CN112522365A - 一种可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法 - Google Patents
一种可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法 Download PDFInfo
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- CN112522365A CN112522365A CN202011578744.3A CN202011578744A CN112522365A CN 112522365 A CN112522365 A CN 112522365A CN 202011578744 A CN202011578744 A CN 202011578744A CN 112522365 A CN112522365 A CN 112522365A
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- reagent
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- uric acid
- calcium dobesilate
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- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 title claims abstract description 48
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 229940116269 uric acid Drugs 0.000 title claims abstract description 48
- HBGOLJKPSFNJSD-UHFFFAOYSA-N Etamsylate Chemical compound CC[NH2+]CC.OC1=CC=C(O)C(S([O-])(=O)=O)=C1 HBGOLJKPSFNJSD-UHFFFAOYSA-N 0.000 title claims abstract description 47
- QGNBTYAQAPLTMX-UHFFFAOYSA-L calcium dobesilate Chemical compound [Ca+2].OC1=CC=C(O)C(S([O-])(=O)=O)=C1.OC1=CC=C(O)C(S([O-])(=O)=O)=C1 QGNBTYAQAPLTMX-UHFFFAOYSA-L 0.000 title claims abstract description 47
- 229960005438 calcium dobesilate Drugs 0.000 title claims abstract description 47
- 229960004817 etamsylate Drugs 0.000 title claims abstract description 47
- 210000002966 serum Anatomy 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 239000003814 drug Substances 0.000 title claims abstract description 26
- 229940079593 drug Drugs 0.000 title claims abstract description 23
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 88
- 239000007853 buffer solution Substances 0.000 claims abstract description 17
- 239000000376 reactant Substances 0.000 claims abstract description 16
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 15
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 15
- 239000003755 preservative agent Substances 0.000 claims abstract description 15
- 230000002335 preservative effect Effects 0.000 claims abstract description 15
- 238000003756 stirring Methods 0.000 claims abstract description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 12
