation treatment, and used for later use.
  • Preparation process of salmon sample solution smash fresh salmon into mashed state, and divide it into 5g portions, one of which is recorded as 0h, and the rest of the samples are accelerated at 45°C and 75% relative humidity. Take the sample marked as 0h, add 10mL of normal saline, magnetically stir and mix for 5min, and then centrifuge at 8000rpm for 10min; take the centrifuged supernatant and add 3mg of activated carbon treated in step 1, fully mix and let stand for 3 minutes, Take the supernatant to obtain 0h salmon sample solution. A sample was then taken out at 4h, 8h, 12h, and 18h, respectively, and processed in the same manner to obtain salmon sample solutions of 4h, 8h, 12h, and 18h.
  • step 3 The results in step 3 are: with time, the purine content increases, and the blue color deepens (from left to right, the salmon sample solution of 0h, 4h, 8h, 12h and 18h).
  • the method of this embodiment can perform qualitative/semi-quantitative detection on the purine content in food.
  • Cotton swab preparation prepared by the following method: first fully immerse the blank cotton swab head in the R4-1 solution, then take it out, vacuum dry at 35-40 °C, and set aside.
  • the cotton swab head can be cotton, sponge, or non-woven fabric, and this embodiment is non-woven fabric.
  • the concentration corresponding to the standard colorimetric card is the concentration of the liquid to be tested. If it is between two color levels, the concentration is estimated.
  • HPLC detection of samples in step 2 Agilent 1260, C18 column (4.6 ⁇ 250mm, 5 ⁇ m); mobile phase consists of mobile phase A and mobile phase B, mobile phase A is 10mM ammonium acetate, mobile phase B is methanol, mobile phase A The volume ratio with mobile phase B is 98:2, the running time is 20min, the flow rate: 1mL/min, and the detection wavelength: 270nm; the test results are shown in the following table.
  • Element concentration Dipotassium phosphate 5g/L POD 20KU/L 4-AAP 1g/L XOD 0.1KU/L MADB 5g/L Urate oxidase 30KU/L BSA 0.5g/L polyethylene glycol 6000 1g/L sucrose 3g/L
  • test paper substrate strip Na Green Polymer Materials Co., Ltd., biological detection test paper
  • R5-1 a mixed solution
  • R5-1 a mixed solution
  • Vacuum dry at 35-40°C cut into test strips 1 cm wide and 5 cm long, and store in a dry, airtight and light-proof plastic bag for low temperature storage.
  • step 2 Immerse the test paper prepared in step 2 in the above standard solution for 2 seconds and take it out immediately;
  • the concentration corresponding to the standard colorimetric card that is closest to the color displayed by the urine sample to be tested is the concentration of the urine sample to be tested. If it is between the two color levels, the concentration is estimated.
  • Agilent 1260 was used for HPLC detection; the conditions were: C18 column (4.6 ⁇ 250mm, 5 ⁇ m), the mobile phase consisted of mobile phase A and mobile phase B, mobile phase A was 10 mM ammonium acetate, mobile phase B was methanol, mobile phase A was The volume ratio of mobile phase B was 98:2. Running time 20min, flow rate: 1mL/min, detection wavelength: 270nm.
  • test paper of this embodiment can more accurately detect the total content of purine and hypoxanthine in the urine to be tested.
  • Reagent 6-1 Reagent 6-2 8g/L dipotassium hydrogen phosphate 8g/L dipotassium hydrogen phosphate 0.35g/L MADB 160KU/L POD 10KU/L Ascorbicase 0.8g/L 4-AAP 3KU/L bilirubin oxidase 1g/L OA / 0.5KU/L XOD 2mL/L Glycerol 2mL/L Glycerol 5g/L polyethylene glycol 6000 5g/L polyethylene glycol 6000 10mL/L ethylene glycol 10mL/L ethylene glycol 15g/L Mannitol 15g/L Mannitol 15g/L trehalose 15g/L trehalose 1g/L BSA 1g/L BSA 1g/L Alkyl Glycoside (APG) 1g/L Alkyl Glycoside (APG) 0.5g/L preservative NaN 3 0.5g/L preservative NaN 3 0.5g/L preservative
  • Kit composition (100T) 25 mL of detection buffer R7-1; 1 bottle each of reagents R7-2, R7-3, R7-4; 1 bottle of xanthine standard (1 ⁇ M).
  • Reagent 7-1 Reagent 7-2 (powder)
  • Reagent 7-3 (powder)
  • Reagent 7-4 (powder) 10g/L dipotassium hydrogen phosphate 0.0003KU XOD 0.0008g TBHBA 0.0007g 4-AAP 1.5g/L OA 0.065KU POD 8mL/L Glycerol 0.018KU Ascorbate 15g/L polyethylene glycol 6000 0.0054KU bilirubin oxidase 5g/L Sorbitol 15g/L glucose 3g/L BSA 2g/L Twwen-20 0.02g/LPoclin 300
  • Preparation method Weigh the corresponding substances according to the above formula, and dilute to the corresponding concentration with 25 mL of pure water to obtain reagent R7-1; dissolve 7-2, 7-3 and 7-4 with 220 ⁇ L of reagent R7-1 to prepare reagent R7 -2, R7-3, R7-4 (currently used)
  • Urine samples were centrifuged at 12,000 rpm for 5 minutes for immediate use; serum/plasma samples were centrifuged at 12,000 rpm for 5 minutes before use; tissue (10 mg) or cells (1x 10 6 ), added 100 ⁇ L of pre-cooled detection buffer (reagent 7-1), Homogenize on ice for 10 min. Centrifuge at 12,000 rpm for 5 min, and collect the supernatant for later use. Add 5 ⁇ L of sample to each well and make up to 50 ⁇ L with buffer.
  • xanthine standard Dissolve 1 ⁇ M xanthine standard in 500 ⁇ L of distilled water until the concentration of xanthine standard is 2.0 mM (2.0 nmol/ ⁇ l). Add 0, 2, 4, 6, 8, and 10 ⁇ L of standards containing 2 mM xanthine to 96-well plates, so that each well contains 0, 4, 8, 12, 16, and 20 nmol/well of xanthine. , make up to 50 ⁇ L per well with detection buffer.
  • reaction solution (reagents should be mixed thoroughly before reaction, including: 44 ⁇ L of R7-1 reagent, 2 ⁇ L of R7-2 reagent, 2 ⁇ L of R7-3 reagent, 2 ⁇ L of R7 -4 reagent), mix well. Incubate at room temperature for 30 min in the dark, and measure the absorbance at (the emission wavelength and excitation wavelength are 520 nm and 550 nm, respectively) with a microplate reader.
  • Kit composition (100T) 200 mL of detection buffer R8-1; 1 bottle of reagents R8-2, R8-3, R8-4; 1 bottle of xanthine standard (20 ⁇ M).
