CN117737192A - Leucine aminopeptidase detection kit and detection method - Google Patents
Leucine aminopeptidase detection kit and detection method Download PDFInfo
- Publication number
- CN117737192A CN117737192A CN202311784396.9A CN202311784396A CN117737192A CN 117737192 A CN117737192 A CN 117737192A CN 202311784396 A CN202311784396 A CN 202311784396A CN 117737192 A CN117737192 A CN 117737192A
- Authority
- CN
- China
- Prior art keywords
- reagent
- leucine
- leucine aminopeptidase
- detection kit
- buffer solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 91
- 102000002704 Leucyl aminopeptidase Human genes 0.000 title claims abstract description 71
- 108010004098 Leucyl aminopeptidase Proteins 0.000 title claims abstract description 71
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 150
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000007853 buffer solution Substances 0.000 claims abstract description 32
- 239000002904 solvent Substances 0.000 claims abstract description 31
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229920000669 heparin Polymers 0.000 claims abstract description 27
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229960002897 heparin Drugs 0.000 claims abstract description 23
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 21
- 239000006184 cosolvent Substances 0.000 claims abstract description 21
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000004094 surface-active agent Substances 0.000 claims abstract description 17
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 12
- 239000003755 preservative agent Substances 0.000 claims abstract description 12
- 230000002335 preservative effect Effects 0.000 claims abstract description 12
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 28
- PKZFFNJDFBBUIS-ZSCHJXSPSA-N (2s)-2-amino-4-methylpentanoic acid;4-nitroaniline Chemical compound CC(C)C[C@H](N)C(O)=O.NC1=CC=C([N+]([O-])=O)C=C1 PKZFFNJDFBBUIS-ZSCHJXSPSA-N 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 16
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 16
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 15
- 239000007995 HEPES buffer Substances 0.000 claims description 15
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 14
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 14
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 14
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 14
- 229920000053 polysorbate 80 Polymers 0.000 claims description 14
- 239000011592 zinc chloride Substances 0.000 claims description 14
- 235000005074 zinc chloride Nutrition 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 229920001223 polyethylene glycol Polymers 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- 238000002835 absorbance Methods 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 6
- 239000002202 Polyethylene glycol Substances 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 4
- DVEKCXOJTLDBFE-UHFFFAOYSA-N n-dodecyl-n,n-dimethylglycinate Chemical compound CCCCCCCCCCCC[N+](C)(C)CC([O-])=O DVEKCXOJTLDBFE-UHFFFAOYSA-N 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 230000000087 stabilizing effect Effects 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- RZQXOGQSPBYUKH-UHFFFAOYSA-N 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound OCC(CO)(CO)NCC(O)CS(O)(=O)=O RZQXOGQSPBYUKH-UHFFFAOYSA-N 0.000 claims description 3
- XCBLFURAFHFFJF-UHFFFAOYSA-N 3-[bis(2-hydroxyethyl)azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound OCCN(CCO)CC(O)CS(O)(=O)=O XCBLFURAFHFFJF-UHFFFAOYSA-N 0.000 claims description 3
- 108700016232 Arg(2)-Sar(4)- dermorphin (1-4) Proteins 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- 239000008118 PEG 6000 Substances 0.000 claims description 3
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 claims description 3
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 3
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 claims description 3
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 3
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 3
- 239000012091 fetal bovine serum Substances 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 235000011147 magnesium chloride Nutrition 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 3
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 3
- 235000011151 potassium sulphates Nutrition 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
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- 235000011152 sodium sulphate Nutrition 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 229960001763 zinc sulfate Drugs 0.000 claims description 3
- 239000000758 substrate Substances 0.000 abstract description 32
- 239000003146 anticoagulant agent Substances 0.000 abstract description 8
- 229940127219 anticoagulant drug Drugs 0.000 abstract description 8
- 238000006911 enzymatic reaction Methods 0.000 abstract description 8
- 239000000126 substance Substances 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 39
- 238000004458 analytical method Methods 0.000 description 17
- 239000000243 solution Substances 0.000 description 17
- 238000012360 testing method Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 9
- 229960003136 leucine Drugs 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 9
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 8
- 150000002500 ions Chemical class 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 108091000069 Cystinyl Aminopeptidase Proteins 0.000 description 5