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 12
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 11
- 239000007800 oxidant agent Substances 0.000 claims abstract description 11
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 10
- 230000001590 oxidative effect Effects 0.000 claims abstract description 10
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 9
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 8
- 235000010323 ascorbic acid Nutrition 0.000 claims abstract description 8
- 239000011668 ascorbic acid Substances 0.000 claims abstract description 8
- 238000005303 weighing Methods 0.000 claims abstract description 7
- GNTDGMZSJNCJKK-UHFFFAOYSA-N divanadium pentaoxide Chemical compound O=[V](=O)O[V](=O)=O GNTDGMZSJNCJKK-UHFFFAOYSA-N 0.000 claims description 20
- 108010092464 Urate Oxidase Proteins 0.000 claims description 19
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 claims description 8
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims description 7
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- ZHXZNKNQUHUIGN-UHFFFAOYSA-N chloro hypochlorite;vanadium Chemical compound [V].ClOCl ZHXZNKNQUHUIGN-UHFFFAOYSA-N 0.000 claims description 6
- 102000007562 Serum Albumin Human genes 0.000 claims description 5
- 108010071390 Serum Albumin Proteins 0.000 claims description 5
- -1 TRTS Chemical compound 0.000 claims description 5
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 claims description 4
- 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 4
- AJTVSSFTXWNIRG-UHFFFAOYSA-N 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid Chemical compound OCC[NH+](CCO)CCS([O-])(=O)=O AJTVSSFTXWNIRG-UHFFFAOYSA-N 0.000 claims description 4
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 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 4
- 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 4
- 108700016232 Arg(2)-Sar(4)- dermorphin (1-4) Proteins 0.000 claims description 4
- 108010008488 Glycylglycine Proteins 0.000 claims description 4
- 239000007995 HEPES buffer Substances 0.000 claims description 4
- OWXMKDGYPWMGEB-UHFFFAOYSA-N HEPPS Chemical compound OCCN1CCN(CCCS(O)(=O)=O)CC1 OWXMKDGYPWMGEB-UHFFFAOYSA-N 0.000 claims description 4
- 239000007993 MOPS buffer Substances 0.000 claims description 4