  • Reagent 8-1 Reagent 8-2 (powder)
  • Reagent 8-3 (powder)
  • Reagent 8-4 (powder) 15g/L dipotassium hydrogen phosphate 0.006KU XOD 0.012g MADB 0.012g 4-AAP 3g/L OA 1.3KU POD 4mL/L Glycerol 0.36KU Ascorbate 5g/L polyethylene glycol 6000 0.108KU bilirubin oxidase 6mL/L ethylene glycol 25g/L Mannitol 15g/L trehalose 1g/L BSA 1g/L Alkyl Glycoside (APG) 0.5g/L preservative NaN 3
  • Preparation method Weigh the corresponding substances according to the above formula, and dilute to the corresponding concentration with 200 mL of pure water to obtain reagent R8-1; dissolve 8-2, 8-3 and 8-4 with 5 mL of reagent R8-1 to prepare reagent R8- 2. R8-3, R8-4.
  • xanthine standard 10 mL of distilled water dissolves 20 ⁇ M xanthine standard, the concentration of the xanthine standard is 2.0 mM (2.0 nmol/ ⁇ l). Add 0, 40, 80, 120, 160, and 200 ⁇ L of 2 mM xanthine-containing standard to 2.5 mL centrifuge tubes, so that the final concentrations of xanthine in each tube are 0, 80, 160, 240, 320, and 400 nmol/ ⁇ L. , make up to 1 mL per tube with detection buffer.
  • Example 9 After animals are administered febuxostat, the total content of xanthine and hypoxanthine in the sample is detected
  • Collection of blood samples and kidney tissue take several rats, and administer febuxostat by intragastric administration at a dose of 6 mg/kg/day.
  • the administration cycle is 7 days, and the administration is administered by intragastric administration for 1 hour on the 7th day.
  • blood was taken from the abdominal aorta, and kidney samples were collected by further dissection.
  • Kidney samples are used after centrifugation. Kidney samples were 10 mg of tissue, 100 ⁇ L of pre-cooled detection buffer was added, and the mixture was homogenized on ice for 10 min. Centrifuge at 12,000 rpm for 5 min, and collect the supernatant for later use.
  • Detection operate according to Example 3 to obtain visual inspection results; operate according to Example 4 to obtain cotton swab method detection results; operate according to Example 5 to obtain test paper method detection results; operate according to Example 6 to obtain biochemical analyzer data; operate according to Example 7 Obtain the data of the microplate reader; obtain the data of the UV-visible spectrophotometer according to the operation in Example 8; HPLC detection: Agilent 1260, C18 column (4.6 ⁇ 250 mm, 5 ⁇ m); the mobile phase is composed of mobile phase A and mobile phase B, mobile phase A is 10 mM ammonium acetate, mobile phase B is methanol, the volume ratio of mobile phase A to mobile phase B is 98:2, the running time is 20 min, the flow rate: 1 mL/min, and the detection wavelength: 270 nm.
  • Example 10 Test kit (quantitative microplate reader) stability comparison test
  • Example 7 For the formulation reagent of Example 7, 13 groups were evenly packed, and 13 groups of commercial xanthine/hypoxanthine detection kits (Sigma Company, product code MAK186) were taken as control; On the same day, a set of reagents were taken out to detect xanthine standard solution (target value 100 ⁇ M/L). The test results showed that the formula reagent of Example 7 was better than the common xanthine/hypoxanthine detection reagent in the market under the storage condition of 2-8°C. The box is more stable. The results are shown in Figure 13.