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 229910052744 lithium Inorganic materials 0.000 description 5
- 230000002195 synergetic effect Effects 0.000 description 5
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 4
- 229960001008 heparin sodium Drugs 0.000 description 4
- AXZJHDNQDSVIDR-NSHDSACASA-N 4178-93-2 Chemical compound CC(C)C[C@H](N)C(=O)NC1=CC=C([N+]([O-])=O)C=C1 AXZJHDNQDSVIDR-NSHDSACASA-N 0.000 description 3
- 102000004400 Aminopeptidases Human genes 0.000 description 3
- 108090000915 Aminopeptidases Proteins 0.000 description 3
- 102100020872 Leucyl-cystinyl aminopeptidase Human genes 0.000 description 3
- 101710153422 Procalin Proteins 0.000 description 3
- 230000003301 hydrolyzing effect Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- 102000030900 Cystinyl aminopeptidase Human genes 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- -1 amide compounds Chemical class 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 210000000232 gallbladder Anatomy 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- RUFPHBVGCFYCNW-UHFFFAOYSA-N 1-naphthylamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1 RUFPHBVGCFYCNW-UHFFFAOYSA-N 0.000 description 1
- XZIIFPSPUDAGJM-UHFFFAOYSA-N 6-chloro-2-n,2-n-diethylpyrimidine-2,4-diamine Chemical compound CCN(CC)C1=NC(N)=CC(Cl)=N1 XZIIFPSPUDAGJM-UHFFFAOYSA-N 0.000 description 1
- 108700023418 Amidases Proteins 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010019754 Hepatitis cholestatic Diseases 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- 235000019454 L-leucine Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 102000005922 amidase Human genes 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000004982 aromatic amines Chemical class 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 231100000838 cholestatic hepatitis Toxicity 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000002774 effect on peptide Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000754 myometrium Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229940035044 sorbitan monolaurate Drugs 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 125000000185 sucrose group Chemical group 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a leucine aminopeptidase detection kit and a detection method, wherein the kit comprises an R1 reagent and an R2 reagent, the R1 reagent comprises a buffer solution, inorganic salt, an enzyme promoter, a surfactant and a preservative, the R2 reagent comprises a buffer solution, an auxiliary solvent, a cosolvent, a substrate and a preservative, the cosolvent is methanol or dimethyl sulfoxide, and the auxiliary solvent is one or more of glycerol, ethylene glycol and PEG; the invention improves the stability of the substrate in the reagent by using the cosolvent and the auxiliary solvent, and eliminates the interference of the heparin anticoagulant on the enzymatic reaction by adding the anti-interference substance, thereby greatly improving the performance of the leucine aminopeptidase kit.
Description
Technical Field
The invention belongs to the technical field of clinical detection products, and particularly relates to a leucine aminopeptidase detection kit and a leucine aminopeptidase detection method, wherein the leucine aminopeptidase detection kit is good in bottle opening stability and high in heparin interference resistance.
Background
Leucine Aminopeptidase (LAP) is widely distributed in animal and plant tissues, and has the main biological effect of hydrolyzing peptide bonds formed by leucine and other amino acids at the N-terminal of a peptide chain, and can hydrolyze amide bonds formed by leucine and ammonia (namely leunamide) or peptide bonds formed by leucine and amine, but has no effect on peptide bonds formed by leucine and amines of benzene or naphthalene, and the LAP has a molecular weight of 75-80kDa. Another aminopeptidase with similar LAP properties and functions is called leucine aromatic amidase (leucine arylamidash, LAA), which can hydrolyze amide compounds formed by amino acids and aromatic amines (such as aniline and naphthylamine containing benzene rings), and hydrolyze leucine paranitroaniline and L-leunamide, and has a molecular weight of 52kDa. In addition, placenta-derived cystine aminopeptidase (cystine aminopeptidase, CAP) or placental leucine aminopeptidase (placental leucine aminopeptidase, P-LAP) also has a hydrolytic effect on both LAP and LAA substrates. At present, the three enzymes are difficult to separate clearly by clinical detection means, so the three enzymes are called LAP.
LAP is widely distributed in tissues such as liver, pancreas, gall bladder, kidney, small intestine and myometrium, and participates in degradation and update of tissue proteins and certain peptides, and is an enzyme reflecting pathological changes of the tissues such as liver, gall bladder and pancreas. The existing LAP detection method mainly comprises a colorimetry, a rate method, an enzyme method, a continuous monitoring method, an L-leucyl-P-nitroaniline substrate method and the like. The method for detecting the activity of leucine aminopeptidase in human serum by using an L-leucyl-p-nitroaniline substrate method is mainly used for monitoring and diagnosing acute and chronic hepatitis, cholestatic hepatitis and liver cirrhosis diseases, and has the characteristics of automatic detection instrument, convenient operation, reliable detection result, high sensitivity, low detection cost and the like.
