CN101055271B - Enzyme method reagent kit for detecting DBil - Google Patents
Enzyme method reagent kit for detecting DBil Download PDFInfo
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- CN101055271B CN101055271B CN 200610025635 CN200610025635A CN101055271B CN 101055271 B CN101055271 B CN 101055271B CN 200610025635 CN200610025635 CN 200610025635 CN 200610025635 A CN200610025635 A CN 200610025635A CN 101055271 B CN101055271 B CN 101055271B
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 39
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 26
- 238000008715 DBil Methods 0.000 title claims description 6
- -1 Potassium ferricyanide Chemical compound 0.000 claims description 11
- 108010015428 Bilirubin oxidase Proteins 0.000 claims description 9
- BJMQOECFBIIVFN-UHFFFAOYSA-N N.[K].C(CCCCCCCCCCCCCCC)Cl Chemical compound N.[K].C(CCCCCCCCCCCCCCC)Cl BJMQOECFBIIVFN-UHFFFAOYSA-N 0.000 claims description 9
- 239000011734 sodium Substances 0.000 claims description 9
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 7
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 5
- 238000013016 damping Methods 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 claims description 3
- 239000007974 sodium acetate buffer Substances 0.000 claims description 3
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 3
- JYQXULADMIJVOA-UHFFFAOYSA-K trisodium 2-hydroxypropane-1,2,3-tricarboxylate 2-hydroxypropanoic acid Chemical compound [Na+].[Na+].[Na+].CC(O)C(O)=O.OC(CC([O-])=O)(CC([O-])=O)C([O-])=O JYQXULADMIJVOA-UHFFFAOYSA-K 0.000 claims description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims 1
- 238000002156 mixing Methods 0.000 claims 1
- 229950006238 nadide Drugs 0.000 claims 1
- LRWJZGCOPMDWFZ-UHFFFAOYSA-N phthalic acid;hydrochloride Chemical compound Cl.OC(=O)C1=CC=CC=C1C(O)=O LRWJZGCOPMDWFZ-UHFFFAOYSA-N 0.000 claims 1
- 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 abstract description 49
- 238000006243 chemical reaction Methods 0.000 abstract description 16
- 239000003112 inhibitor Substances 0.000 abstract description 6
- 239000002131 composite material Substances 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 2
- 238000008789 Direct Bilirubin Methods 0.000 abstract 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 16
- 230000008569 process Effects 0.000 description 13
- 235000009508 confectionery Nutrition 0.000 description 9
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 8
- 239000001103 potassium chloride Substances 0.000 description 8
- 235000011164 potassium chloride Nutrition 0.000 description 8
- 239000012954 diazonium Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-O diazynium Chemical compound [NH+]#N IJGRMHOSHXDMSA-UHFFFAOYSA-O 0.000 description 7
- 206010023126 Jaundice Diseases 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 4
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical group Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 210000000941 bile Anatomy 0.000 description 2
- 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 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000013329 compounding Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 235000010288 sodium nitrite Nutrition 0.000 description 2
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Chemical compound [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- WQEVDHBJGNOKKO-UHFFFAOYSA-K vanadic acid Chemical compound O[V](O)(O)=O WQEVDHBJGNOKKO-UHFFFAOYSA-K 0.000 description 2
- SCJLWMXOOYZBTH-BTVQFETGSA-N (2s,3s,4s,5r,6s)-6-[3-[2-[[3-[3-[(2s,3r,4s,5s,6s)-6-carboxy-3,4,5-trihydroxyoxan-2-yl]oxy-3-oxopropyl]-5-[(z)-(3-ethenyl-4-methyl-5-oxopyrrol-2-ylidene)methyl]-4-methyl-1h-pyrrol-2-yl]methyl]-5-[(z)-(4-ethenyl-3-methyl-5-oxopyrrol-2-ylidene)methyl]-4-meth Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(=O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@H](O2)C(O)=O)O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(=O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@H](O2)C(O)=O)O)N1 SCJLWMXOOYZBTH-BTVQFETGSA-N 0.000 description 1
- ZAJAQTYSTDTMCU-UHFFFAOYSA-N 3-aminobenzenesulfonic acid Chemical compound NC1=CC=CC(S(O)(=O)=O)=C1 ZAJAQTYSTDTMCU-UHFFFAOYSA-N 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 206010019755 Hepatitis chronic active Diseases 0.000 description 1