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Abstract

一种检测或辅助检测嘌呤类物质的试剂盒，包括试剂1、试剂2和试剂3，试剂1为显色剂，试剂2为反应酶，试剂3为弱酸盐；所述反应酶包括过氧化物酶和黄嘌呤氧化酶。所述试剂盒还包括维生素C和抗坏血酸酶；所述维生素C和抗坏血酸酶分开包装。该试剂盒检测待测样本时，快速、直观，当样本体量很大，操作也不受限；该试剂盒用于检测嘌呤类物质不需任何仪器和专业训练人员即可得到定性或半定量的结果。

Description

一种检测嘌呤类物质的试剂盒及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种检测嘌呤类物质的试剂盒及其应用。
背景技术
黄嘌呤/次黄嘌呤是一种广泛分布于人和其他生物体的器官和体液中的嘌呤碱，其正常的生理浓度在血清中为0.5～2.5μmol/L，在尿液中为40～160μmol/L。通常在黄嘌呤氧化酶(xo)的作用下生成尿酸，并随尿液排出。人体中黄嘌呤、次黄嘌呤和尿酸的浓度变化可以直接反映人体免疫和代谢等机能的状况，提示与嘌呤代谢有关的疾病。
体内黄嘌呤累积易发展成黄嘌呤尿症，最终导致肾衰；服用别嘌呤醇、非布司他等黄嘌呤氧化酶抑制剂后，在肾脏和输尿管发现了黄嘌呤结晶，提醒患者在服药期间需要监测肾脏功能和尿中黄嘌呤含量(信息来源非布司他申报资料)；此外，在脑积水治疗过程中，脑脊液(CSF)黄嘌呤/次黄嘌呤水平已被用作治疗指导指标和疾病进展标志物。
食品工业对肉类的新鲜程度要求很高。鱼死亡后，体内的核苷酸被降解为次黄嘌呤及黄嘌呤，其含量随着储存时间的增加而增加，因此，鱼肉中黄嘌呤的含量也可用于评估鱼肉的新鲜程度。总而言之，黄嘌呤的检测无论是在食品领域还是在医疗领域都非常重要。
目前常规的检测方法，例如高效液相色谱、分光光度法、化学发光，毛细管电泳等都能高灵敏度和高选择性地检测次黄嘌呤和黄嘌呤，然而这些检测方法的步骤非常繁琐，且需要相对昂贵的设备以及专业熟练的技术人员来进行操作，增加了检测的成本和时间。
商品化XOD活性检测试剂盒，多为科研用途性质，主要是通过酶标仪手段进行黄嘌呤含量的检测，操作复杂且检测通量较小；而生化分析仪检测能够直接应用于临床检测，操作简便且通量高。另外，研发的直接显色判断产品，能够进一步简化操作，不受时间场地的限制，非专业人员也可以自行操作，判断黄嘌呤含量的高低。
因此，开发一种能快捷、直观、稳定的检测黄嘌呤的方法，既可满足生化分析仪等高通量的定量检测，又可满足家用便携定性半定性检测是非常必要的。
发明内容
因此，本发明要解决的技术问题在于克服现有技术中的嘌呤类物质检测的操作复杂、成本高、用时长的缺陷，从而提供一种能够快捷、直观、稳定的检测嘌呤类物质的试剂盒及其制备方法和应用、用所述试剂盒检测待测一种检测或辅助检测待测样品中的嘌呤类物质的方法。
一种检测或辅助检测嘌呤类物质的试剂盒，包括试剂1、试剂2和试剂3，试剂1为显色剂，试剂2为反应酶，试剂3为弱酸盐；所述反应酶包括过氧化物酶和黄嘌呤氧化酶。
可选的，所述试剂盒还包括维生素C和抗坏血酸酶；所述维生素C和抗坏血酸酶分开包装。
可选的，所述显色剂由显色剂A和显色剂B组成，显色剂A为4-AAP，显色剂B为MADB或TBHBA；可选的，所述显色剂A和显色剂B均独立包装。
可选的，所述反应酶独立包装；所述弱酸盐为磷酸氢二钾或磷酸二氢钾。
过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA为20-600：0.1-15：0.1-5：0.1-10，比例关系为KU：KU：g：g。
可选的，过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶为20-1300：0.1-18：0.1-5：0.1-10：1-12：1-360，比例关系为：KU：KU：g:g:mM：KU；
或，磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶为0.2-35：20-1300：0.1-18：0.1-5：0.1-10：1-12：1-360，比例关系为：g：KU：KU：g:g:mM：KU。
可选的，所述试剂盒中还包括稳定剂、表面活性剂和防腐剂中的一种或两种或三种。
可选的，稳定剂选自BSA、醇类或糖类中的一种或几种；可选的，醇类为丙三醇、甘露醇、乙二醇、聚乙二醇6000、山梨醇中的任一一种或几种；可选的，糖类为海藻糖、葡萄糖、蔗糖中的任意一一种或 几种；表面活性剂选自Triton X-100、Twwen-20或烷基糖苷中的任一一种或几种；防腐剂为NaN 3或Proclin300。
可选的，所述试剂盒还包括尿酸氧化酶抑制剂；可选的所述尿酸氧化酶抑制剂为氧嗪酸钾；
可选的，所述试剂盒还包括胆红素氧化酶；
可选的，所述试剂盒还包括独立包装的尿酸氧化酶。
可选的，过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶:氧嗪酸钾为20-1300：0.1-18：0.1-5：0.1-10：1-12：1-360：0.5-5，比例关系为：KU：KU：g:g:mM：KU：g；
磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶:氧嗪酸钾为0.2-45：20-1300：0.1-18：0.1-5：0.1-10：1-12：1-360：0.5-5，比例关系为：g：KU：KU：g:g:mM：KU：g；
可选的，磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶:氧嗪酸钾：胆红素氧化酶为0.2-35：20-600：0.1-18：0.1-5：0.1-10：1-12：1-360：1-4：1-108，比例关系为：g：KU：KU：g:g:mM：KU：g：KU；
可选的，磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：尿酸氧化酶为0.2-45：20-1300：0.1-18：0.1-5：0.1-10：30，比例关系为：g：KU：KU：g:g:KU。
可选的，所述盒还包括辅料，所述辅料选自乳糖、柠檬酸、碳酸氢钠、羧甲基淀粉钠、PVP-K30、PEG6000和微粉硅胶中的一种或几种。
一种检测或辅助检测嘌呤类物质的试剂盒，所述试剂盒包括试纸或棉签；所述试纸或棉签上含有权利要求1-7任一所述试剂盒中的试剂。
可选的，所述试剂盒还包括标准比色卡和/或黄嘌呤标准品。
一种制备检测或辅助检测嘌呤类物质的试剂盒的方法，包括根据试剂盒称取各个成分，用水溶解后定容，制备为溶液，分装的步骤；
或，包括根据所述试剂盒称取各个成分，制备成目标剂型的步骤。
一种检测或辅助检测嘌呤类物质的系统，所述系统包括所述的试剂盒或所述方法制备得到的试剂盒。
可选的，所述系统还包括生化分析仪、酶标仪或紫外可见分光光度计。
试剂盒，所述方法制备得到的试剂盒，系统在检测或者辅助检测待测样本中的嘌呤类物质中的应用。
一种检测或辅助检测待测样品中的嘌呤类物质的方法，包括用试剂盒或方法制备得到的试剂盒或系统对待测样本进行检测的步骤；所述方法为非疾病诊断方法。