However, the current assay kits for leucine aminopeptidase on the market have the following drawbacks:
1) Usually, L-leucine-p-nitroaniline (also called L-leucine paranitroaniline or L-leucine-4-nitroaniline) is adopted as a substrate, but currently, the L-leucine-p-nitroaniline is adopted as the substrate, the calculated reference value (XLogP) of the hydrophobic parameter is 1.7, the substrate is extremely slightly dissolved (0.24 g/L,25 ℃), and the high analysis sensitivity requirement of the reagent cannot be met by the substrate with the solubility; the prepared reagent has low solubility in purified water, and can be separated out along with the change of temperature due to the obvious change of the solubility of the L-leucine-p-nitroaniline, so that the reagent has the defects of poor stability, low repeatability and the like.
2) In general, only serum samples can be tested, and plasma samples for testing heparin sodium and heparin lithium anticoagulants are easy to interfere, so that the measured value is inaccurate; in addition, the measured values of heparin sodium and heparin lithium plasma samples are normal when reagents are just uncapped in the market, but after the reagents are placed in a reagent bin at the temperature of 2-8 ℃ and uncapped for two weeks, the test results of the reagent-testing heparin plasma samples on a biochemical analyzer are obviously raised, namely the accuracy of the reagent bottle opening stability test is affected.
Disclosure of Invention
Aiming at the problems of low solubility of substrate leucine aminopeptidase and poor stability of reagent bottle opening in the prior art, the invention develops a leucine aminopeptidase detection kit and a detection method with good stability, good repeatability and strong anti-interference capability.
The technical scheme of the invention is as follows:
the invention provides an aminopeptidase detection kit for stabilizing heparin interference, which comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises an R1 buffer solution, inorganic salt, an enzyme promoter, a surfactant and a preservative, the R2 reagent comprises an R2 buffer solution, an auxiliary solvent, a cosolvent, L-leucine-p-nitroaniline and the preservative, the cosolvent is methanol or dimethyl sulfoxide, and the auxiliary solvent is one or more of glycerol, ethylene glycol and polyethylene glycol (PEG).
In the kit, the R2 reagent effectively increases the stability and solubility of a substrate (namely L-leucine-p-nitroaniline) in the reagent by utilizing the synergistic effect of the auxiliary solvent and the cosolvent, and the problems of low substrate consumption and easy precipitation in the R2 reagent caused by low substrate solubility are avoided, so that the phenomena of poor stability and low repeatability of the existing aminopeptidase detection reagent are improved.
Preferably, in the above detection kit, the R1 reagent comprises 50-200mmol/L R1 buffer solution, 100-500mmol/L inorganic salt, 2-100g/L enzyme promoter, 5-30g/L surfactant and preservative, and the pH is 8.0-8.5;
the R2 reagent comprises 50-300mmol/L R buffer solution, 10-100g/L auxiliary solvent, 40-70g/L auxiliary solvent, 10-60 mmol/LL-leucine-p-nitroaniline and preservative, and the pH value of the reagent is 3.0-5.5; the auxiliary solvent is one or more of glycerol, ethylene glycol, PEG6000, PEG8000 and PEG20000.
More preferably, in the above detection kit, the preservative is procalin 300, and the concentration of procalin 300 in the R1 reagent is 2 to 5ml/L and the concentration of procalin 300 in the R2 reagent is 0.5 to 5ml/L.
More preferably, in the above detection kit, the R1 buffer is one of TAPSO, tris, DIPSO, TAPS and HEPES.
More preferably, in the above-described detection kit, the inorganic salt is one of sodium chloride, potassium chloride, zinc sulfate, zinc chloride, potassium sulfate, sodium sulfate, magnesium chloride, and magnesium sulfate.
More preferably, in the above detection kit, the enzyme promoter is one or more of sucrose, trehalose, mannitol and fetal bovine serum.
More preferably, in the above detection kit, the surfactant is one of dodecyl polyethylene glycol ether, polyoxyethylene (80) sorbitan monolaurate (tween 80), emulgen a90, triton-100, GENAPOLX-080, flower king 709 and dodecyl betaine (BS-12).
More preferably, in the above detection kit, the R2 buffer is one of acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, and disodium hydrogen phosphate-citric acid.
Based on the leucine aminopeptidase detection kit provided by the invention, the second aspect of the invention provides a leucine aminopeptidase detection method, which specifically comprises the following steps: and uniformly mixing the sample to be tested with the R1 reagent, adding the R2 reagent after incubation, uniformly mixing, and measuring absorbance after incubation.
Preferably, in the above method, the sample to be tested is a serum sample or a heparin anticoagulated plasma sample.
In one embodiment of the present invention, the volume ratio of the sample to be tested to the R1 reagent and the R2 reagent is 1:16:4.
