CN112986584A - Method for making total bilirubin determination reagent kit - Google Patents
Method for making total bilirubin determination reagent kit Download PDFInfo
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- CN112986584A CN112986584A CN202110200924.6A CN202110200924A CN112986584A CN 112986584 A CN112986584 A CN 112986584A CN 202110200924 A CN202110200924 A CN 202110200924A CN 112986584 A CN112986584 A CN 112986584A
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 84
- 238000008050 Total Bilirubin Reagent Methods 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 13
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 81
- 239000000758 substrate Substances 0.000 claims abstract description 40
- 239000007853 buffer solution Substances 0.000 claims abstract description 39
- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229910000166 zirconium phosphate Inorganic materials 0.000 claims abstract description 23
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims abstract description 22
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims abstract description 22
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims abstract description 22
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 22
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 22
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 22
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims abstract description 22
- 238000002156 mixing Methods 0.000 claims abstract description 21
- 238000004519 manufacturing process Methods 0.000 claims abstract description 17
- 239000004094 surface-active agent Substances 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- 102000012011 Isocitrate Dehydrogenase Human genes 0.000 claims abstract description 13
- 108010075869 Isocitrate Dehydrogenase Proteins 0.000 claims abstract description 13
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims abstract description 13
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims abstract description 13
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 13
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 13
- 239000005515 coenzyme Substances 0.000 claims abstract description 13
- 239000003381 stabilizer Substances 0.000 claims abstract description 13
- 102000004190 Enzymes Human genes 0.000 claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 9
- 238000003756 stirring Methods 0.000 claims abstract description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 18
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 18
- 229940088598 enzyme Drugs 0.000 claims description 10
- 108010015776 Glucose oxidase Proteins 0.000 claims description 6
- 239000004366 Glucose oxidase Substances 0.000 claims description 6
- 239000012295 chemical reaction liquid Substances 0.000 claims description 6
- 229940116332 glucose oxidase Drugs 0.000 claims description 6
- 235000019420 glucose oxidase Nutrition 0.000 claims description 6
- 238000003149 assay kit Methods 0.000 claims description 5
- 101710130006 Beta-glucanase Proteins 0.000 claims description 3
- 108010059892 Cellulase Proteins 0.000 claims description 3
- 108010084185 Cellulases Proteins 0.000 claims description 3
- 102000005575 Cellulases Human genes 0.000 claims description 3
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 3
- 229940106157 cellulase Drugs 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims 1
- 239000007979 citrate buffer Substances 0.000 claims 1
- 230000002255 enzymatic effect Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 24
- 230000009286 beneficial effect Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 4
- 238000006701 autoxidation reaction Methods 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000000536 complexating effect Effects 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- GWZYPXHJIZCRAJ-UHFFFAOYSA-N Biliverdin Natural products CC1=C(C=C)C(=C/C2=NC(=Cc3[nH]c(C=C/4NC(=O)C(=C4C)C=C)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O GWZYPXHJIZCRAJ-UHFFFAOYSA-N 0.000 description 1
- RCNSAJSGRJSBKK-NSQVQWHSSA-N Biliverdin IX Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(\C=C/2C(=C(C)C(=C/C=3C(=C(C=C)C(=O)N=3)C)/N\2)CCC(O)=O)N1 RCNSAJSGRJSBKK-NSQVQWHSSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- QBUVFDKTZJNUPP-UHFFFAOYSA-N biliverdin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(C=C2C(=C(C)C(C=C3C(=C(C=C)C(=O)N3)C)=N2)CCC(O)=O)N1 QBUVFDKTZJNUPP-UHFFFAOYSA-N 0.000 description 1
- BPYKTIZUTYGOLE-UHFFFAOYSA-N billirubin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(C=C3C(=C(C=C)C(=O)N3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-UHFFFAOYSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/728—Bilirubin; including biliverdin
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention is applicable to the technical field of reagent kit preparation, and provides a method for manufacturing a total bilirubin determination reagent kit, which comprises the following steps: A. preparing a reagent 1, wherein the reagent 1 comprises a citric acid buffer solution, a surfactant and triton, and the components of the citric acid buffer solution comprise isocitrate dehydrogenase, lactate dehydrogenase, a stabilizer and coenzyme; B. preparing a single reagent, wherein the single reagent comprises disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium vanadate and disodium ethylene diamine tetraacetate; C. stirring and mixing the citric acid buffer solution and the single reagent, uniformly mixing, keeping the temperature for 3-10 minutes at 37 ℃, adding the enzyme reagent, and uniformly mixing to obtain a reaction solution; D. the reaction solution is poured into a reagent kit, and a substrate reagent is loaded in the reagent kit, wherein the substrate reagent comprises an oxidase substrate and a substrate buffer solution. Therefore, the invention can eliminate the influence of the turbidity of the fat on the detection, thereby improving the detection accuracy.