- 206010023129 Jaundice cholestatic Diseases 0.000 description 1
- 206010023138 Jaundice neonatal Diseases 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
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- 201000005267 Obstructive Jaundice Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- OMJQSUYLFOVYNZ-UHFFFAOYSA-N [Na].C(C1=CC=CC=C1)OO Chemical compound [Na].C(C1=CC=CC=C1)OO OMJQSUYLFOVYNZ-UHFFFAOYSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
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- 230000008901 benefit Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000739 chaotic effect Effects 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
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- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
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- 238000005286 illumination Methods 0.000 description 1
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- 229910000358 iron sulfate Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 229940071125 manganese acetate Drugs 0.000 description 1
- UOGMEBQRZBEZQT-UHFFFAOYSA-L manganese(2+);diacetate Chemical compound [Mn+2].CC([O-])=O.CC([O-])=O UOGMEBQRZBEZQT-UHFFFAOYSA-L 0.000 description 1
- HVHKMUMXERBUAN-IFADSCNNSA-N mesobilirubin IXalpha Chemical compound N1C(=O)C(CC)=C(C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C)C(=O)N\3)CC)N2)CCC(O)=O)N1 HVHKMUMXERBUAN-IFADSCNNSA-N 0.000 description 1
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- 230000000630 rising effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an enzyme method detection direct bilirubin kit, which accelerates reaction of the bilirubin by adding enzyme composite accelerant, ensures specificity of reaction by selecting composite inhibitor, and increases stability of reagent by adding enzyme protectant. The kit possesses better clinic application future.
Description
Technical field:
The invention belongs to the biological reagent technical field.Be specifically related to a kind of enzyme method reagent kit for detecting DBil.
Background technology:
Cholerythrin is an important evidence of judging jaundice clinically, also is the important indicator of liver function.The cholerythrin total amount increases, indirect bilirubin increases and shows: hemolytic anemia, incompatible blood transfusion, malignant disease, icterus neonatorum etc.Directly all increase with indirect bilirubin and show: acute icteric hepatitis, chronic active hepatitis, cirrhosis, toxic hepatitis can cause that the cholerythrin total amount increases, direct and indirect bilirubin all increases; The cholerythrin total amount increases, bilirubin direct increases and shows: intrahepatic and extrahepatic obstructive jaundice, cancer of pancreas, bile capillaries type hepatitis and other bile stasis of bloods syndrome that stagnates.
Normal serum total bilirubin concentration is 1.7～17.1 μ mol/L, and wherein bilirubin direct is lower than 3.4 μ mol/L.When total bilirubin reaches 34 μ mol/L, can find jaundice clinically; As the serum total bilirubin overrun and naked eyes are not seen jaundice, then be called jaundice occult, jaundice is most commonly in disease in the liver and gallbladder, but the other system disease also can occur.
Cholerythrin (bilirubin) is the metabolic product of protoheme, mainly transforms, drains through combination in liver; It can exist with three kinds of forms in serum: unconjugated bilirubin (BU, i.e. unconjugated bilirubin), combined with bilirubin (Bc: single glucuronic acid Bcm and two bilirubin glucuronide Bcc sum) and cholerythrin (the covalently bound cholerythrin of albumin).Cholerythrin is a kind of effective anti-oxidants, has the ability of catching oxygen radical, can protect lipid and lipoprotein not oxidized.Synergy is educated by it and interior other anti-oxidative defense system of human body; with the myocyte position that is present in ventricle after the albumin bound; stop this position to produce oxygen radical, scalable cardiac muscle cell's cholerythrin antioxidation activity, the protection ventricular muscle cell is not damaged by oxygen radical.Therefore when bilirubin concentration reduced, the danger of coronary heart disease increased.