In the detection kit and the detection method, the basic detection principle is as follows: leucine aminopeptidase in a sample to be detected catalyzes and hydrolyzes a substrate L-leucyl-p-nitroaniline to generate leucine and paranitroaniline, the absorbance of the paranitroaniline is increased at 405nm, and the activity of the leucine aminopeptidase in the sample can be calculated by measuring the rate of the increase of absorbance.
Compared with the prior art, the invention has the beneficial effects that:
(1) Aiming at the problem that the substrate L-leucine-p-nitroaniline is not dissolved in water, the invention solves the problem through the synergistic effect of the specific cosolvent and the auxiliary solvent, so that the stability and repeatability of the reagent are obviously improved, and the stability, the anti-interference capability and the sensitivity of the reagent are greatly improved.
(2) The inorganic salt, the enzyme promoter and the surfactant are only present in the R1 reagent, and the invention utilizes the synergistic effect of the inorganic salt, the enzyme promoter and the surfactant to help eliminate the interference of heparin anticoagulant on enzymatic reaction, thereby realizing the detection of heparin anticoagulant plasma samples.
(3) The detection kit developed by the invention not only has good analysis sensitivity, but also has excellent bottle opening stability, and experimental data show that the anti-interference capability is still very strong after bottle opening for one month, and the fluctuation of measured values is not obvious, so the kit has good clinical detection application value.
Drawings
FIG. 1 is a graph showing the results of the stability analysis of leucine aminopeptidase detection kit of example 1 of the present invention in open bottle.
FIG. 2 is a graph showing the results of the stability analysis of leucine aminopeptidase detection kit of example 2 of the present invention in open bottle.
FIG. 3 is a graph showing the results of the stability analysis of leucine aminopeptidase detection kit of example 3 of the present invention in open bottle.
FIG. 4 is a graph showing the results of the stability analysis of leucine aminopeptidase detection kit of example 4 of the present invention in open bottle.
FIG. 5 is a graph showing the results of the stability analysis of leucine aminopeptidase detection kit of example 5 of the present invention in open bottle.
FIG. 6 is a graph showing the results of the stability analysis of the leucine aminopeptidase detection kit of comparative example 1 of the present invention in open bottle.
FIG. 7 is a graph showing the results of the stability analysis of the leucine aminopeptidase detection kit of comparative example 2 of the present invention in open bottle.
FIG. 8 is a graph showing the results of the stability analysis of the leucine aminopeptidase detection kit of comparative example 3 of the present invention in open bottle.
FIG. 9 is a graph showing the results of the stability analysis of the leucine aminopeptidase detection kit of comparative example 4 of the present invention in open bottle.
FIG. 10 is a graph showing the results of the stability analysis of the leucine aminopeptidase detection kit of comparative example 5 of the present invention in open bottle.
FIG. 11 is a graph showing the results of the stability analysis of the leucine aminopeptidase detection kit of comparative example 6 of the present invention in open bottle.
FIG. 12 is a graph showing the results of the stability analysis of the leucine aminopeptidase detection kit of comparative example 7 of the present invention in open bottle.
FIG. 13 is a graph showing the results of the stability analysis of the leucine aminopeptidase detection kit of comparative example 8 of the present invention in open bottle.
FIG. 14 is a graph showing the results of the stability analysis of the leucine aminopeptidase detection kit of comparative example 9 of the present invention in open bottle.
Detailed Description
The technical scheme of the present invention will be clearly and completely described below with reference to the detailed description and the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terms "comprising," "having," and any variations thereof, as used in the description and claims of the invention, are intended to cover a non-exclusive inclusion.
Aiming at the following problems in the prior art: the problem of poor stability and low repeatability of a leucine aminopeptidase detection reagent caused by poor solubility of a substrate L-leucine-p-nitroaniline in water, and the problem of inaccurate detection value when detecting a plasma sample of heparin sodium or heparin lithium anticoagulant caused by poor anti-interference capability of the leucine aminopeptidase detection reagent; the invention provides a leucine aminopeptidase detection kit and a leucine aminopeptidase detection method, and detection accuracy and repeatability of the kit are greatly improved by optimizing detection reagents, adding cosolvent, auxiliary solvent and the like.
The leucine aminopeptidase detection kit provided by the invention mainly comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 50-200mmol/L R1 buffer solution, 100-500mmol/L inorganic salt, 2-100g/L enzyme promoter, 5-30g/L surfactant and 2-5ml/L PC-300, and the pH value is 8.0-8.5;
the R2 reagent consists of 50-300mmol/L R2 buffer solution, 10-100g/L auxiliary solvent, 40-70g/L auxiliary solvent, 10-60 mmol/LL-leucine-p-nitroaniline and 0.5-5ml/L PC-300, and the pH value is 3.0-5.5.
The R1 buffer solution is selected from TAPSO, tris, DIPSO, TAPS, HEPES.