一种检测或辅助检测待测样品中的嘌呤类物质的方法，具体步骤为：
1)将待测样本与试剂盒中的试剂混合，形成混合液；
2)观察混合液的颜色，判断待测样本中次黄嘌呤/黄嘌呤的含量高低。
试剂为泡腾片时，1g泡腾片检测时所需样品体积大于等于10mL；
步骤1)中的试剂为液体时，所需的待测样品体积为0.3-3mL；
可选的，还包括先后加入抗坏血酸酶和Vc的步骤。
或，可选的，还包括加入氧嗪酸钾的步骤。
所述待测样本为食品、血液、尿液、汗液、唾液、泪液或组织；所述血液、尿液、汗液、唾液、泪液或组织来自人或动物。
待测样本为血液、尿液、汗液、唾液、泪液或组织；所述血液、尿液、汗液、唾液、泪液或组织来自实验动物；还包括加尿酸氧化酶抑制剂；可选的所述尿酸氧化酶抑制剂为氧嗪酸钾。
待测样品为食品时，还包括用纯化试剂对待测样品进行预处理的步骤。
一种检测或辅助检测待测样品中的嘌呤类物质的方法，具体步骤为：
将待测样本与试剂盒中的试纸或棉签接触，根据试纸或者棉签的颜色变化，判断待测样本中黄嘌呤和次黄嘌呤的浓度大小。
试纸法所需的样品体积0.5-1mL；
接触前试纸上，磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA为：1-20：5-100： 0.01-5：0.1-5：0.1-10，浓度比例关系为：g/L：KU/L：KU/L：g/L:g/L。
接触前试纸上，磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：尿酸氧化酶为：1-20：5-100：0.01-5：0.1-5：0.1-10：30，浓度比例关系为：g/L：KU/L：KU/L：g/L:g/L：KU/L。
接触前棉签上，磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：抗坏血酸酶为：10-30：20-400：0.1-15：0.1-5：0.1-10：1-10，浓度比例关系为：g/L：KU/L：KU/L：g/L:g/L：KU/L。
棉签法所需的样品体积0.5-1mL。
一种检测或辅助检测待测样品中的嘌呤类物质的方法，具体步骤为：
(b1)绘制标准曲线
配制系列浓度的嘌呤类物质标准品溶液，将所得的若干份含有不同浓度的标准品溶液分别与试剂盒中的试剂混合，形成混合液；然后将所述混合液室温孵育30分后，使用酶标仪或紫外可见分光光度计测定混合液的吸光度；以所述嘌呤类物质的标准品溶液的浓度为横坐标，以所述混合液的的吸光度的为纵坐标绘制标准曲线图，得到标准曲线方程；
(b2)待测样本检测
将所述待测样本与试剂盒中的试剂混合，得到混合液；使用酶标仪、生化分析仪或紫外可见分光光度计测定混合液的吸光度，将所述待测样本的吸光度的值代入步骤b1)所得标准曲线方程，计算得到所述待测样本中嘌呤类物质(黄嘌呤和次黄嘌呤)的浓度值。
生化仪分析时，所需的样品体积0.06-0.2mL；
酶标仪分析时，所需的样品体积0.005mL；
紫外分光光度计分析时，所需的样品体积0.1mL。
将试剂盒中的试剂制备为单剂、双剂、三剂或四剂；
所述试剂盒中的试剂为固体时，剂型可为片剂、胶囊或泡腾片；
单剂即为将所有成分混合。
还可以将酶、稳定剂、显色剂和维生素C混合在一起，制备为制成冻干制剂，从而保证酶的活性；
所述试剂盒中的试剂均为固体反应剂，具体为全粉末，普通片剂/胶囊/颗粒或泡腾片。
纯化剂活性炭(型号JH303，粉末状)预处理步骤为：经10％的NaOH浸泡3h，滤出，在105℃烘烤24小时进行活化处理，备用。
本发明技术方案，具有如下优点：
1、本发明提供一种检测或辅助检测嘌呤类物质的试剂盒，包括试剂1、试剂2和试剂3，试剂1为显色剂，试剂2为反应酶，试剂3为弱酸盐；所述反应酶包括过氧化物酶和黄嘌呤氧化酶。所述试剂盒还包括维生素C和抗坏血酸酶；所述维生素C和抗坏血酸酶分开包装。该试剂盒在检测嘌呤类物质的试剂中加入一定量的维生素C，将尿液中的背景消除(正常人体内有一定量的黄嘌呤，实现正常人无色)，检测时增加了尿液中嘌呤类物质(黄嘌呤/次黄嘌呤)超标与正常人尿液的区分度；
2、本发明提供一种检测或辅助检测待测样品中的嘌呤类物质的方法，包括用试剂盒或所述方法制备得到的试剂盒或系统对待测样本进行检测的步骤；所述方法为非疾病诊断方法。所述方法检测待测样本时，快速(反应时间短，仅需3分钟)、方便(便于携带，适于流动检测)、直观(肉眼即可辨别)；该方法用于检测嘌呤类物质(黄嘌呤和次黄嘌呤)不需任何仪器和专业训练人员即可得到定性或半定量的结果；当样本体量很大，操作也不受限，易操作、提升用户体验感(如尿液样本，可以直接法测定，减少接触及取样)。
3、本发明试剂盒中的试剂可以分成双剂、三剂或四剂，提高了产品的稳定性和准确性；还可以将酶制成冻干制剂，现配现用；
4、本发明首次将嘌呤类物质检测用于相关疾病的辅助筛查和诊断；而且对病人用药过程也可以起到一定的监控作用；
5、本发明提供一种检测或辅助检测嘌呤类物质的系统，所述系统包括所述的试剂盒或所述方法制备得到的试剂盒；所述系统还包括生化分析仪、酶标仪或紫外可见分光光度计。市场上不存在同类型的，可用于高通量检测的产品；
6、本发明提供的本发明的试剂盒、系统和方法，检测范围较广，如相关的生物样本/组织：血液、尿液、汗液、唾液、泪液、脏器、其他体液；
7、动物科研中，如将动物解剖后检测评价脏器的功能，如肝肾黄嘌呤结晶、关节积液中黄嘌呤含量、脑脊液中黄嘌呤含量，可以运用本发明的试剂盒、系统和方法更直观的检测，且精准度高；
8、本发明的试剂盒、系统和方法可适用于食品如鱼、啤酒、蔬菜等含有嘌呤的食物的检测。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1(a)是本发明实施例1中实验组的结果；(b)是本发明实施例1中对照组结果；
图2是实施例2中试验结果；
图3是实施例3中试验结果；
图4是实施例4中的棉签法的标准比色卡；
图5是实施例4中的棉签法的结果；
图6是实施例5中的试纸法的标准比色卡；
图7是实施例5中的试纸法的结果；
图8是实施例6中的标准曲线；
图9是实施例7中的酶标仪法的标准曲线；
图10是实施例8中的紫外可见分光光度计法的标准曲线；
图11是实施例9中的肾脏样本黄嘌呤及次黄嘌呤总含量试纸法的结果；
图12是尿液样本黄嘌呤及次黄嘌呤总含量结果；
图13是试剂盒(酶标仪定量)稳定性对比试验结果；
图14是(a)黄嘌呤标准品单标溶；(b)次黄嘌呤标准品单标溶液；(c)腺嘌呤标准品单标溶液；(d)鸟嘌呤标准品单标溶液。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明，并不局限于所述最佳实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品，均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
试剂规格及厂家见下表。
表0
Figure PCTCN2021074330-appb-000001
Figure PCTCN2021074330-appb-000002
实施例1目测法
表1试剂配方
试剂1-1 试剂1-2 试剂1-3
25g/L磷酸氢二钾 15g/L磷酸氢二钾 15g/L磷酸氢二钾
15mL/L丙三醇 8g/L MADB 400KU/L POD
20g/L聚乙二醇6000 10KU/L抗坏血酸酶 2g/L 4-AAP
3mL/L乙二醇   10KU/L XOD
25g/L甘露醇   10mM/L Vc