The bilirubin direct method for measuring mainly contains diazonium method, enzyme process and vanadate oxidizing process.Diazo reagent method history at most, and is still most widely used so far.Most popular have J-G method and a M-E method.The former makes it to become green by redness under alkali condition at the diazonium cholerythrin of cholerythrin and diazo reagent reaction back generation.The transfer of spectrum has increased the sensitivity and the specificity of reaction, do not disturb substantially in green environment, but this change has increased the difficulty of robotization.Therefore adopt this method often have to manual operations.M-E method generates back direct color comparison (redness) at the diazonium cholerythrin, and formality is simple, and being easy to robotization is advantage, but poor specificity, the result of haemolysis sample is seriously disturbed.Because the diazonium method is with a long history, medically relevant bilirubinic various argumentations are all based on this law.But there is defective in it: first diazo reagent instability, and it must be generated by sodium nitrite and aminobenzenesulfonic acid temporarily, at most can only be with 2 days (general 6 hours) after the generation; It two is that the atopic of diazo reagent and a courage and straight courage is not good, surveys straight courage under certain condition, and courage participates in reaction between a part, if the reaction time prolongs, participation cause the inaccurate of measured value more.At above-mentioned 2 points, people did countless improvement, but did not still have satisfied result so far.And the operation of this method is complicated, reagent is unstable, the reaction is not single-minded.
In addition, chemical oxidization method promptly uses chemical oxidization method to survey bilirubinic origin very early, and the cholerythrin when just adopting the seventies potassium ferricyanide to remove titrimetry survey calcium disturbs, and also once has report to survey total courage with potassium ferricyanide method later on.But also all only rest in the methodological discussion both at home and abroad, and do not have actual application value.Japanese scholar To-kuda in 1993 delivers the vanadic acid oxidizing process and surveys serum mesobilirubin.This kit in 1998 has developed numerous users through clinical practice is very fast, and well received, thinks that linearity, specificity and enzyme process relevant of this method all reaches more satisfactory level.Bilirubinic tetrapyrrol(e) structure has reductibility, various theoretically low molecular inorganic oxidizer and have the various transition metals of oxidisability under certain condition can the oxidation cholerythrin, thereby can be used for measuring cholerythrin.But must guarantee the selectivity of reaction, also will prevent interference and subsidiary reaction that early, middle and late phase of oxidation reaction may occur hand and foot.This compounds also has benzylhydroperoxide sodium, sodium peroxydisulfate, manganese acetate (III), copper sulphate, iron sulfate etc. except that vanadic acid and sodium nitrite.Therefore developing these class methods also has wide space.The practicality of these class methods has obtained proof, but makes little progress.
From relatively above-mentioned, the enzymatic assays cholerythrin has simple, special characteristics.The enzyme process oxidizing process is better, linear broad, precision height, disturbed less.Ot-suji in 1988 reports a kind of new survey total, straight courage enzyme process, and the total courage of its effect is also passable, and straight courage is measured pH is dropped to 3.7, and uses CuSO4 to do the promoter of enzyme, but this method is not promoted yet.Nineteen ninety 1hara etc. is reported in during straight courage after neonate's phototherapy measures, and enzyme process is the result obviously raise than diazonium method.Reason is that the photobilirubin enzyme process that produces after the illumination can decompose by analysis, thereby measured value is higher, and the diazonium rule is not.This false rising take place chaotic when may cause differentiating neonatal physiology or pathological jaundice clinically.
Thereby, though at present enzyme process is surveyed the cholerythrin technology and can be entered clinical practice. go back imperfection, in the measuring process of bilirubin direct, exist reaction velocity slow excessively, prevent that the interference of indirect bilirubin is poor, the activity of enzyme fast or the like the defective that descends.
Summary of the invention:
Technical matters to be solved by this invention is to overcome above-mentioned weak point, and a kind of acceleration bilirubin direct reaction is provided, and the enzyme process that improves reagent stability detects the reagent of DBil.
The present invention is based on following principle:
(1) select for use effective substances to quicken the reaction of bilirubin direct.Wherein the compound accelerant of enzyme is HgCl2, Cetyl Chloride potassium ammonia, TrionX-405 (surfactant);
(2) inhibitor of the effective indirect bilirubin of increase is guaranteed the specificity of reacting.Available composite inhibitor is: potassium chloride, reduced diphosphopyridine nucleotide, the potassium ferricyanide etc.;
(3), added special enzymatic protective reagent, sweet mellow wine and Na for the stability of guarantee reagent
2CO
3
The invention provides a kind of enzyme method reagent kit for detecting DBil, this kit is made up of following reagent 1 and reagent 2:
Reagent 1:
Damping fluid 10-200mmol/L
HgCl
2 10-100mmol/L
Triton?x-405 0.01-20ml/L
Cetyl Chloride potassium ammonia 20-100mmol/L
Reagent 2:
Na
2CO
3 10-200mmol/L
NaHCO
3 10-200mmol/L
Bilirubin oxidase 2000-4000U/L
Reduced diphosphopyridine nucleotide 10-200mmol/L
KCl 10-500mmol/L
Sweet mellow wine 10-200mmol/L
proclin300 0.01-0.5ml/L
Potassium ferricyanide 10-200mmol/L
Damping fluid described in the mentioned reagent is: sodium citrate-lactic acid buffer; Glycocoll-hydrochloride buffer; Acetate-sodium acetate buffer.
The preparation method:
1. elder generation adds 80% deionized water;
2. add different agent dissolves in water;
3. reagent 1 mixes with the 3:1 ratio with 2, regulates required pH value;
4. add enzyme at last, add remaining water.
It is as follows that kit of the present invention detects effect: sodium citrate-lactic acid buffer; Glycocoll-hydrochloride buffer; Acetate-sodium acetate buffer;
(1) 0.1M HgCl
2Cetyl Chloride potassium ammonia, Triton x-405 are as the influence of compounding activation agent to reaction:
Activator is to the influence of reaction
| Concentration | Do not add activator and reach the reaction end required time | Add the compounding activation agent and reach the required time of reaction end |
| 19.1μmol/L | 5min | 1.8min |
| 31.4μmol/L | 13min | 2.1min |
| 58μmol/L | 24min | 3.2min |
| 80μmol/L | 30min | 3.9min |
(2) influence of inhibitor to reacting: the potassium chloride of 0.1M, reduced diphosphopyridine nucleotide, the potassium ferricyanide suppress bilirubin direct as composite inhibitor
Inhibitor is to the influence of reaction
(3) different enzymatic protective reagents are to the influence of bilirubin oxidase: the sweet mellow wine and the Na that add 0.1M
2CO
3Protective effect to enzyme
| Time | Live with the remaining enzyme of protective agent | Add 0.1mol/L sweet mellow wine 0.1mol/LNa 2CO 3Remaining enzyme is lived |
| 0 | 100％ | 100％ |
| 1 month | 95％ | 98％ |
| 3 |
80％ | 95％ |
| 6 |
50％ | 88％ |
| 12 |
20％ | 77％ |
Above-mentioned testing result shows that kit of the present invention has very strong protective effect to bilirubin oxidase; As time goes on; protective effect meeting to enzyme is more obvious, adds protectant reagent after 12 months and does not compare with not adding protectant reagent, and remaining enzyme motility rate raises 57%.
Description of drawings:
Fig. 1 be clinical relevance ratio.Ordinate is the diazonium method, and abscissa is an enzyme process.
Embodiment:
Example 1
Reagent 1
Sodium citrate 300mmol/L
Lactic acid 50mmol/L
HgCl
2 50mmol/L
Triton?x-405 5ml/L
Cetyl Chloride potassium ammonia 50mmol/L
Reagent 2
Na
2CO
3 200mmol/L
NaHCO
3 200mmol/L
Bilirubin oxidase 4000U/L
Reduced diphosphopyridine nucleotide 100mmol/L
KCl 20mmol/L
Sweet mellow wine 20mmol/L
proclin300 0.3ml/L
Potassium ferricyanide 50mmol/L
This example kit has been made clinical correlation relatively: the clinical sample of getting 30 routine variable concentrations is with 3 kinds of parallel detections of method
3 kinds of method test results
Example 2
Reagent 1
Glycocoll 100mmol/L
Hydrochloric acid 50mmol/L
HgCl2 50mmol/L
Triton?x-405 5ml/L
Cetyl Chloride potassium ammonia 50mmol/L
Reagent 2
Na
2CO
3 200mmol/L
NaHCO
3 200mmol/L
Bilirubin oxidase 4000U/L
Reduced diphosphopyridine nucleotide 100mmol/L
KCl 20mmol/L
Sweet mellow wine 20mmol/L
proclin300 0.3ml/L
Potassium ferricyanide 50mmol/L
Example 3
Reagent 1
Phthalic acid 100mmol/L
Hydrochloric acid 50mmol/L
HgCl2 50mmol/L
Triton?x-405 5ml/L
Cetyl Chloride potassium ammonia 50mmol/L
Reagent 2
Na
2CO
3 100mmol/L
NaHCO
3 100mmol/L
Bilirubin oxidase 4000U/L
Reduced diphosphopyridine nucleotide 100mmol/L
KCl 20mmol/L
Sweet mellow wine 20mmol/L
proclin300 0.4ml/L
Potassium ferricyanide 50mmol/L
Example 4
Reagent 1
Acetate 100mmol/L
Sodium acetate 100mmol/L
HgCl2 50mmol/L
Triton?x-405 5ml/L
Cetyl Chloride potassium ammonia 50mmol/L
Reagent 2
Na
2CO
3 200mmol/L
NaHCO
3 200mmol/L
Bilirubin oxidase 4000U/L
Reduced diphosphopyridine nucleotide 100mmol/L
KCl 20mmol/L
Sweet mellow wine 20mmol/L
proclin300 0.3ml/L
Potassium ferricyanide 50mmol/L
Example 5
Reagent 1
Acetate 200mmol/L
Sodium acetate 150mmol/L
HgCl
2 50mmol/L
Triton?x-405 5ml/L
Cetyl Chloride potassium ammonia 50mmol/L
Reagent 2:
Na
2CO
3 100mmol/L
NaHCO
3 100mmol/L
Bilirubin oxidase 4000U/L
Reduced diphosphopyridine nucleotide 50mmol/L
KCl 20mmol/L
Sweet mellow wine 20mmol/L
proclin300 0.2ml/L
Potassium ferricyanide 50mmol/L
Claims (1)
1. enzyme method reagent kit for detecting DBil is characterized in that this kit is made up of following reagent 1 and reagent 2:
Reagent 1:
Damping fluid 10-200mmol/L
HgCl
2 10-100mmol/L
Triton?x-405 0.01-20ml/L
Cetyl Chloride potassium ammonia 20-100mmol/L;
Described damping fluid is: sodium citrate-lactic acid buffer; Glycocoll-hydrochloride buffer; Acetate-sodium acetate buffer; Phthalic acid-hydrochloride buffer;
Reagent 2:
Na
2CO
3 10-200mmol/L
NaHCO
3 10-200mmol/L
Bilirubin oxidase 2000-4000U/L
Reduced coenzyme I 10-200mmol/L
KCl 10-500mmol/L
Sweet mellow wine 10-200mmol/L
proclin300 0.01-0.5ml/L
Potassium ferricyanide 10-200mmol/L;
The blending ratio of described reagent 1 and reagent 2 is 3: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 200610025635 CN101055271B (en) | 2006-04-12 | 2006-04-12 | Enzyme method reagent kit for detecting DBil |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610025635 CN101055271B (en) | 2006-04-12 | 2006-04-12 | Enzyme method reagent kit for detecting DBil |
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| CN101055271A CN101055271A (en) | 2007-10-17 |
| CN101055271B true CN101055271B (en) | 2011-01-26 |
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| CN 200610025635 Active CN101055271B (en) | 2006-04-12 | 2006-04-12 | Enzyme method reagent kit for detecting DBil |
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Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101963611B (en) * | 2009-07-23 | 2014-08-13 | 深圳迈瑞生物医疗电子股份有限公司 | Clinical assay reagent, kit and method |
| CN102226769A (en) * | 2011-03-17 | 2011-10-26 | 郑州兰森生物技术有限公司 | Reagents used for measuring direct bilirubin through sodium nitrite oxidation method |
| CN102944683A (en) * | 2012-11-16 | 2013-02-27 | 李立和 | Double reagent method for detecting indirect bilirubin kit and preparation method |
| FR3022257B1 (en) * | 2014-06-16 | 2018-03-30 | Roquette Freres | PROCESS FOR PRODUCING A STABLE AQUEOUS SOLUTION OF BETA-AMYLASE, AQUEOUS SOLUTION OBTAINED AND USES THEREOF |
| CN106353512A (en) * | 2016-08-15 | 2017-01-25 | 山东博科生物产业有限公司 | Novel reagent for detecting blood total bilirubin through synthetic chemistry oxidation method |
| CN106404686B (en) * | 2016-08-27 | 2019-03-29 | 山东博科生物产业有限公司 | A kind of serum total bilirubin of antiheparin (Vanadic acid oxidation) detection kit |
| CN109991177A (en) * | 2017-12-30 | 2019-07-09 | 济南宇鑫生物科技有限公司 | A kind of stabilization, bilirubin direct (enzymatic measurement) detection reagent of strong antijamming capability and detection method |
| CN109541238A (en) * | 2018-09-21 | 2019-03-29 | 武汉中太生物技术有限公司 | Direct bilirubin detecting method and kit |
| CN109374884B (en) * | 2018-12-24 | 2021-10-22 | 四川沃文特生物技术有限公司 | PCT concentration detection kit and preparation method thereof |
| CN111455018B (en) * | 2020-03-03 | 2023-05-23 | 天津大学 | Direct bilirubin detection kit containing bacillus subtilis laccase |
| CN111424070B (en) * | 2020-03-03 | 2023-01-20 | 天津大学 | Total bilirubin detection kit containing bacillus subtilis laccase |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0686849A2 (en) * | 1990-10-30 | 1995-12-13 | Wako Pure Chemical Industries Ltd | Method for measuring bilirubin |
| CN1155583A (en) * | 1995-10-27 | 1997-07-30 | 协和梅迪克斯株式会社 | Method of determining amount of bilirubin |
| WO1999004258A1 (en) * | 1997-07-17 | 1999-01-28 | Synermed International Inc. | Assay for total and direct bilirubin |
-
2006
- 2006-04-12 CN CN 200610025635 patent/CN101055271B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0686849A2 (en) * | 1990-10-30 | 1995-12-13 | Wako Pure Chemical Industries Ltd | Method for measuring bilirubin |
| CN1155583A (en) * | 1995-10-27 | 1997-07-30 | 协和梅迪克斯株式会社 | Method of determining amount of bilirubin |
| WO1999004258A1 (en) * | 1997-07-17 | 1999-01-28 | Synermed International Inc. | Assay for total and direct bilirubin |
Non-Patent Citations (1)
| Title |
|---|
| 宋克征.胆红素及检测方法研究的进展.《国外医学临床生物化学》.2002,第23卷(第5期),283-284. * |
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| CN101055271A (en) | 2007-10-17 |
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