The inorganic salts are selected from sodium chloride, potassium chloride, zinc sulfate, zinc chloride, potassium sulfate, sodium sulfate, magnesium chloride and magnesium sulfate, and form ions in the R1 reagent, so that interference of IgM proteins in serum samples on reagent tests can be effectively eliminated. IgM is a macromolecular polymer, and needs to have an ion-supported IgM protein molecular structure, so that the IgM is easy to crosslink and aggregate to generate turbidity after losing the ion support. In the process of catalyzing and hydrolyzing a substrate L-leucyl-p-nitroaniline by leucine aminopeptidase, turbidity generated by cross-linking aggregation of IgM can influence the catalytic reaction, so that the reaction is difficult to go on. It should be noted that these ions need to be present in the R1 reagent to directly eliminate interference caused by IgM cross-linked aggregation during the incubation process of the sample to be tested and the R1 reagent, and these ions do not affect the main reaction (i.e., enzymatic reaction) of the detection, but rather promote the enzymatic reaction. The ions can be stably and uniformly dissolved in the reagent, are not affected by abrupt temperature change (such as abrupt change from 2-8 ℃ to 37 ℃), and can play a role in regulating osmotic pressure.
The enzyme promoter is selected from sucrose, trehalose, mannitol and fetal bovine serum, and can be added into the R1 reagent only in view of the function of the enzyme promoter. The combined use of one or more enzyme promoters can increase the density of the solution, play a role in stabilizing the reagent, and can protect the biological activity of leucine aminopeptidase and promote the enzymatic reaction in the reaction process of the reagent and a sample to be tested.
The surfactant is selected from dodecyl polyethylene glycol ether, tween 80, emulgen A90, triton-100, GENAPOLX-080, flower king 709 and BS-12.
The inorganic salt (ions), the enzyme promoter and the surfactant in the R1 reagent are synergistic, so that interference of anticoagulants such as heparin sodium and heparin lithium on enzymatic reaction is eliminated, and detection of a plasma sample to be detected is realized. The principle is as follows: heparin plays a role similar to an accelerator in the LAP catalytic reaction process, so that the false detection result is increased, and in view of the physicochemical properties of heparin with strong negative charges, a large amount of free ions in the R1 reagent can neutralize the charge of the heparin, and then the enzyme accelerator and the surfactant wrap the heparin with the charge neutralized, so that the influence of the heparin on enzymatic reaction is shielded. More importantly, after the R1 reagent is placed for a long time under the condition of 2-8 ℃ bottle opening (substances in the air can be dissolved into the reagent in the bottle opening process, so that the detection result of the reagent is changed), the synergistic effect of ions, enzyme promoters and surfactants on eliminating heparin interference is not affected, namely, the R1 reagent has good stability, and the interference of heparin on detection false positive can be continuously eliminated after the bottle opening for a period of time.
In addition, after the R1 reagent and the sample to be tested are mixed and the R2 reagent is added, the surfactant in the R1 reagent can promote the substrate in the R2 reagent to react with other components quickly, so that dissolution is promoted.
The R2 buffer solution is selected from acetic acid-sodium acetate buffer solution (0.05-0.3 mol/L), citric acid-sodium citrate buffer solution (0.05-0.3 mol/L) and disodium hydrogen phosphate-citric acid (0.05-0.3 mol/L).
The cosolvent is selected from methanol and dimethyl sulfoxide. Dimethyl sulfoxide has the characteristics of high polarity, high boiling point, good thermal stability, aprotic property, water miscibility, compatibility with aromatic hydrocarbon compounds and the like. The saturated monohydric alcohol with the simplest structure is very easy to dissolve in water, and can dissolve L-leucine-p-nitroaniline. Compared with other organic solvents, the methanol and the dimethyl sulfoxide can be dissolved in water and L-leucine-p-nitroaniline, and the structural formula of the L-leucine-p-nitroaniline cannot be changed, so that the use of the methanol and the dimethyl sulfoxide can effectively improve the solubility of the L-leucine-p-nitroaniline.
In the kit of the invention, the amount of the cosolvent (i.e., methanol or dimethyl sulfoxide) is critical, because: the two materials are easy to volatilize, and after the reagent is opened, the cosolvent volatilizes to cause the precipitation of the substrate, so that the opening stability is obviously affected; in addition, excessive use of the cosolvent can influence sample denaturation when a test sample is tested, and too low use of the cosolvent can cause the substrate to be effectively dissolved; moreover, because of the low solubility of the substrate in water, the uniformity of the substrate in solution is poor and the reagent reproducibility is also significantly affected. In this regard, the present invention further solves this problem by using an auxiliary solvent.