Description
Technical Field
The invention relates to the technical field of preparation of kits, in particular to a manufacturing method of a total bilirubin determination kit.
Background
Total bilirubin is a general term of combined bilirubin and unconjugated bilirubin produced by heme metabolism in human bodies, abnormal high total bilirubin is often found in liver and gall diseases, and in recent years, some researches show that bilirubin in blood lower than normal value is often related to coronary heart disease. Therefore, the determination of total bilirubin in serum, plasma or urine of a human body is one of the most common items in medical testing. The total bilirubin in the sample is oxidized into biliverdin by sodium vanadate serving as an oxidant under the conditions that the pH of the reagent is about 3.00 and a surfactant and an accelerator exist, the change before and after absorbance is measured, and the content of the total bilirubin in the sample is calculated.
The conventional method for measuring bilirubin adopts a diazo reagent method, which has long history and accurate measurement, and has the main defects of poor reagent stability, poor reaction specificity and the like, and although countless improvements are made, no satisfactory result is obtained so far. The enzymatic method for determining total bilirubin has the characteristic of specificity, but the kit is not beneficial to popularization and application due to poor enzyme thermal stability, short preservation time and overhigh price.
The existing kit has inaccurate measuring value when detecting a sample with serious fat turbidity. At present, the content of fat in diet of people is high, and blood is not always fasting every night, so the probability of fat turbidity in a serum sample is more and more, the blank of the sample is higher, and the bilirubin determination is influenced.
In view of the above, the prior art is obviously inconvenient and disadvantageous in practical use, and needs to be improved.
Disclosure of Invention
In view of the above-mentioned drawbacks, an object of the present invention is to provide a method for manufacturing a total bilirubin determination kit, which can eliminate the influence of turbidity on the detection and thereby improve the accuracy of the detection.
In order to achieve the above object, the present invention provides a method for manufacturing a total bilirubin assay kit, comprising the steps of:
A. preparing a reagent 1, wherein the reagent 1 comprises a citric acid buffer solution, a surfactant and triton, the components of the citric acid buffer solution comprise isocitrate dehydrogenase, lactate dehydrogenase, a stabilizer and coenzyme, and the ratio of the isocitrate dehydrogenase to the lactate dehydrogenase to the stabilizer to the coenzyme is 200:180:3: 1;
B. preparing a single reagent, wherein the single reagent comprises disodium hydrogen phosphate, monopotassium phosphate, sodium vanadate and disodium ethylene diamine tetraacetate, and the ratio of the disodium hydrogen phosphate to the monopotassium phosphate to the sodium vanadate to the disodium ethylene diamine tetraacetate is 1:4:7: 2;
C. stirring and mixing the citric acid buffer solution and the single reagent, uniformly mixing, keeping the temperature for 3-10 minutes at 37 ℃, adding the enzyme reagent, and uniformly mixing to obtain a reaction solution;
D. pouring the reaction liquid into a reagent box, wherein a substrate reagent is loaded in the reagent box, the substrate reagent comprises an oxidase substrate and a substrate buffer solution, and the ratio of the oxidase substrate to the substrate buffer solution is 12: 7.
According to the manufacturing method of the total bilirubin determination kit, the pH value of the citric acid buffer solution is 2.6-3.4, the content of the surfactant is 0.2-1.2 g/L, the content of the triton is 100, and the content of the triton is 1.5-7.5 ml/L.
According to the manufacturing method of the total bilirubin determination kit, the content of disodium hydrogen phosphate is 1-5.5 g/L, the content of monopotassium phosphate is 0.25-1.5 g/L, the content of sodium vanadate is 0.15-0.7 g/L, and the content of disodium ethylene diamine tetraacetate is 0.2-1.0 g/L.