5g/L海藻糖    
4g/LBSA    
5g/L烷基糖苷(APG)    
2g/L防腐剂NaN 3    
4g/L氧嗪酸钾(OA)    
1.配制：按上表配方1-1称取相应物质，以纯水定容至1L，得试剂1-1，同理，得试剂1-2、1-3。将试剂1-2、1-3分别冻干成粉末状态，备用。
2.样本：以正常尿液作为空白对照，在该尿液中添加4种不同浓度的黄嘌呤标准溶液，使其终浓度分别为：200μM、400μM、700μM、1000μM。
3.操作：临用前，粉末状态试剂1-2中加入1L试剂1-1溶解混匀，记为试剂R1-A；粉末状态试剂1-3中加入1L试剂1-1溶解混匀，记为试剂R1-B，备用。
取尿液与上述试剂R1-A以体积比20:1混合，放置3min后，混合液与试剂R1-B以体积比5:1混合，摇匀，放置3-5min后观察颜色变化(在5-30min内观察颜色，超时影响结果判断)。
4.结果观察：正常尿液、黄嘌呤浓度低于200μM不显色，黄嘌呤浓度高于200μM的尿液显蓝色
如图1(a)所示，从左到右依次为，空白对照尿液显色前、含黄嘌呤终浓度分别为：200μM、400μM、700μM、1000μM的尿液显色后。
对照组：将上述步骤中的维生素C去除，其它步骤不变结果见，图1(b)。
由上图可知，加维生素C可以将尿液中的背景消除(正常人体内黄嘌呤含量为160μM以下)，加VC后正常人尿液显示为无色，检测时增加了尿液中黄嘌呤/次黄嘌呤超标与正常人尿液的区分度；不加VC，尿液显色后需要测试者根据颜色深浅，主观来判断体内黄嘌呤是否超标，而这极容易因为光线、操作、尿液本身的颜色等干扰造成误判。
实施例2.泡腾片法检测
表2配方
Figure PCTCN2021074330-appb-000003
Figure PCTCN2021074330-appb-000004
注：上述百分数为质量百分数；每千克R2-A中含有抗坏血酸酶10KU，每千克R2-B中含有POD 500KU，XOD 18KU。
1.配制：按上表中的配方分别混匀压制成片剂，每片1g，即得泡腾片R2-A、R2-B；
2.样本：以正常尿液作为空白对照，取5份正常尿液，分别在该尿液中添加黄嘌呤标准溶液，使尿液中黄嘌呤的终浓度分别为：150μM、300μM、500μM、700μM和900μM；
3.操作：取步骤2中的尿液样本150mL(实际使用时对样本量要求不高，超过10mL即可将泡腾片溶解，也可以直接放2-3片至马桶内观察显色)，加入1片泡腾片R2-A，放置2min后，加入泡腾片R2-B，放置5min后，在30min内观察颜色，结果见图2，从左到右黄嘌呤浓度分别为150μM、300μM、500μM、700μM、900μM的尿液，从图2中可以看出，黄嘌呤浓度为150μM不显色，黄嘌呤高于150μM的尿液变为红色，尿液中黄嘌呤浓度越大，颜色越深。
实施例3.目测法
表3试剂配方
成分 浓度
磷酸氢二钾 30g/L
POD 600KU/L
4-AAP 5g/L
XOD 15KU/L
MADB 10g/L
1.配制：按上述配方称取相应物质，以纯水定容至1L，得配方试剂R3-1，现配现用。
纯化剂活性炭(型号JH303，粉末状)预处理：经10g/ml的NaOH浸泡3h，滤出，在105℃烘烤24小时进行活化处理，备用。
2.三文鱼样品溶液制备过程：将新鲜三文鱼搅碎成为肉泥状态，分为5g每份，其中1份记为0h，其余样品于45℃，相对湿度75％环境下加速腐败。取标记为0h的样品，加入10mL生理盐水，磁力搅拌混匀5min后在8000rpm的条件下离心10min；取离心后的上清液加入步骤1处理后的3mg活性炭，充分混匀静置3分钟，取上清，获得0h的三文鱼样品溶液。再分别于4h、8h、12h、18h取出一份样品，按照同样的方式处理，获得4h、8h、12h、18h的三文鱼样品溶液。
3.操作：不同时间点处理后样品和上述试剂R3-1以体积比为10：1混合，室温放置10min，观察混合液颜色，结果见图3。
4.HPLC检测数据对比：
检测条件：安捷伦1260，C18柱(4.6×250mm，5μm)，流动相：10mM乙酸铵水溶液：甲醇＝体积比98：2，运行时间20min，流速：1mL/min，检测波长：270nm
精确定量显示：0h、4h、8h、12h和18h的三文鱼样品溶液中嘌呤含量分别为：26.9μM、89.7μM、221μM、436μM、605μM。
5.显色结果观察：步骤3中结果为：随时间放置，嘌呤含量增加，蓝色加深(从左往右依次为0h、4h、8h、12h和18h的三文鱼样品溶液)。
实施例中鱼肉放置4h外观无明显差异，但加入检测试剂后，可见溶液蓝色明显加深，以此可判断其嘌呤含量升高；且随时间延长，蓝色持续加深，由液相数据可知嘌呤含量与其颜色深浅正相关，可做半定量由于预估食物中嘌呤含量。
说明本实施例的方法能够对食品中的嘌呤含量进行定性/半定量检测。
实施例4.棉签法
表4试剂配方
磷酸氢二钾 15g/L
MADB 2g/L
POD 80KU/L
4-AAP 0.5g/L
OA 2g/L
XOD 5KU/L
抗坏血酸酶 2KU/L
丙三醇 4mL/L
聚乙二醇6000 10g/L
乙二醇 15mL/L
甘露醇 5g/L
海藻糖 50g/L
BSA 2g/L
一、棉签的制备
1.配制，按上述配方称取相应物质，以纯水定容至1L，得配方试剂R4-1，备用。
2.棉签准备，通过以下方法制备得到的：先将空白棉签头充分浸入R4-1液后取出，35-40℃真空干燥，备用。棉签头可以是棉花、海绵、无纺布，本实施例为无纺布。
3.标准比色卡的制备
1)配制浓度为100、200、300、400、600、800μmol/L的黄嘌呤标准溶液；
2)将按照步骤2制备好的棉签分别浸于上述6种标准溶液中1秒立即取出；
3)根据各棉签5分钟后的显色情况制备比色卡，见图4。
二、棉签法检测待测样本
1)样本：健康人尿液样本记为Co1、Co2；服用别嘌醇200mg/d的痛风患者尿液样本记为A1、服用别嘌醇600mg/d的尿液样本记为A2、A3；服用非布司他40mg/d的痛风患者尿液样本记为F1、服用非布司他80mg/d的尿液样本记为F2、F3；
2)操作方法
使用时，将上述棉签浸入被测液中1秒立即取出(或将被测液滴加到棉签上)，等待5分钟，与标准比色卡(图4)比较，与测试样颜色最接近的标准比色卡所对应的浓度即为待测液的浓度，如果在两个色阶之间，则估读浓度。
三、HPLC检测待测样本
HPLC检测步骤二中的样本：安捷伦1260，C18柱(4.6×250mm，5μm)；流动相由流动相A和流动相B组成，流动相A为10mM乙酸铵，流动相B为甲醇，流动相A与流动相B的体积比为98：2，运行时间20min，流速：1mL/min，检测波长：270nm；试验结果见下表。
表5
样本 显色 比色卡估读浓度 HPLC检测数据
健康人Co-1 图5(a) ＜100μmol/L 37.2μmol/L
健康人Co-2 图5(b) ＜100μmol/L 27.5μmol/L
别嘌醇A-1 图5(c) 200-300μmol/L 278.2μmol/L
别嘌醇A-2 图5(d) 400-600μmol/L 598.4μmol/L
别嘌醇A-3 图5(e) 约800μmol/L 759.2μmol/L
非布司他F-1 图5(f) 约200μmol/L 197.1μmol/L
非布司他F-2 图5(g) 约600μmol/L 585.2μmol/L
非布司他F-3 图5(h) 600-800μmol/L 683.9μmol/L
由上表和图5可以看出，本实施例的棉签法的结果可信。
实施例5.试纸法
表6
成分 浓度
磷酸氢二钾 5g/L
POD 20KU/L
4-AAP 1g/L
XOD 0.1KU/L
MADB 5g/L
尿酸氧化酶 30KU/L
BSA 0.5g/L
聚乙二醇6000 1g/L
蔗糖 3g/L
一、试纸的制备
1)配制：按上述配方称取相应物质，以纯水定容至1L，得配方试剂R5-1，备用。
2)试纸准备
将试纸基材条(九绿高分子材料有限公司，生物检测试纸)浸泡上述混合溶液R5-1中，振摇30-60min，使得各组分被共同吸附到基材上，将基材取出低温35-40℃真空干燥，剪成1厘米宽、5厘米长大小的试纸条，并置于干燥密闭和避光的塑料袋中低温保存。
3)标准比色卡的制备方法
1)配制浓度为100、200、300、400、600、800μmol/L的黄嘌呤标准溶液；
2)将步骤2制备好的试纸分别浸于上述标准溶液中2秒立即取出；
3)根据各试纸5-15分钟内的显色情况制备比色卡，见图6。
二、试纸法检测待测样本
1)样本：健康人尿液样本Co1-2、高嘌呤尿症患者尿液样本H1-6
2)操作方法
使用时，将上述步骤制备好的试纸浸入待测尿液样本中2秒立即取出(或将待测尿液滴加到试纸上)，等待5分钟，与标准比色卡(图6)比较，与待测尿液样本显示的颜色最接近的标准比色卡所对应的浓度即为待测尿液样本的浓度，如果在两个色阶之间，则估读浓度。
三、HPLC法检测数据对比
HPLC法检测步骤二中的待测样本。
HPLC法检测运用安捷伦1260；条件为：C18柱(4.6×250mm，5μm)，流动相由流动相A和流动相B组成，流动相A为10mM乙酸铵，流动相B为甲醇，流动相A与流动相B的体积比为98：2。运行时间20min，流速：1mL/min，检测波长：270nm。
结果见表7和图7。
表7
样本 图 比色卡估读浓度 HPLC检测数据
健康人Co-1 图7(a) ＜100μmol/L 28.7μmol/L
健康人Co-2 图7(b) ＜100μmol/L 35.3μmol/L
高嘌呤尿症-1 图7(c) 约400μmol/L 362.2μmol/L
高嘌呤尿症-2 图7(d) 600-800μmol/L 685.7μmol/L
高嘌呤尿症-3 图7(e) 600-800μmol/L 655.9μmol/L
高嘌呤尿症-4 图7(f) 约300μmol/L 275.2μmol/L
高嘌呤尿症-5 图7(g) 约300μmol/L 298.4μmol/L
高嘌呤尿症-6 图7(h) 约800μmol/L 783.5μmol/L
由上述结果可以看出，本实施例的试纸能够较为准确的检测待测尿液的嘌呤及次黄嘌呤的总含量。
实施例6.生化分析仪用试剂盒
表8
试剂6-1 试剂6-2
8g/L磷酸氢二钾 8g/L磷酸氢二钾
0.35g/L MADB 160KU/L POD
10KU/L抗坏血酸酶 0.8g/L 4-AAP
3KU/L胆红素氧化酶 1g/L OA
/ 0.5KU/L XOD
2mL/L丙三醇 2mL/L丙三醇
5g/L聚乙二醇6000 5g/L聚乙二醇6000
10mL/L乙二醇 10mL/L乙二醇
15g/L甘露醇 15g/L甘露醇
15g/L海藻糖 15g/L海藻糖
1g/L BSA 1g/L BSA
1g/L烷基糖苷(APG) 1g/L烷基糖苷(APG)
0.5g/L防腐剂NaN 3 0.5g/L防腐剂NaN 3
一、试剂盒的制备
1)配制方法：按上述配方称取相应物质，以纯水定容至相应浓度，分别得配方试剂R6-1、R6-2；
2)样本制备：取尿液样本12000rpm离心5min，取上清进行检测；血清/血浆样本12000rpm离心5min后取上清进行检测；组织(10mg)或细胞(1x 10 6个)，加入预冷检测缓冲液(生理盐水)100μL，在冰上匀浆10min，12000rpm离心5min，收集上清液进行检测。
3)标准曲线的制备
精密称定4.6mg黄嘌呤标准品至50mL容量瓶，纯水超声溶解并定容，得604.8μmol/L的黄嘌呤母液，母液依次稀释1，2，4，8，16，32，64倍，制成X1-X6水标，浓度范围为9.45-604.8μM。
二、生化分析仪检测
生化分析仪参数设置
表9
  样本/试剂体积(μL) 稀释体积
样本 5.6 0
R6-1 48 72
R6-2 20 10
以X1-X6为检测点，在贝克曼AU480生化分析仪中制作多点法标曲，发射波长和激发波长分别为660nm和800nm。标准曲线见图8。
三、黄嘌呤、次黄嘌呤、腺嘌呤和鸟嘌呤标准品单标溶液的测定
配制终浓度分别为50、150、300、600μmol/L的黄嘌呤标准品单标溶液；配制终浓度分别为50、150、
300、600μmol/L的次黄嘌呤标准品单标溶液；
配制终浓度分别为50、150、300、600μmol/L的腺嘌呤标准品单标溶液；配制终浓度分别为50、150、
300、600μmol/L的鸟嘌呤标准品单标溶液；
分别吸取纯水或各标准品单标溶液50μL加入R6-1试剂500μL，混匀静置3min，再加入R6-2试剂200μL，混匀静置10min，观察颜色。见图14，图14(a)从左到右依次为0、50、150、300、600μmol/L；图14(b)从左到右依次为0、50、150、300、600μmol/L；图14(c)从左到右依次为0、50、150、300、600μmol/L；图14(d)从左到右依次为0、50、150、300、600μmol/L。
实施例7.酶标仪用试剂盒
试剂盒组成(100T)：检测缓冲液R7-1共25mL；试剂R7-2、R7-3、R7-4各1瓶；黄嘌呤标准品(1μM)1瓶。
表10
试剂7-1 试剂7-2(粉末) 试剂7-3(粉末) 试剂7-4(粉末)
10g/L磷酸氢二钾 0.0003KU XOD 0.0008g TBHBA 0.0007g 4-AAP
1.5g/L OA 0.065KU POD    
8mL/L丙三醇 0.018KU抗坏血酸酶    
15g/L聚乙二醇6000 0.0054KU胆红素氧化酶    
5g/L山梨醇      
15g/L葡萄糖      
3g/L BSA      
2g/L Twwen-20      
0.02g/LProclin 300      
配制方法：按上述配方称取相应物质，以25mL纯水定容至相应浓度，得试剂R7-1；分别用220μL试剂R7-1溶解7-2、7-3和7-4配制得到试剂R7-2、R7-3、R7-4(现配现用)
样本制备：尿液样本12000rpm离心5min直接使用；血清/血浆样本12000rpm离心5min后备用；组织(10mg)或细胞(1x 10 6个)，加入预冷检测缓冲液(试剂7-1)100μL，在冰上匀浆10min。12000rpm离心5min，收集上清液备用。加入5μL的样本至每孔，采用缓冲液补足至50μL。
黄嘌呤标曲制作：500μL蒸馏水溶解1μM的黄嘌呤标准品，至黄嘌呤标准品浓度为2.0mM(2.0nmol/μl)。在96孔板上分别加入0、2、4、6、8和10μL含浓度为2mM黄嘌呤的标准品，使每孔含黄嘌呤终浓度为0、4、8、12、16和20nmol/孔，采用检测缓冲液补足至每孔50μL。
检测：在含标准品的小孔内，加入50μL反应液(试剂在反应前需充分混合，包括：44μL的R7-1试剂，2μL的R7-2试剂，2μL的R7-3试剂，2μL的R7-4试剂)，充分混合。室温，避光孵育30min，酶标仪在(发射波长和激发波长分别为520nm和550nm)处测定吸光度。
得到的标准曲线见图9。
实施例8.紫外可见分光光度计用试剂盒
试剂盒组成(100T)：检测缓冲液R8-1共200mL；试剂R8-2、R8-3、R8-4各1瓶；黄嘌呤标准品(20μM)1瓶。
表11
试剂8-1 试剂8-2(粉末) 试剂8-3(粉末) 试剂8-4(粉末)
15g/L磷酸氢二钾 0.006KU XOD 0.012g MADB 0.012g 4-AAP
3g/L OA 1.3KU POD    
4mL/L丙三醇 0.36KU抗坏血酸酶    
5g/L聚乙二醇6000 0.108KU胆红素氧化酶    
6mL/L乙二醇      
25g/L甘露醇      
15g/L海藻糖      
1g/L BSA      
1g/L烷基糖苷(APG)      
0.5g/L防腐剂NaN 3      
配制方法：按上述配方称取相应物质，以200mL纯水定容至相应浓度，得试剂R8-1；分别用5mL试剂R8-1溶解8-2、8-3和8-4配制试剂R8-2、R8-3、R8-4。
2、样本制备：尿液样本12000rpm离心5min直接使用；血清/血浆样本12000rpm离心5min后备用；组织(10mg)或细胞(1x 10 6)，加入预冷检测缓冲液(试剂8-1)100μL，在冰上匀浆10min。12000rpm离心5min，收集上清液备用。加入100μL的样本，采用缓冲液补足至1mL。
3、黄嘌呤标曲制作：10mL蒸馏水溶解20μM的黄嘌呤标准品，该黄嘌呤标准品浓度为2.0mM(2.0nmol/μl)。在2.5mL离心管中分别加入0、40、80、120、160和200μL的含2mM黄嘌呤的标准品，使每管含黄嘌呤终浓度为0、80、160、240、320和400nmol/μL，采用检测缓冲液补足至每管1mL。
4、标准曲线的制作：在含标准品的小孔内，加入1mL反应液(反应液中试剂在反应前需充分混合，包括：880μL的R8-1试剂，40μL的R8-2试剂，40μL的R8-3试剂，40μL的R8-4试剂)，充分混合，室温，避光孵育30min。紫外可见分光光度计(发射波长和激发波长分别为660nm和800nm)测定吸光度。得到的标曲见图10。
实施例9.动物给药非布司他后，样本中黄嘌呤及次黄嘌呤总含量检测
1.血液样本及肾脏组织的采集：取大鼠若干只，灌胃给药非布司他，给药剂量6mg/kg/天，给药周期为7天，于第7天灌胃给药1h后麻醉，腹主动脉取血，进一步解剖采集肾脏样本
2.尿液样本的采集：取大鼠若干只，灌胃给药非布司他，给药剂量6mg/kg/天，给药周期为7天，于第7天灌胃给药后，迅速将大鼠置于大鼠代谢笼内，收集给药后0-6h及6-24h的尿液
3.样本制备：尿液/血液样本离心后备用。肾脏样本按照组织10mg，加入预冷检测缓冲液100μL，在冰上匀浆10min。12000rpm离心5min，收集上清液备用。
检测：按实施例3操作获得目测法结果；按实施例4操作获得棉签法检测结果；按实施例5操作获得试纸法检测结果；按实施例6操作获得生化分析仪数据；按实施例7操作获得酶标仪数据；按实施例8操作获得紫外可见分光光度仪数据；HPLC检测：安捷伦1260，C18柱(4.6×250mm，5μm)；流动相由流动相A和流动相B组成，流动相A为10mM乙酸铵，流动相B为甲醇，流动相A与流动相B的体积比为98：2，运行时间20min，流速：1mL/min，检测波长：270nm。
4.黄嘌呤及次黄嘌呤总含量结果对比，见下表；肾脏样本黄嘌呤及次黄嘌呤总含量结果见下表，及图11，尿液样本黄嘌呤及次黄嘌呤总含量结果对比见下表，及图12。结果证明：血液样本的生化分析仪数据、酶标仪数据和HPLC数据没有统计学差异(P<0.05)；肾脏样本紫外可见分光光度计用试剂盒测定数据和HPLC数据没有统计学差异；尿液样本试纸法、棉签法趋势相同，生化分析仪数据和HPLC数据没有统计学差异(P<0.05)。
表12
血液样本 生化分析仪数据 酶标仪数据 HPLC数据
Co-1 17.8μM 17.2μM 18.4μM
Co-2 23.1μM 23.6μM 23.5μM
Feb-1 77.5μM 78.6μM 80.6μM
Feb-2 72.9μM 73.4μM 75.8μM
Feb-3 69.4μM 69.2μM 72.9μM
表13
Figure PCTCN2021074330-appb-000005
实施例10.试剂盒(酶标仪定量)稳定性对比试验
对实施例7配方试剂，均匀分装13组，并取13组商品化黄嘌呤/次黄嘌呤检测试剂盒(sigma公司，产品代码MAK186)作为对照；放置到2-8℃冰箱中，每月的同一天取出一组试剂检测黄嘌呤标准品溶液(靶值100μM/L)，检测结果显示，实施例7配方试剂在2-8℃储存条件下比市场常见的黄嘌呤/次黄嘌呤检测试剂盒更加稳定。结果见图13。
对比例1
样本处理：取正常尿液添加黄嘌呤标准溶液，使黄嘌呤在尿液中的终浓度分别为500μM和1000μM，在该尿液中添加活性炭，添加量0.5g/mL，混匀，离心取滤液HPLC检测，每个浓度各平行10份，HPLC检测结果见下表：
表4
实验组序号 处理前 1 2 3 4 5 6 7 8 9 10
浓度(单位μM) 500 103 86 70 140 89 93 110 96 124 108
浓度黄嘌呤 1000 337 258 285 261 207 167 199 223 186 460
上表表明，活性炭随机吸附黄嘌呤，没有规律性，不能实现定量去除，不同浓度活性炭添加量结果一致。

Claims (25)

  1. 一种检测或辅助检测嘌呤类物质的试剂盒，其特征在于，包括试剂1、试剂2和试剂3，试剂1为显色剂，试剂2为反应酶，试剂3为弱酸盐；所述反应酶包括过氧化物酶和黄嘌呤氧化酶。
  2. 根据权利要求1所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，所述试剂盒还包括维生素C和抗坏血酸酶；所述维生素C和抗坏血酸酶分开包装。
  3. 根据权利要求1或2任一所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，所述显色剂由显色剂A和显色剂B组成，显色剂A为4-AAP，显色剂B为MADB或TBHBA；可选的，所述显色剂A和显色剂B均独立包装。
  4. 根据权利要求1-3任一所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，所述反应酶独立包装；所述弱酸盐为磷酸氢二钾或磷酸二氢钾。
  5. 根据权利要求4所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，所述试剂盒中，过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA为20-600：0.1-15：0.1-5：0.1-10，比例关系为KU：KU：g：g。
  6. 根据权利要求4所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶为20-1300：0.1-18：0.1-5：0.1-10：1-12：1-360，比例关系为：KU：KU：g:g:mM：KU；
    或，磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶为0.2-35：20-1300：0.1-18：0.1-5：0.1-10：1-12：1-360，比例关系为：g：KU：KU：g:g:mM：KU。
  7. 根据权利要求1-6任一所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，所述试剂盒中还包括稳定剂、表面活性剂和防腐剂中的一种或两种或三种。
  8. 根据权利要求7所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，
    稳定剂选自BSA、醇类或糖类中的一种或几种；可选的，醇类为丙三醇、甘露醇、乙二醇、聚乙二醇6000、山梨醇中的任一一种或几种；可选的，糖类为海藻糖、葡萄糖、蔗糖中的任意一一种或几种；表面活性剂选自Triton X-100、Twwen-20或烷基糖苷中的任一一种或几种；防腐剂为NaN 3或Proclin 300。
  9. 根据权利要求1-8任一所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，所述试剂盒还包括尿酸氧化酶抑制剂；可选的所述尿酸氧化酶抑制剂为氧嗪酸钾；
    可选的，所述试剂盒还包括胆红素氧化酶；
    可选的，所述试剂盒还包括独立包装的尿酸氧化酶。
  10. 根据权利要求1-9任一所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶:氧嗪酸钾为20-1300：0.1-18：0.1-5：0.1-10：1-12：1-360：0.5-5，比例关系为：KU：KU：g:g:mM：KU：g；
    磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶:氧嗪酸钾为0.2-45：20-1300：0.1-18：0.1-5：0.1-10：1-12：1-360：0.5-5，比例关系为：g：KU：KU：g:g:mM：KU：g；
    可选的，磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：维生素C：抗坏血酸酶:氧嗪酸钾：胆红素氧化酶为0.2-35：20-600：0.1-18：0.1-5：0.1-10：1-12：1-360：1-4：1-108，比例关系为：g：KU：KU：g:g:mM：KU：g：KU；
    可选的，磷酸氢二钾：过氧化物酶：黄嘌呤氧化酶：4-AAP：MADB或TBHBA：尿酸氧化酶为0.2-45：20-1300：0.1-18：0.1-5：0.1-10：30，比例关系为：g：KU：KU：g:g:KU。
  11. 根据权利要求1-7任一所述检测或辅助检测嘌呤类物质的试剂盒，其特征在于，所述盒还包括辅料，所述辅料选自乳糖、柠檬酸、碳酸氢钠、羧甲基淀粉钠、PVP-K30、PEG6000和微粉硅胶中的一种或几种。
  12. 一种检测或辅助检测嘌呤类物质的试剂盒，其特征在于，所述试剂盒包括试纸或棉签；所述试纸或棉签上含有权利要求1-11任一所述试剂盒中的试剂。
  13. 根据权利要求12所述的所述试剂盒，其特征在于，所述试剂盒还包括标准比色卡和/或黄嘌呤标准品。
  14. 一种制备权利要求1-13任一所述检测或辅助检测嘌呤类物质的试剂盒的方法，其特征在于，包括根据权利要求1-13所述试剂盒称取各个成分，用水溶解后定容，制备为溶液，分装的步骤；
    或，包括根据权利要求1-13所述试剂盒称取各个成分，制备成目标剂型的步骤。
  15. 一种检测或辅助检测嘌呤类物质的系统，所述系统包括权利要求1-13任一所述的试剂盒或权利要求14所述方法制备得到的试剂盒。
  16. 根据权利要求15所述的系统，其特征在于，所述系统还包括生化分析仪、酶标仪或紫外可见分光光度计。
  17. 权利要求1-13任一所述的试剂盒，权利要求14所述方法制备得到的试剂盒，权利要求15或16所述系统在检测或者辅助检测待测样本中的嘌呤类物质中的应用。
  18. 一种检测或辅助检测待测样品中的嘌呤类物质的方法，其特征在于，包括用1-13任一所述的试剂盒或权利要求14所述方法制备得到的试剂盒或权利要求15或16所述系统对待测样本进行检测的步骤；所述方法为非疾病诊断方法。
  19. 一种检测或辅助检测待测样品中的嘌呤类物质的方法，其特征在于，具体步骤为：
    1)将待测样本与权利要求1-13任一所述试剂盒中的试剂混合，形成混合液；
    2)观察混合液的颜色，判断待测样本中嘌呤类物质的含量高低。
  20. 根据权利要求19所述的方法，其特征在于，所述待测样本为食品、血液、尿液、汗液、唾液、泪液或组织；所述血液、尿液、汗液、唾液、泪液或组织来自人或动物。
  21. 根据权利要求20所述的方法，其特征在于，待测样本为血液、尿液、汗液、唾液、泪液或组织；所述血液、尿液、汗液、唾液、泪液或组织来自实验动物；还包括加尿酸氧化酶抑制剂；可选的所述尿酸氧化酶抑制剂为氧嗪酸钾。
  22. 根据权利要求19或20所述的方法，其特征在于，待测样品为食品时，还包括用纯化试剂对待测样品进行预处理的步骤。
  23. 一种检测或辅助检测待测样品中的嘌呤类物质的方法，其特征在于，具体步骤为：
    将待测样本与权利要求12或13所述试剂盒中的试纸或棉签接触，根据试纸或者棉签的颜色变化，判断待测样本中黄嘌呤和次黄嘌呤的浓度大小；
    或，将待测样本与权利要求12或13所述试剂盒中的试纸或棉签接触，与标准比色卡比较，判断待测样本中黄嘌呤和次黄嘌呤的浓度大小。
  24. 一种检测或辅助检测待测样品中的嘌呤类物质的方法，其特征在于，具体步骤为：
    (b1)绘制标准曲线
    配制系列浓度的嘌呤类物质标准品溶液，将所得的若干份含有不同浓度的标准品溶液分别与权利要求1-13任一所述试剂盒中的试剂混合，形成混合液；然后将所述混合液室温孵育30分后，使用酶标仪、生化分析仪或紫外可见分光光度计测定混合液的吸光度；以所述嘌呤类物质的标准品溶液的浓度为横坐标，以所述混合液的吸光吸光度的为纵坐标绘制标准曲线图，得到标准曲线方程；
    (b2)待测样本检测
    将所述待测样本与权利要求1-13任一所述试剂盒中的试剂混合，得到混合液；使用酶标仪、生化分析仪或紫外可见分光光度计测定混合液的吸光度，将所述待测样本的吸光度的值代入步骤b1)所得标准曲线方程，计算得到所述待测样本中嘌呤类物质的浓度值。
  25. 根据权利要求1-13任一所述的试剂盒，权利要求14所述方法，权利要求15或16所述系统，权利要求17-24任一所述方法，其特征在于，所述嘌呤类物质为黄嘌呤及其衍生物、次黄嘌呤及其衍生物、腺嘌呤及其衍生物、鸟嘌呤及其衍生物。
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CN106282309A (zh) * 2016-08-31 2017-01-04 广东省中医院 一种腺苷脱氨酶检测方法
CN106855508A (zh) * 2017-01-06 2017-06-16 北京物资学院 一种检测鱼中次黄嘌呤含量的方法
CN111662955A (zh) * 2020-07-26 2020-09-15 武汉中太生物技术有限公司 一种腺苷脱氨酶测定试剂盒

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CN106282309A (zh) * 2016-08-31 2017-01-04 广东省中医院 一种腺苷脱氨酶检测方法
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