The auxiliary solvent is selected from glycerol, ethylene glycol, PEG6000, PEG8000 and PEG20000. The auxiliary solvent and the auxiliary solvent can be mutually dissolved, and the auxiliary solvent and the substrate dissolved in the auxiliary solvent are dissolved in the purified water solvent by utilizing the specific auxiliary solvent, so that the volatilization of the auxiliary solvent is avoided, and the effect of stabilizing the reagent is achieved. Meanwhile, the auxiliary solvent can also act together with the surfactant in the R1 reagent to promote the substrate to quickly participate in the reaction in the main reaction process, accelerate the reagent reaction, further improve the stability and further prolong the shelf life of the reagent.
It should be noted that the kit of the present invention has stringent requirements for the choice of co-solvent and co-solvent, which is not only compatible with water and substrate, but also is unreactive with the substrate; the auxiliary solvent is compatible with water, cosolvent and substrate, and can not react with cosolvent and substrate; meanwhile, experiments are needed to detect whether the cosolvent and the auxiliary solvent meeting the conditions can be applied to the whole detection system.
In the detection kit, the substrate L-leucine-p-nitroaniline needs to be placed in the R2 reagent to maintain the stability of the whole set of reagent and the reaction system, and the substrate is more stable in a proper pH range (namely 3.0-5.5), so that the stability period of the kit is prolonged.
The R1 reagent and the R2 reagent in the leucine aminopeptidase detection kit provided by the invention can be prepared by the following steps:
preparation of R1 reagent: adding each component in the reagent into a buffer solution according to the prepared concentration at normal temperature, then adjusting the PH value, and preparing the components according to the above-mentioned components, adding the components, and uniformly mixing the components at a constant volume for later use;
preparation of R2 reagent: adding the components in the reagent into buffer solution according to the prepared concentration at normal temperature, then adjusting the PH value, and preparing the components according to the above-mentioned components, adding the components, and uniformly mixing the components at a constant volume for later use.
The following examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the product specifications; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 80g/L ethylene glycol, 50g/L methanol, 30mmol/L L-leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Example 2
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 100g/L glycerol, 50g/L methanol, 30mmol/L L-leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Example 3
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 80g/L ethylene glycol, 70g/L dimethyl sulfoxide, 30mmol/L leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Example 4
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 20g/LPEG6000, 50g/L methanol, 30mmol/L L-leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Example 5
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 15g/LPEG20000, 70g/L dimethyl sulfoxide, 30mmol/L leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Comparative example 1
The leucine aminopeptidase detection kit of the example is derived from CN115469099A, and the R1 reagent and the R2 reagent respectively comprise the following components:
the R1 reagent consists of 50mmol/L Tris-diglycolide buffer solution, 3mmol/L EDTA, 5g/L NP-9, 30g/L NaCl and 0.5g/L LProclin300, and the pH is 7.5;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 1mmol/L EDTA, 15mmol/L L-leucine-p-nitroaniline and 0.5g/LProclin300, and the pH is 4.5.
Comparative example 2
The leucine aminopeptidase detection kit of the example is derived from CN102864206A, and the R1 reagent and the R2 reagent respectively comprise the following components:
the R1 reagent consists of 50mmol/L of tris buffer solution (pH 7.0), 5ml/L of Tween 20 and 0.1g/L of sodium azide;
the R2 reagent consists of 100 mmol/L2-morpholinoethanesulfonic acid buffer solution (pH 4.0), 100mmol/L L-leucine-p-nitroaniline, 5g/L sucrose and 0.1g/L sodium azide.
Comparative example 3
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 80g/L ethylene glycol, 50g/L dimethyl sulfoxide, 30mmol/L leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Comparative example 4
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 30mmol/L L-leucine-p-nitroaniline and 2ml/LProclin300, and the pH value is 4.5.
Comparative example 5
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 80g/L ethylene glycol, 30mmol/L L-leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Comparative example 6
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 50g/L methanol, 30mmol/L L-leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Comparative example 7
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 80g/L ethylene glycol, 50g/L isopropanol, 30mmol/L L-leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Comparative example 8
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 80g/L n-butanol, 50g/L methanol, 30mmol/L L-leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Comparative example 9
The leucine aminopeptidase detection kit of the embodiment comprises an R1 reagent and an R2 reagent, wherein,
the R1 reagent consists of 150mmol/L HEPES buffer solution, 350mmol/L zinc chloride, 20g/L trehalose, 15g/L tween 80 and 2ml/L Proclin300, and the pH value is 8.0;
the R2 reagent consists of 20mmol/L citric acid-sodium citrate buffer solution, 80g/L n-butanol, 50g/L isopropanol, 30mmol/L L-leucine-p-nitroaniline and 2ml/L Proclin300, and the pH value is 4.5.