According to the manufacturing method of the total bilirubin determination kit, the enzyme reagent comprises 20% -30% (by weight) of cellulase and 1% -4% (by weight) of glucose oxidase.
According to the manufacturing method of the total bilirubin determination kit, the glucose oxidase comprises 30% of endo-cellulase, 45% of neutral xylanase and 25% of beta-glucanase.
According to the manufacturing method of the total bilirubin determination kit, the pH value of the citric acid buffer solution is 3.0, the content of the surfactant is 0.7g/L, the triton is 100, and the content of the triton is 5.2 ml/L.
According to the manufacturing method of the total bilirubin determination kit, the content of disodium hydrogen phosphate is 3.5g/L, the content of monopotassium phosphate is 0.86g/L, the content of sodium vanadate is 0.43g/L, and the content of disodium ethylene diamine tetraacetate is 0.67 g/L.
The invention provides a method for preparing a total bilirubin determination kit, which comprises the following steps:
A. preparing a reagent 1, wherein the reagent 1 comprises a citric acid buffer solution, a surfactant and triton, the components of the citric acid buffer solution comprise isocitrate dehydrogenase, lactate dehydrogenase, a stabilizer and coenzyme, and the ratio of the isocitrate dehydrogenase to the lactate dehydrogenase to the stabilizer to the coenzyme is 200:180:3: 1;
B. preparing a single reagent, wherein the single reagent comprises disodium hydrogen phosphate, monopotassium phosphate, sodium vanadate and disodium ethylene diamine tetraacetate, and the ratio of the disodium hydrogen phosphate to the monopotassium phosphate to the sodium vanadate to the disodium ethylene diamine tetraacetate is 1:4:7: 2;
C. stirring and mixing the citric acid buffer solution and the single reagent, uniformly mixing, keeping the temperature for 3-10 minutes at 37 ℃, adding the enzyme reagent, and uniformly mixing to obtain a reaction solution;
D. pouring the reaction liquid into a reagent box, wherein a substrate reagent is loaded in the reagent box, the substrate reagent comprises an oxidase substrate and a substrate buffer solution, and the ratio of the oxidase substrate to the substrate buffer solution is 12: 7.
The invention has the beneficial effects that: the reaction of total bilirubin is accelerated by utilizing a surfactant, the reaction rate is improved, the triton promotes various substances in a sample to be dissolved, the influence of turbidity of the sample on a measurement result is reduced, the oxidation reaction of bilirubin can be promoted, disodium hydrogen phosphate and potassium dihydrogen phosphate are favorable for the dispersion stability of various components in a reagent, sodium vanadate and disodium ethylene diamine tetraacetate can be used for complexing divalent metal ions in a test sample, the autoxidation of bilirubin in the sample is reduced, the influence of turbidity of the fat on detection is eliminated, and therefore the detection accuracy is improved.
Detailed Description
The present invention will be described in further detail with the aim of making the objects, technical solutions and advantages thereof more apparent, and it should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.
The invention provides a method for preparing a total bilirubin determination kit, which comprises the following steps:
A. preparing a reagent 1, wherein the reagent 1 comprises a citric acid buffer solution, a surfactant and triton, the components of the citric acid buffer solution comprise isocitrate dehydrogenase, lactate dehydrogenase, a stabilizer and coenzyme, and the ratio of the isocitrate dehydrogenase to the lactate dehydrogenase to the stabilizer to the coenzyme is 200:180:3: 1;
B. preparing a single reagent, wherein the single reagent comprises disodium hydrogen phosphate, monopotassium phosphate, sodium vanadate and disodium ethylene diamine tetraacetate, and the ratio of the disodium hydrogen phosphate to the monopotassium phosphate to the sodium vanadate to the disodium ethylene diamine tetraacetate is 1:4:7: 2;
C. stirring and mixing the citric acid buffer solution and the single reagent, uniformly mixing, keeping the temperature for 3-10 minutes at 37 ℃, adding the enzyme reagent, and uniformly mixing to obtain a reaction solution;
D. pouring the reaction liquid into a reagent box, wherein a substrate reagent is loaded in the reagent box, the substrate reagent comprises an oxidase substrate and a substrate buffer solution, and the ratio of the oxidase substrate to the substrate buffer solution is 12: 7.
Preferably, the pH value of the citric acid buffer solution is 2.6-3.4, the content of the surfactant is 0.2-1.2 g/L, the triton is 100, the content of the triton is 1.5-7.5 ml/L, and the pH value of the citric acid buffer solution (pH 2.6-3.4) is too high to promote the bilirubin to undergo autooxidation, so that the reaction pH value cannot be too low. The surfactant is 0.2-1.2 g/L to accelerate the reaction of the total bilirubin and improve the reaction rate. The triton is 1001.5-7.5 ml/L to promote the dissolution of various substances in a sample, reduce the influence of sample lipid turbidity and the like on a measurement result, and promote the oxidation reaction of bilirubin.
In addition, the content of the disodium hydrogen phosphate is 1-5.5 g/L, the content of the monopotassium phosphate is 0.25-1.5 g/L, the content of the sodium vanadate is 0.15-0.7 g/L, the content of the disodium ethylene diamine tetraacetate is 0.2-1.0 g/L, the content of the disodium hydrogen phosphate is 1-5.5 g/L, 0.25-1.5 g/L of monopotassium phosphate is beneficial to the dispersion stability of various components in the reagent, the content of the sodium vanadate is 0.15-0.7 g/L, and the content of the disodium ethylene diamine tetraacetate is 0.5g/L, so that divalent metal ions in a test sample can be complexed, and the autoxidation of bilirubin in the sample is reduced.
Further, the enzyme reagent comprises 20-30% by weight of cellulase and 1-4% by weight of glucose oxidase, hydrolysis of part of starchy polysaccharide and non-starchy polysaccharide in the raw materials is realized by using the enzyme reagent, carbohydrate beneficial to the fermentation process is obtained, the biological fermentation level is improved, and residues of soluble polysaccharide and monosaccharide are effectively reduced, so that COD (chemical oxygen demand) in the wastewater is reduced.
Preferably, the citric acid buffer solution has a pH value of 3.0, the surfactant content of 0.7g/L, the triton is 100, and the triton content is 5.2ml/L, so that various substances in a sample can be effectively dissolved, the influence of sample lipid turbidity and the like on a measurement result can be reduced, and the oxidation reaction of bilirubin can be promoted.
The invention provides a method for preparing a total bilirubin determination kit, which comprises the following steps:
A. preparing a reagent 1, wherein the reagent 1 comprises a citric acid buffer solution, a surfactant and triton, the components of the citric acid buffer solution comprise isocitrate dehydrogenase, lactate dehydrogenase, a stabilizer and coenzyme, and the ratio of the isocitrate dehydrogenase to the lactate dehydrogenase to the stabilizer to the coenzyme is 200:180:3: 1;
B. preparing a single reagent, wherein the single reagent comprises disodium hydrogen phosphate, monopotassium phosphate, sodium vanadate and disodium ethylene diamine tetraacetate, and the ratio of the disodium hydrogen phosphate to the monopotassium phosphate to the sodium vanadate to the disodium ethylene diamine tetraacetate is 1:4:7: 2;
C. stirring and mixing the citric acid buffer solution and the single reagent, uniformly mixing, keeping the temperature for 3-10 minutes at 37 ℃, adding the enzyme reagent, and uniformly mixing to obtain a reaction solution;
D. pouring the reaction liquid into a reagent box, wherein a substrate reagent is loaded in the reagent box, the substrate reagent comprises an oxidase substrate and a substrate buffer solution, and the ratio of the oxidase substrate to the substrate buffer solution is 12: 7. The glucose oxidase comprises 30% of endo-cellulase, 45% of neutral xylanase and 25% of beta-glucanase. The content of the disodium hydrogen phosphate is 3.5g/L, the content of the monopotassium phosphate is 0.86g/L, the content of the sodium vanadate is 0.43g/L, and the content of the disodium ethylene diamine tetraacetate is 0.67 g/L.
In summary, the invention provides a method for preparing a total bilirubin determination kit, which comprises the following steps:
A. preparing a reagent 1, wherein the reagent 1 comprises a citric acid buffer solution, a surfactant and triton, the components of the citric acid buffer solution comprise isocitrate dehydrogenase, lactate dehydrogenase, a stabilizer and coenzyme, and the ratio of the isocitrate dehydrogenase to the lactate dehydrogenase to the stabilizer to the coenzyme is 200:180:3: 1;
B. preparing a single reagent, wherein the single reagent comprises disodium hydrogen phosphate, monopotassium phosphate, sodium vanadate and disodium ethylene diamine tetraacetate, and the ratio of the disodium hydrogen phosphate to the monopotassium phosphate to the sodium vanadate to the disodium ethylene diamine tetraacetate is 1:4:7: 2;
C. stirring and mixing the citric acid buffer solution and the single reagent, uniformly mixing, keeping the temperature for 3-10 minutes at 37 ℃, adding the enzyme reagent, and uniformly mixing to obtain a reaction solution;
D. pouring the reaction liquid into a reagent box, wherein a substrate reagent is loaded in the reagent box, the substrate reagent comprises an oxidase substrate and a substrate buffer solution, and the ratio of the oxidase substrate to the substrate buffer solution is 12: 7.
The invention has the beneficial effects that: the reaction of total bilirubin is accelerated by utilizing a surfactant, the reaction rate is improved, the triton promotes various substances in a sample to be dissolved, the influence of turbidity of the sample on a measurement result is reduced, the oxidation reaction of bilirubin can be promoted, disodium hydrogen phosphate and potassium dihydrogen phosphate are favorable for the dispersion stability of various components in a reagent, sodium vanadate and disodium ethylene diamine tetraacetate can be used for complexing divalent metal ions in a test sample, the autoxidation of bilirubin in the sample is reduced, the influence of turbidity of the fat on detection is eliminated, and therefore the detection accuracy is improved.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A method for manufacturing a total bilirubin determination kit is characterized by comprising the following steps:
A. preparing a reagent 1, wherein the reagent 1 comprises a citric acid buffer solution, a surfactant and triton, the components of the citric acid buffer solution comprise isocitrate dehydrogenase, lactate dehydrogenase, a stabilizer and coenzyme, and the ratio of the isocitrate dehydrogenase to the lactate dehydrogenase to the stabilizer to the coenzyme is 200:180:3: 1;
B. preparing a single reagent, wherein the single reagent comprises disodium hydrogen phosphate, monopotassium phosphate, sodium vanadate and disodium ethylene diamine tetraacetate, and the ratio of the disodium hydrogen phosphate to the monopotassium phosphate to the sodium vanadate to the disodium ethylene diamine tetraacetate is 1:4:7: 2;
C. stirring and mixing the citric acid buffer solution and the single reagent, uniformly mixing, keeping the temperature for 3-10 minutes at 37 ℃, adding the enzyme reagent, and uniformly mixing to obtain a reaction solution;
D. pouring the reaction liquid into a reagent box, wherein a substrate reagent is loaded in the reagent box, the substrate reagent comprises an oxidase substrate and a substrate buffer solution, and the ratio of the oxidase substrate to the substrate buffer solution is 12: 7.
2. The method for producing a total bilirubin assay kit according to claim 1, wherein the pH of the citric acid buffer is 2.6 to 3.4, the surfactant content is 0.2 to 1.2g/L, the triton is 100, and the triton content is 1.5 to 7.5 ml/L.
3. The method for producing a total bilirubin assay kit according to claim 1, wherein the content of disodium hydrogen phosphate is 1 to 5.5g/L, the content of monopotassium phosphate is 0.25 to 1.5g/L, the content of sodium vanadate is 0.15 to 0.7g/L, and the content of disodium ethylenediaminetetraacetate is 0.2 to 1.0 g/L.
4. The method for manufacturing the total bilirubin assay kit according to claim 1, wherein the enzymatic reagents include 20% -30% by weight cellulase and 1% -4% by weight glucose oxidase.
5. The method for manufacturing the total bilirubin determination kit according to claim 4, wherein the glucose oxidase includes 30% of an endo-cellulase, 45% of a neutral xylanase and 25% of a beta-glucanase.
6. The method of manufacturing a total bilirubin assay kit according to claim 2, wherein the pH of the citrate buffer is 3.0, the surfactant content is 0.7g/L, the triton is 100, and the triton content is 5.2 ml/L.
7. The method of manufacturing a total bilirubin determination kit according to claim 3, wherein the content of disodium hydrogen phosphate is 3.5g/L, the content of potassium dihydrogen phosphate is 0.86g/L, the content of sodium vanadate is 0.43g/L, and the content of disodium ethylenediaminetetraacetate is 0.67 g/L.
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| CN202110200924.6A Pending CN112986584A (en) | 2021-02-23 | 2021-02-23 | Method for making total bilirubin determination reagent kit |
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