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 claims description 4
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 claims description 4
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- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 claims description 4
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- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 claims description 4
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- 239000010452 phosphate Substances 0.000 claims description 4
- YDBHVMTTYXWHLI-UHFFFAOYSA-N 2,4,6-tribromo-3-hydroxybenzoic acid Chemical compound OC(=O)C1=C(Br)C=C(Br)C(O)=C1Br YDBHVMTTYXWHLI-UHFFFAOYSA-N 0.000 claims description 3
- WXNZTHHGJRFXKQ-UHFFFAOYSA-N 4-chlorophenol Chemical compound OC1=CC=C(Cl)C=C1 WXNZTHHGJRFXKQ-UHFFFAOYSA-N 0.000 claims description 3
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 229910021538 borax Inorganic materials 0.000 claims description 3
- DPXDJGUFSPAFJZ-UHFFFAOYSA-L disodium;4-[3-methyl-n-(4-sulfonatobutyl)anilino]butane-1-sulfonate Chemical compound [Na+].[Na+].CC1=CC=CC(N(CCCCS([O-])(=O)=O)CCCCS([O-])(=O)=O)=C1 DPXDJGUFSPAFJZ-UHFFFAOYSA-L 0.000 claims description 3
- 229940043257 glycylglycine Drugs 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
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- 239000004328 sodium tetraborate Substances 0.000 claims description 3
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 3
- SVLRFMQGKVFRTB-UHFFFAOYSA-M sodium;3-(3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].COC1=CC(NCC(O)CS([O-])(=O)=O)=CC(OC)=C1 SVLRFMQGKVFRTB-UHFFFAOYSA-M 0.000 claims description 3
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 claims description 2
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- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 claims 2
- NMWCVZCSJHJYFW-UHFFFAOYSA-M sodium;3,5-dichloro-2-hydroxybenzenesulfonate Chemical compound [Na+].OC1=C(Cl)C=C(Cl)C=C1S([O-])(=O)=O NMWCVZCSJHJYFW-UHFFFAOYSA-M 0.000 claims 2
- 238000005259 measurement Methods 0.000 abstract description 18
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 abstract description 14
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- FVYXIJYOAGAUQK-UHFFFAOYSA-N honokiol Chemical compound C1=C(CC=C)C(O)=CC=C1C1=CC(CC=C)=CC=C1O FVYXIJYOAGAUQK-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/62—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving uric acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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Abstract
本发明的一种可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,其技术方案要点是包括以下步骤:R1试剂配制:称取缓冲液→加入防腐剂→搅拌溶解→用氢氧化钠或盐酸调PH至8.0→加入氧化剂→搅拌溶解→调PH至8.0→加入Trinder’s反应剂A和非离子表面活性剂,混合均匀→2~8℃隔夜→加入抗坏血酸氧化酶和过氧化物酶→搅拌溶解→2~8℃隔夜→检测、分装;所述的Trinder’s反应剂A为4-氨基安替比林;R2试剂配制:本发明克服现在各医院临床检验测定肌酐时,由于血清标本中含羟苯磺酸钙、酚磺乙胺造成的负偏差,使肌酐测定时不准确的缺陷,适用于全自动生化分析仪血清肌酐的检测。
Description
本案为分案申请,具体为申请号:2020108215098,名称为:一种可消除血清中羟苯磺酸钙,酚磺乙胺药物干扰的尿酸试剂盒及其制备方法,申请日为:2020.08.15的分案申请。
技术领域
本发明属于医学检验技术领域,本发明涉及一种可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法。
背景技术
尿酸(Uric Acid,UA)是体内嘌呤类化合物代谢的终产物,呈弱酸性。尿酸既可以来自体内,也可以来自于食物中嘌呤的分解代谢。在体内,尿酸主要在肝脏中生成,小部分可经肝脏随胆汁排泄,其余大部分均从肾脏排泄。尿酸的溶解度较小,因而在体内浓度偏高时容易析出结晶。尿酸被认为与痛风、心脑血管疾病、代谢综合征、输尿管结石和肾脏疾病等的发生、发展密切相关。近年来,随着人们生活水平的提高和包含结构的改变,特别是富含蛋白质和嘌呤食物摄入的增加,高尿酸血症和痛风发病人数呈上升趋势。因此,临床检测血清尿酸浓度具有非常重要的参考价值和意义,其检测原理如下:
现行市售检测试剂盒均为双试剂,基于Trinder反应的尿酸酶-过氧化物酶偶联法具有灵敏度、适用于自动生化分析仪等优点,试剂R1内含过氧化物酶及Trinder’s A试剂,加入R2后,R2里含尿酸酶及Trinder’s试剂B,尿酸酶将尿酸水解成尿囊素及H2O2,H2O2再与R1里的过氧化物酶及Trinder’s A试剂共同存在下呈色进行测定。此反应过程均利用了酶的专一性,但生成的H2O2为强氧化剂,极易被血清中的还原性药物消耗而产生负干扰。
尿酸为嘌呤的最终代谢产物,当嘌呤代谢异常或肾脏对尿酸的排泄障碍均可引起血液中尿酸浓度升高或降低;痛风、肾功能障碍,恶性肿瘤,镉、铅等重金属中毒均可能引起血液尿酸浓度升高,药物如水杨酸、嘌呤醇的服用,严重肝病,肾脏排泄增加均会使血液尿酸含量降低。羟苯磺酸钙作为微血管保护及治疗的常用药,酚磺乙胺作为止血的常用药,这些药物均为强还原剂,当服用这些药物的病人在测定血浆尿酸含量时,这些还原性药物会与尿酸酶分解尿酸产生的H2O2直接反应,消耗掉H2O2造成尿酸值会严重的负偏差,造成医师诊断治疗时的误判。
发明内容
本发明公开一种可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,在试剂R1中加入高氧化还原电位之氧化型上述金属酸盐或五氧化二钒或三氯氧钒之一种或一种以上混合,利用所述金属酸盐或五氧化二钒或三氯氧钒之氧化特性,先将羟苯磺酸钙及酚磺乙胺氧化,避免了在加入试剂R2后尿酸酶分解尿酸产生的H2O2被消耗掉,操作简单,迅速得到准确的尿酸测定值。
本发明还公开一种可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒,包括试剂R1和试剂R2,所述试剂R1中包含缓冲液、过氧化物酶、抗坏血酸氧化酶、防腐剂、Trinder’s反应剂A、金属酸盐;所述试剂R2包含缓冲液、血清白蛋白、尿酸酶、防腐剂、及Trinder’s反应剂B。选择合适的金属酸盐,做为氧化剂来消除尿酸测定时血清中羟苯磺酸钙及酚磺乙胺的干扰造成的负偏差,同时不影响试剂R1及试剂R2中各组分的稳定性,可以在加入尿酸酶(试剂R2)之前先将血清中的羟苯磺酸钙及酚磺乙胺氧化,避免其消耗在尿酸测定时产生的H2O2,使尿酸测定值准确。
优选的,所述金属酸盐包括氧化态镍酸盐、铁酸盐、钴酸盐、铬酸盐、锰酸盐、钒酸盐中的一种或几种,所述钒酸盐为偏钒酸盐、多聚钒酸盐、五氧化二钒或三氯氧钒一种或一种以上混合,所述的高氧化还原电位金属酸盐,包含所述含金属酸的钠、钾、铵盐。在多次试验中发现了采用元素周期表中23、24、25、26、27、28位各金属元素酸盐,选择氧化还原电位高的氧化态镍酸盐、钴酸盐、铬酸盐、锰酸盐、钒酸盐、偏钒酸盐、多聚钒酸盐、五氧化二钒或三氯氧钒均可做为氧化剂来消除尿酸测定时血清中羟苯磺酸钙及酚磺乙胺的干扰造成的负偏差,同时不影响试剂R1及试剂R2中各组分的稳定性。
优选的,所述的Trinder’s反应剂A为4-氨基安替比林(4-Aminoantipyrine,简称4-AAP)。本发明在一系列试验中发现必须将4-AAP加在试剂R1中才可与上述金属酸盐或五氧化二钒及三氯氧钒及试剂R1中其他组分共同存在,Trinder’s反应另一试剂苯酚类化合物必须与尿酸酶共同存在试剂R2中,才可使本尿酸试剂盒除了可消除羟苯磺酸钙及酚磺乙胺的干扰外,才可使本试剂盒在保持2~8℃一年以上的稳定,而抗坏血酸氧化酶的量在上述范围内时可以有效地降低血液标本中抗坏血酸对尿酸测定所造成的干扰。
优选的,所述试剂R1中,缓冲液浓度为5~200mM,pH6.5~9.5;
过氧化物酶含量1.0~10ku/升;
抗坏血酸氧化酶1.0~10ku/升;
Trinder’s反应剂A0.3~5mM/升;
防腐剂重量比为0.01%-0.5%时,采用含量在0.1~10mM的金属酸盐氧化剂或五氧化二钒或三氯氧钒氧化剂。
优选的,所述试剂R1的缓冲液为BES、BICINE、DIPSO、EPPS、PIPES、HEPES、MOPS、TAPS、TAPSO、TES、Tricine、Tris、双甘肽、磷酸盐中的一种或一种以上混合;试剂R2所述的缓冲液为BES、BICINE、DIPSO、EPPS、PIPES、HEPES、MOPS、TAPS、TAPSO、TES、TRICINE、TRIS、双甘肽、磷酸盐中的一种或一种以上混合。
优选的,所述试剂R1及试剂R2中还含有非离子表面活性剂,其重量比含量为0.01%~0.3%,所述非离子表面活性剂具体为BRIJ,EMULGEN,TRITON,TWEEN中的一种或两种。
优选的,所述防腐剂为Proclin、硫酸庆大霉素、PARABEN、氯霉素、叠氮钠、硼砂之一种或两种。防腐剂能够阻却试剂中生物酶的作用,使试剂在一定时间内保持功效稳定,如硼砂、叠氮钠等。
优选的,所述试剂R2中,缓冲液浓度为10~200mM、pH6.5~9.0;尿酸酶含量5-20ku/升;Trinder’s反应剂B含量为0.1~20mM。
优选的,所述试剂R2内之Trinder’s反应剂B为TOOS、DHBS、DAOS、HDAOS、TBHBA、TOPS、TODB、4-氯酚的一种或两种以上混合。
一种可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法:
试剂R1配制:
称取缓冲液→加入防腐剂→搅拌溶解→用氢氧化钠或盐酸调pH至8.0→加入金属酸盐→搅拌溶解→调pH至8.0→加入Trinder’s反应剂A和非离子表面活性剂,混合均匀→2~8℃隔夜→加入抗坏血酸氧化酶和过氧化物酶→搅拌溶解→2~8℃隔夜→检测、分装;
试剂R2配制:
称取缓冲液→加入防腐剂→搅拌溶解→调pH至8.0→加入Trinder’s反应剂B和表面活性剂→混合均匀→2~8℃隔夜→加入血清白蛋白,尿酸酶→混合均匀→2~8℃隔夜→检测、分装。
本尿酸试剂盒由试剂R1及试剂R2组成,以试剂R1先与标本混合,如标本中含治疗药物羟苯磺酸钙或酚磺乙胺时,试剂R1中的上述氧化态金属酸盐或五氧化二钒或三氯氧钒可先将其氧化,避免了加入试剂R2中含有的尿酸酶反应产生的H2O2被上述药物消耗掉,同时试剂R2中含有Trinder’s反应剂B即苯酚类化合物与试剂R1中4-AAP与尿酸酶反应产生的H2O2呈色,测定其吸光度,计算其含量。
本发明的有益效果是:
本发明的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒,稳定性好,操作简单,检测反应迅速,特别是将过氧化物酶和尿酸酶分别制备于试剂R1和试剂R2中,区别于市场上现有的试剂盒组成,能够在一段时间内延长试剂盒的稳定性,持续消除羟苯磺酸钙及酚磺乙胺的负干扰,避免医师在临床诊断时的误判,可以精准用药及治疗,适用于全自动生化分析仪。
本发明的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,试剂盒稳定性好,操作简单迅速,消除了羟苯磺酸钙及酚磺乙胺的负干扰,准确度高,适用于全自动生化分析仪。
具体实施方式
下面结合具体实施例对本发明的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒做进一步描述:
以目前市售具中国国家药监局批准之尿酸酶法试剂盒测定羟苯磺酸钙及酚磺乙胺之干扰,结果如表一:
其试剂组成依说明书所述为:
[主要组成成份]由试剂R1、试剂R2和校准品组成。
试剂R1:二氯羟基苯磺酸(DHBS)、磷酸盐缓冲液;
试剂R2:尿酸酶、过氧化物酶(交底中过氧化物酶在R1的组分中)、4-氨基安替比林。
校准品:含肌酐水溶液,浓度见标签,可溯源至Randox CAL3校准品。
[测定方法]
(1)双试剂无需配制,直接使用。
(2)试验条件:样本(S):3μl试剂1(R1):240μl试剂2(R2):60μl温度:37℃
测定类型:终点法主波长:505nm副波长:660nm反应方向:上升
方法:先将标准品或样本与R1混合,37℃5分钟后加入试剂R2,然后测定加入试剂R2后5分钟的反应吸光度。
测定空白吸光度(A1)测定反应吸光度(A2)
样本:3μl
R1:240μl R2:60μl
(3)校准程序:使用校准品进行校准,每次更换试剂批次时都应进行校准。校准后,各实验室要用质控品验证。如果质控结果不在可接受范围值内,则需要进行重新校准。
(4)质量控制程序:选用适当的质控品进行质量控制。各实验室建立各自的质控频率和可接受范围值。当测定结果超出可接受范围时,有必要采取相应措施。
(5)计算
测定仪器及参数:
单位:μmol/L,正常值90~420μmol/L
测定仪器:日立7180
测定参数:R1:R2:S(标本量)=240:60:3μl
主/副波长:505/660nm
2POINT END,INC.
表1现有双试剂肌酐酶法试剂盒测定干扰结果:(单位:μmol/L)
结论:测定结果显示羟苯磺酸钙及酚磺乙胺均对尿酸测定结果造成负偏差。
具体结合实施例1-4说明本申请的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的作用:
表2实施例1-4的试剂组分:
依照本发明的肌酐试剂具体组份描述如下:
实施例1:
试剂R1组份:
试剂R2组份:
实施例2:
试剂R1组份:
试剂R2组份:
实施例3:
试剂R1组份:
试剂R2组份:
实施例4:
试剂R1组份:
试剂R2组份:
实施例1-4的制备方法包括如下步骤:
试剂R1配制:
称取缓冲液→加入防腐剂→搅拌溶解→用氢氧化钠或盐酸调pH至8.0→加入金属酸盐→搅拌溶解→调pH至8.0→加入Trinder’s反应剂A和非离子表面活性剂,混合均匀→2~8℃隔夜→加入抗坏血酸氧化酶和过氧化物酶→搅拌溶解→2~8℃隔夜→检测、分装;
试剂R2配制:
称取缓冲液→加入防腐剂→搅拌溶解→调pH至8.0→加入Trinder’s反应剂B和表面活性剂→混合均匀→2~8℃隔夜→加入血清白蛋白,尿酸酶→混合均匀→2~8℃隔夜→检测、分装。
测定方法:
将实施例1-4制备得试剂R1及试剂R2放置于全自动凝血分析仪(日立7180)相应试剂位置,吸取试剂R1 210μl,试剂R2 70μl,定标液或标本10μl,定标液浓度300μM,主/副波长540/660nm,二点终点法,反应方向:INC,以定标液定标测定含不同浓度羟苯磺酸钙或酚磺乙胺各标本肌酐值。
表3:实施例1测定结果:(单位:μmol/L)
测定 | 1 | 2 | 3 | 平均值 | 相对偏差 |
母液 | 356 | 355 | 356 | 355.7 | |
羟苯磺酸钙8ml/L | 357 | 355 | 356 | 356 | 0.1% |
羟苯磺酸钙16ml/L | 357 | 358 | 356 | 357 | 0.4% |
羟苯磺酸钙32ml/L | 356 | 356 | 355 | 355.7 | 0.0% |
羟苯磺酸钙64ml/L | 349 | 348 | 349 | 348.7 | 2.0% |
酚磺乙胺12.5mg/L | 357 | 358 | 358 | 357.7 | 0.6% |
酚磺乙胺25mg/L | 357 | 357 | 357 | 357.0 | 0.4% |
酚磺乙胺50mg/L | 357 | 356 | 355 | 356.0 | 0.1% |
酚磺乙胺100mg/L | 358 | 357 | 358 | 357.7 | 0.6% |
表4:实施例2测定结果:(单位:μmol/L)
表5:实施例3测定结果:(单位:μmol/L)
测定 | 1 | 2 | 3 | 平均值 | 相对偏差 |
母液 | 360 | 360 | 362 | 361.0 | |
羟苯磺酸钙8ml/L | 360 | 360 | 362 | 360.7 | -0.1% |
羟苯磺酸钙16ml/L | 361 | 361 | 361 | 361 | 0.0% |
羟苯磺酸钙32ml/L | 360 | 360 | 359 | 359.7 | -0.4% |
羟苯磺酸钙64ml/L | 359 | 359 | 360 | 359.3 | -0.2% |
酚磺乙胺12.5mg/L | 360 | 360 | 360 | 360.0 | -0.3% |
酚磺乙胺25mg/L | 360 | 361 | 359 | 360.0 | -0.3% |
酚磺乙胺50mg/L | 358 | 360 | 359 | 359 | -0.6% |
酚磺乙胺100mg/L | 359 | 359 | 358 | 358.7 | -0.6% |
表6:实施例4测定结果:(单位:μmol/L)
测定 | 1 | 2 | 3 | 平均值 | 相对偏差 |
母液 | 360 | 360 | 360 | 360.0 | |
羟苯磺酸钙8ml/L | 361 | 360 | 361 | 360.7 | 0.2% |
羟苯磺酸钙16ml/L | 360 | 361 | 358 | 359.7 | -0.1% |
羟苯磺酸钙32ml/L | 359 | 359 | 358 | 358.7 | -0.4% |
羟苯磺酸钙64ml/L | 358 | 357 | 357 | 357.3 | -0.8% |
酚磺乙胺12.5mg/L | 361 | 360 | 361 | 360.7 | 0.2% |
酚磺乙胺25mg/L | 361 | 360 | 359 | 360.0 | 0% |
酚磺乙胺50mg/L | 359 | 359 | 360 | 359.3 | -0.2% |
酚磺乙胺100mg/L | 359 | 357 | 359 | 358.3 | -0.5% |
上面各实施例1-4测定结果可知,本发明的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒,加入氧化还原电位高的氧化态高铁酸盐,或多聚偏钒酸盐,或偏钒酸盐,或五氧化二钒或三氯氧钒,均可有效的消除尿酸测定时羟苯磺酸钙及酚磺乙胺的干扰,而本试剂盒稳定性好,操作简单,检测反应迅速,特别是将过氧化物酶和尿酸酶分别制备于试剂R1和R2中,区别于市场上现有的试剂盒组成,能够在一段时间内延长试剂盒的稳定性,持续消除羟苯磺酸钙及酚磺乙胺的负干扰,避免医师在临床诊断时的误判,可以精准用药及治疗,适用于全自动生化分析仪。
Claims (8)
1.一种可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,其特征在于包括以下步骤:
R1试剂配制:
称取缓冲液→加入防腐剂→搅拌溶解→用氢氧化钠或盐酸调PH至8.0→加入氧化剂→搅拌溶解→调PH至8.0→加入Trinder’s反应剂A和非离子表面活性剂,混合均匀→2~8℃隔夜→加入抗坏血酸氧化酶和过氧化物酶→搅拌溶解→2~8℃隔夜→检测、分装;所述的Trinder’s反应剂A为4-氨基安替比林,所述氧化剂包括五氧化二钒、高铁酸钠、偏钒酸钠、三氯氧钒中的任一种;
R2试剂配制:
称取缓冲液→加入防腐剂→搅拌溶解→调PH至8.0→加入Trinder’s反应剂B和表面活性剂→混合均匀→2~8℃隔夜→加入血清白蛋白,尿酸酶→混合均匀→2~8℃隔夜→检测、分装,所述Trinder’s反应剂B为TOOS、DHBS、HDAOS、TBHBA、TOPS、TODB、4-氯酚的一种或两种以上混合。
2.根据权利要求1所述的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,其特征在于:所述试剂盒包括试剂R1和试剂R2,所述试剂R1中包含缓冲液、过氧化物酶、抗坏血酸氧化酶、防腐剂、Trinder’s反应剂A、氧化剂和非离子表面活性剂;所述试剂R2包含缓冲液、血清白蛋白、尿酸酶、防腐剂、Trinder’s反应剂B和非离子表面活性剂;所述的Trinder’s反应剂A为4-氨基安替比林,所述氧化剂包括五氧化二钒、高铁酸钠、偏钒酸钠、三氯氧钒中的任一种,所述Trinder’s反应剂B为TOOS、DHBS、HDAOS、TBHBA、TOPS、TODB、4-氯酚的一种或两种以上混合。
3.根据权利要求1所述的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,其特征在于:所述过氧化物酶是与尿酸酶结合作用的。
4.根据权利要求1所述的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,其特征在于:
所述R1中,缓冲液浓度为5~200mM,PH6.5~9.5;
过氧化物酶含量1.5~9ku/升;
抗坏血酸氧化酶1~10ku/升;
Trinder’s反应剂A 0.3~5mM/升;
防腐剂重量比为0.01%-0.5%,采用含量在0.1~10mM的氧化剂。
5.根据权利要求1所述的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,其特征在于:所述R1的缓冲液为BES、BICINE、DIPSO、EPPS、PIPES、HEPES、MOPS、TAPS、TAPSO、TES、Tricine、Tris、双甘肽、磷酸盐中的一种或一种以上混合;试剂R2所述的缓冲液为BES、BICINE、DIPSO、EPPS、PIPES、HEPES、MOPS、TAPS、TAPSO、TES、TRICINE、TRTS、双甘肽、磷酸盐中的一种或一种以上混合。
6.根据权利要求1所述的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,其特征在于:所述试剂R1及试剂R2中还含有非离子表面活性剂,其重量比含量为0.01%~0.3%,所述非离子型表面活性剂具体为BRIJ,EMULGEN,TRITON,TWEEN中的一种或两种。
7.根据权利要求1所述的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,其特征在于:所述防腐剂为Proclin、硫酸庆大霉素、PARABEN、氯霉素、叠氮钠、硼砂之一种或两种。
8.根据权利要求1所述的可消除血清中羟苯磺酸钙、酚磺乙胺药物干扰的尿酸试剂盒的制备方法,其特征在于:所述R2中,缓冲液浓度为20~200mM、PH6.5~9.5;尿酸酶含量5-20ku/升;Trinder’s反应剂B含量为0.1~20mM。
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