Example 6
The leucine aminopeptidase test kits of examples 1 to 5 and comparative examples 1 to 9 were used, respectively, using a biochemical analyzer with a dominant wavelength of 405nm.
The detection process specifically comprises the following steps: the ratio of the dosage of the sample to be tested to the dosage of the R1 reagent and the dosage of the R2 reagent are 1:16:4 (volume ratio), the sample to be tested and the R1 reagent are firstly mixed uniformly, the mixture is incubated at 37 ℃ for five minutes and then the R2 reagent is added for uniform mixing, the initial absorbance is tested after incubation at 37 ℃ for two minutes, and then the change rate of the average absorbance per minute is accurately measured within 3 minutes, so that the analysis sensitivity of each group of reagents is measured. Simultaneously, a quality control product, a serum sample and a heparin lithium anticoagulant plasma sample are respectively used as samples to be tested, and the stability of the sample is synchronously tested after 31 days of bottle opening.
The results of the measurements are shown in tables 1-15 and FIGS. 1-14.
TABLE 1 analytical sensitivity data for examples 1-5 and comparative examples 1-9
| Project | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 | Comparative example 1 | Comparative example 2 |
| Analytical sensitivity | 0.0258 | 0.0257 | 0.0262 | 0.0257 | 0.0261 | 0.0156 | 0.0158 |
| Project | Comparative example 3 | Comparative example 4 | Comparative example 5 | Comparative example 6 | Comparative example 7 | Comparative example 8 | Comparative example 9 |
| Analytical sensitivity | 0.0256 | 0.0090 | 0.0091 | 0.0201 | 0.0103 | 0.0198 | 0.0087 |
As can be seen from Table 1, the analytical sensitivities of examples 1 to 5 were significantly better than those of comparative examples 1 to 9.
Table 2 data on stability to opening of examples 1-3
Table 3 data on stability to opening of examples 4-5
Table 4 data on stability to opening of comparative examples 1 to 3
Table 5 data on stability to opening of comparative examples 4 to 6
Table 6 data on stability to opening of comparative examples 7 to 9
As can be seen from tables 2 to 6 and fig. 1 to 14, the interference ability of heparin plasma samples is significantly reduced after the reagent is stabilized for a period of time in the opening of the bottle in comparative example 1, comparative example 2 and comparative example 3; comparing the plasma value measured in the test process of comparative example 3 with the test results of examples 1-5, the measured value of comparative example 3 is much higher, which indicates that the test result is high in false positive and the fluctuation difference is very obvious; after the bottles of comparative examples 4 to 9 are opened, the test results are always reduced, which indicates that the bottle opening effect is poor; in examples 1 to 5, the anti-interference ability remained after one month of the test for the stability of the reagent in opening the bottle, and the fluctuation of the measured value was not obvious.
In summary, the leucine aminopeptidase detection scheme provided by the invention still uses L-leucine-p-nitroaniline as a substrate, the solubility and the solvent uniformity of the substrate are improved through a cosolvent and an auxiliary solvent, and the interference of heparin anticoagulant on enzymatic reaction is eliminated through the interaction of a surfactant, inorganic salt and an enzyme promoter, so that the accuracy and the repeatability of reagent testing are greatly improved, and the reagent has good clinical detection application prospects.
It should be noted that the above-mentioned embodiments are only some embodiments of the present invention, but not all embodiments, and are only used for illustrating the technical scheme of the present invention, not limiting; all other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
Claims (10)
1. The leucine aminopeptidase detection kit for stabilizing heparin interference is characterized by comprising an R1 reagent and an R2 reagent, wherein the R1 reagent comprises an R1 buffer solution, inorganic salt, an enzyme promoter, a surfactant and a preservative, the R2 reagent comprises an R2 buffer solution, an auxiliary solvent, a cosolvent, L-leucine-p-nitroaniline and the preservative, the cosolvent is methanol or dimethyl sulfoxide, and the auxiliary solvent is one or more of glycerol, ethylene glycol and PEG.
2. The leucine aminopeptidase detection kit according to claim 1, wherein,
the R1 reagent comprises 50-200mmol/L R1 buffer solution, 100-500mmol/L inorganic salt, 2-100g/L enzyme promoter, 5-30g/L surfactant and preservative, and the pH value is 8.0-8.5;
the R2 reagent comprises 20-300mmol/L R buffer solution, 10-100g/L auxiliary solvent, 40-70g/L auxiliary solvent, 10-60mmol/L L-leucine-p-nitroaniline and preservative, and the pH value is 3.0-5.5, and the PEG comprises PEG6000, PEG8000 and PEG20000.
3. The leucine aminopeptidase detection kit according to claim 2 wherein the R1 buffer is one of TAPSO, tris, DIPSO, TAPS and HEPES and the R2 buffer is one of acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer and disodium hydrogen phosphate-citric acid.
4. The leucine aminopeptidase detection kit according to claim 2 wherein the inorganic salt is one or more of sodium chloride, potassium chloride, zinc sulfate, zinc chloride, potassium sulfate, sodium sulfate, magnesium chloride, magnesium sulfate.
5. The leucine aminopeptidase detection kit according to claim 2 wherein the enzyme promoter is one or more of sucrose, trehalose, mannitol and fetal bovine serum.
6. The leucine aminopeptidase detection kit according to claim 2 wherein the surfactant is one of dodecyl polyethylene glycol ether, tween 80, emulgen a90, triton-100, GENAPOLX-080, flower king 709 and BS-12.
7. The leucine aminopeptidase detection kit according to claim 2 wherein the preservative is Proclin300, the Proclin300 being present in the R1 reagent at a concentration of 2-5ml/L and the R2 reagent at a concentration of 0.5-5ml/L.
8. A leucine aminopeptidase detection method, characterized in that the leucine aminopeptidase detection kit according to any one of claims 1 to 7 is used for detection, specifically: and uniformly mixing the sample to be tested with the R1 reagent, adding the R2 reagent after incubation, uniformly mixing, and measuring absorbance after incubation.
9. The method according to claim 8, wherein the volume ratio of the sample to be tested to the R1 reagent and the R2 reagent is 1:16:4.
10. The method according to claim 8, wherein the sample to be tested is a serum sample or a heparin anticoagulated plasma sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311784396.9A CN117737192A (en) | 2023-12-21 | 2023-12-21 | Leucine aminopeptidase detection kit and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311784396.9A CN117737192A (en) | 2023-12-21 | 2023-12-21 | Leucine aminopeptidase detection kit and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117737192A true CN117737192A (en) | 2024-03-22 |
Family
ID=90252483
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311784396.9A Pending CN117737192A (en) | 2023-12-21 | 2023-12-21 | Leucine aminopeptidase detection kit and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117737192A (en) |
-
2023
- 2023-12-21 CN CN202311784396.9A patent/CN117737192A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bogorad | [122] Porphyrin synthesis | |
| Auld et al. | Kinetics of carboxypeptidase A. pH dependence of tripeptide hydrolysis catalyzed by zinc, cobalt, and manganese enzymes | |
| Watanabe et al. | Determination of hypoxanthine in fish meat with an enzyme sensor | |
| ES2502481T3 (en) | Composition for glycosylated protein analysis | |
| CN109239059B (en) | Glycated serum protein assay kit and preparation method and application thereof | |
| US5508202A (en) | Method of determining blood coagulation factor XIII activity and kit of reagents for the determination | |
| CN109239061A (en) | A kind of liquid-type Antiprothrombin antibodies assay kit | |
| JP7056577B2 (en) | Method for measuring the saccharification rate of hemoglobin | |
| CN105203472A (en) | Stable NEFA (non-esterified fatty acid) measuring kit | |
| CN112051354B (en) | Lipase determination kit and preparation method thereof | |
| CN107870170B (en) | A kind of kit of luminol chemiluminescence analysis measurement glycated albumin | |
| Tuengler et al. | Enhanced heat, alkaline and tryptic stability of acetamidinated pig heart lactate dehydrogenase | |
| CN111808921A (en) | Trinder reaction-based detection kit and application thereof | |
| US5204240A (en) | Method and a kit containing means for the kinetic determination of factor xiii | |
| Fossati et al. | A step forward in enzymatic measurement of creatinine | |
| CN111944872A (en) | Reagent combination, reagent or kit for measuring creatinine content | |
| SU515471A3 (en) | Method for quantitative determination of hydrolytic enzymes | |
| JPH0365958B2 (en) | ||
| CN117737192A (en) | Leucine aminopeptidase detection kit and detection method | |
| CN101403691A (en) | Melamine or tricyanic acid measuring method and its reagent kit or test paper strip | |
| JP5192647B2 (en) | Stable chromogenic test reagents and their use in coagulation diagnostic tests | |
| Lederer et al. | Acetoacetate decarboxylase. Subunits and properties | |
| CN109307720B (en) | Composition, composite calibrator and application of composite calibrator | |
| CA1198040A (en) | Stabilized enzymatic solutions and methods for determining total cholesterol in human serum | |
| US20220357334A1 (en) | Glycated protein assay reagent containing stabilizer of protease that increases oxidation-reduction potential of ferrocyanide, method for assaying glycated protein, method for preserving glycated protein assay reagent, and method for stabilizing glycated protein assay reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |