CN117269503A - 可溶性生长刺激表达基因2蛋白(st2) 检测试剂盒 - Google Patents
可溶性生长刺激表达基因2蛋白(st2) 检测试剂盒 Download PDFInfo
- Publication number
- CN117269503A CN117269503A CN202311000592.2A CN202311000592A CN117269503A CN 117269503 A CN117269503 A CN 117269503A CN 202311000592 A CN202311000592 A CN 202311000592A CN 117269503 A CN117269503 A CN 117269503A
- Authority
- CN
- China
- Prior art keywords
- reagent
- protein
- detection
- soluble growth
- antibody
- 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
- 101710122194 Gene 2 protein Proteins 0.000 title claims abstract description 15
- 101710137426 Replication-associated protein G2P Proteins 0.000 title claims abstract description 15
- 230000000638 stimulation Effects 0.000 title claims abstract description 12
- 238000001514 detection method Methods 0.000 title abstract description 42
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 54
- 239000006249 magnetic particle Substances 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 239000000427 antigen Substances 0.000 claims abstract description 12
- 102000036639 antigens Human genes 0.000 claims abstract description 12
- 108091007433 antigens Proteins 0.000 claims abstract description 12
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 239000004005 microsphere Substances 0.000 claims abstract description 9
- 239000004793 Polystyrene Substances 0.000 claims abstract description 7
- 239000000872 buffer Substances 0.000 claims abstract description 7
- 229920002223 polystyrene Polymers 0.000 claims abstract description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 5
- 239000003792 electrolyte Substances 0.000 claims abstract description 4
- 239000012634 fragment Substances 0.000 claims abstract description 4
- 239000003755 preservative agent Substances 0.000 claims abstract description 4
- 230000002335 preservative effect Effects 0.000 claims abstract description 4
- 239000004094 surface-active agent Substances 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 13
- 244000309466 calf Species 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 4
- 241001494479 Pecora Species 0.000 claims description 3
- 239000004098 Tetracycline Substances 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- PGBHMTALBVVCIT-VCIWKGPPSA-N framycetin Chemical compound N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CN)O2)N)O[C@@H]1CO PGBHMTALBVVCIT-VCIWKGPPSA-N 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229940053050 neomycin sulfate Drugs 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 229960002180 tetracycline Drugs 0.000 claims description 3
- 229930101283 tetracycline Natural products 0.000 claims description 3
- 235000019364 tetracycline Nutrition 0.000 claims description 3
- 150000003522 tetracyclines Chemical class 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000003149 assay kit Methods 0.000 claims 4
- 230000004936 stimulating effect Effects 0.000 claims 3
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 206010019280 Heart failures Diseases 0.000 description 10
- 238000003317 immunochromatography Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 206010007556 Cardiac failure acute Diseases 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000012123 point-of-care testing Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 206010007558 Cardiac failure chronic Diseases 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 239000007987 MES buffer Substances 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 206010003658 Atrial Fibrillation Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000019223 Interleukin-1 receptor Human genes 0.000 description 1
- 108050006617 Interleukin-1 receptor Proteins 0.000 description 1
- 206010028594 Myocardial fibrosis Diseases 0.000 description 1
- 102400001263 NT-proBNP Human genes 0.000 description 1
- 108020001621 Natriuretic Peptide Proteins 0.000 description 1
- 102000004571 Natriuretic peptide Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical class C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000001648 catalysed chemiluminescence detection Methods 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000692 natriuretic peptide Substances 0.000 description 1
- 239000000101 novel biomarker Substances 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 108010008064 pro-brain natriuretic peptide (1-76) Proteins 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
- G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及免疫医学检测技术领域,具体是指可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒,包括所述试剂盒包含第一试剂,第二试剂,校准品,抗试剂、磁微粒试剂、发光底物,其中所述第一试剂包含磁微粒偶联的ST2蛋白抗体、30g/L的电解质、20‑70g/L的促进剂、0.5‑10g/L防腐剂、1‑20g/L表面活性剂和pH为6.5‑9.5的1‑100mmol/L的缓冲液;所述第二试剂包含酶标记的ST2蛋白抗体或其抗原结合片段,按w/v计0.05%‑5%的带有羧基的聚苯乙烯微球,和pH为5.5~9.5的1‑200mmol/L的缓冲液;优点在于:使得检测灵敏度、精密性大大提高、检测范围扩大,准确度高,特异性强,精确度和检测效率有了较大的提高。
Description
技术领域
本发明涉及免疫医学检测技术领域,具体是指可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒。
背景技术
生长刺激表达基因2蛋白(growth STimulation expressed gene 2,ST2)是白介素1受体家族的成员,其中可溶性ST2(sST2)和跨膜型ST2(ST2L)是心力衰竭(HF)的主要研究对象。ST2在免疫和炎症反应中起到关键作用。2013ACC/AHA/HFSA心衰指南指出,可溶性ST2为一个心肌纤维化的标志物,可以预测心衰患者的入院和死亡概率,还可以为利钠肽类增加额外的预测价值。ST2有一些独特的优点,如检测结果不受原有心血管疾病、房颤、年龄、提质量指数等影响,而NT-proBNP的浓度值随着肾功能的衰减显著升高(Bayes-Genisetal.2013JCF)。研究发现,当急性失代偿性心衰患者ST2>35ng/ml时,死亡和因心衰再住院风险显著增高。PRIDE研究表明,ST2可以预测急性心衰患者30天的死亡率。在急性发病后前30天就可通过检测ST2识别出高风险患者,预测价值达一年以上。ST2动态检测有助于预测住院心衰患者出院后因心衰再住院的风险。慢性心衰患者ST2浓度与一年内死亡率成正相关,其测定值在最高十分位人群一年死亡率超过50%。可溶性ST2作为HF的一种新型生物标志物,目前已被纳入美国心衰管理指南和《中国心心力衰竭诊断和治疗指南2014》,主要用于评估急性心力衰竭和慢性心力衰竭病人的危险分级、预后并指导医生药物治疗。
目前市售的可溶性ST2的免疫检测方法主要是酶联免疫法。酶联免疫法(ELISA)检测原理,以双抗体夹心法为例,是在固相载体上包被一种特异性抗体,加入待测样本后,再加入酶标记的另一种抗体,形成双抗体夹心复合物,再通过发光底物的显色作用,使仪器可以定量检测出的免疫反应。国内已申请专利的可溶性ST2检测技术包括胶体金免疫层析法(公开号CN204514934Μ)和磁微粒化学发光法(公开号CN110208549A、CN108152486A)等。
免疫层析(ICA)作为一种快速诊断技术,其原理是将特异的抗体先固定于硝酸纤维素膜的某一区带,当该干燥的硝酸纤维素一端浸入样品(尿液或血清)后,由于毛细管作用,样品将沿着该膜向前移动,当移动至固定有抗体的区域时,样品中相应的抗原即与该抗体发生特异性结合从而实现特异性的免疫诊断。目前主要的检测方法为胶体金法、荧光免疫层析、荧光微球层析、时间分辨荧光免疫测定等。而胶体金免疫层析法作为即时检验(point-of-care testing,POCT)的一种方法,具有上样量少、简便快速的优点,适用于床旁检验。其原理如下,仍以双抗体夹心法为例,当含有抗原的样本滴加到吸收孔后,抗原先与胶体金标记的抗体结合形成抗原-抗体复合物,然后固定在样品垫上的另一种抗原会捕获该复合物,形成双抗体夹心复合物,红色的检测线就会显示出来,并且检测线上复合物越多,光密度越大。
磁微粒化学发光法作为近年来逐渐兴起的一种免疫检测技术,是将免疫反应系统、磁性分离技术和化学发光技术紧密结合的产物,主要分为直接化学发光和间接化学发光。其中磁微粒酶促化学发光技术属于典型的间接化学发光,是以酶标记抗体,与样本中的抗原进行免疫反应形成复合物,再连接在磁微粒上,在外加磁场中将免疫复合物与未结合的其他物质分离,免疫复合物上的酶再作用于其相应的底物,从而实现化学发光。其中,常用的标记酶主要有碱性磷酸酶(ALP)和辣根过氧化物酶(HRP)。直接化学发光是标记物直接标记抗体,与待测物形成复合物,在碱性/酸性条件下,加入强氧化剂,瞬间发光,产生的光子能与待测物的量成正比的一种技术。常用的标记物有吖啶酯等。
酶联免疫吸附法(ELISA),存在着检测周期较长、灵敏度低、操作繁琐和线性范围较窄等缺点。目前市售的ELISA试剂盒,其检测时长约4-6小时,灵敏度为1.8ng/ml,线性范围为3-200ng/ml。这些缺陷导致其无法及时有效的帮助医生判断病人的情况。
胶体金免疫层析由于是人工操作,因此操作误差大,精密度和重复性相对较差,灵敏度和线性范围较差,检测结果易受外界环境影响。
酶促化学发光(CN107991485A)由于常用的辣根过氧化物酶和碱性磷酸酶的成本较高,而导致检测成本较高;且酶容易受外界因素如温度等影响,从而干扰检测结果。以及底物到达平台期时间较长,从而降低了检测速度。目前国内已申请的专利磁微粒酶促化学发光法(公开号CN110208549A),其灵敏度为1ng/ml,检测范围为1-300ng/ml,批内变异系数在±10%范围内。国内已申请的专利磁微粒化学发光检测试剂(CN108152486A)是人工操作,存在一定的操作误差。
因此,亟需一种能够快速、准确且能够高灵敏度、高特异性的磁珠化学发光检测试剂盒。
发明内容
本发明要解决的技术问题是克服以上技术缺陷,提供可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒。
为解决上述技术问题,本发明提供的技术方案为:包括所述试剂盒包含第一试剂,第二试剂,校准品,抗试剂、磁微粒试剂、发光底物。
其中所述第一试剂包含磁微粒偶联的ST2蛋白抗体、30g/L的电解质、20-70g/L的促进剂、0.5-10g/L防腐剂、1-20g/L表面活性剂和pH为6.5-9.5的1-100mmol/L的缓冲液;
所述第二试剂包含酶标记的ST2蛋白抗体或其抗原结合片段,按w/v计0.05%-5%的带有羧基的聚苯乙烯微球,和pH为5.5~9.5的1-200mmol/L的缓冲液。
所述校准品是通过在1L的新生牛血清中加入0.03g~0.06g的四环素和0.5g~0.8g的硫酸新霉素,完全溶解后经过0.3μm滤膜处理制备而成。
作为优选的,所述酶标记的ST2蛋白抗体的浓度为大于或等于2.5ng/ml,且小于或等于4.5ng/ml。
作为优选的,所述抗试剂缓冲液的制备:5~10Na 2PO 3H·12H 2O、2~4的NaPO3H2·12H2O、2~56的绵羊血清、35~15的新生牛血清、1g~3g的马血清加入1L纯化水中,充分搅拌至完全溶解,用4M HC1调节pH至5~6,制得所述抗试剂缓冲液。
作为优选的,所述第二试剂的终浓度为0.1~3μg/ml;优选为1.25~2μg/ml;更优选为1.5~1μg/ml;
作为优选的,所述的可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒的应用:包括以下步骤:
(1)依次混合待测样品、第一试剂、第二试剂,依次混合校准品、抗试剂、磁微粒试剂、,分别孵育并洗涤后加入发光底物;
(2)检测所述发光底物的相对发光强度,通过检测得到的相对发光强度,计算ST2蛋白的浓度;其中,所述裸ST2蛋白抗体与所述酶标记的ST2蛋白抗体用以竞争性地结合待测抗原。
有益效果,本发明与现有的技术相比的优点在于:
1、该试剂盒将化学发光技术与磁微粒相结合,提供了一种接近均相的反应体系,并且采用了一步法反应模式,使得检测灵敏度、精密性大大提高、检测范围扩大,反应时间大大缩短,从开始加样到检测结果,时间少于20min,明显快于同类试剂盒;
2、试剂盒中校准品、质控品、发光底物液和浓缩洗液均是该反应体系下的最优配方,给该试剂盒的使用效期及检测性能提供了有力保障;
3、本发明可以在全自动化学发光仪上同时测定多个样本,实现可溶性生长刺激表达基因2蛋白的高通量快速化测定,准确度高,特异性强,精确度和检测效率有了较大的提高。
附图说明
图1是本发明ELISA试剂盒检测样本符合率图。
图2是本发明试剂盒的线性范围检测结果图。
图3是本发明试剂盒的范围检测结果表。
具体实施方式
下面对发明作进一步说明。
可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒,其特征在于,包括所述试剂盒包含第一试剂,第二试剂,校准品,抗试剂、磁微粒试剂、发光底物。
其中所述第一试剂包含磁微粒偶联的ST2蛋白抗体、30g/L的电解质、20-70g/L的促进剂、0.5-10g/L防腐剂、1-20g/L表面活性剂和pH为6.5-9.5的1-100mmol/L的缓冲液;
所述第二试剂包含酶标记的ST2蛋白抗体或其抗原结合片段,按w/v计0.05%-5%的带有羧基的聚苯乙烯微球,和pH为5.5~9.5的1-200mmol/L的缓冲液。
所述校准品是通过在1L的新生牛血清中加入0.03g~0.06g的四环素和0.5g~0.8g的硫酸新霉素,完全溶解后经过0.3μm滤膜处理制备而成。
一种新的FITC抗体与磁微粒偶联方法,该方法偶联效率高,结合牢固,且工艺稳定,在提高产品性能的同时,大大降低了产品成本。
所述酶标记的ST2蛋白抗体的浓度为大于或等于2.5ng/ml,且小于或等于4.5ng/ml。
所述抗试剂缓冲液的制备:5~10Na 2PO 3H·12H 2O、2~4的NaPO 3H 2·12H2O、2~56的绵羊血清、35~15的新生牛血清、1g~3g的马血清加入1L纯化水中,充分搅拌至完全溶解,用4M HCl调节pH至5~6,制得所述抗试剂缓冲液。
所述第二试剂的终浓度为0.1~3μg/ml;优选为1.25~2μg/ml;更优选为1.5~1μg/ml;
所述的可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒的应用:包括以下步骤:
(1)依次混合待测样品、第一试剂、第二试剂,依次混合校准品、抗试剂、磁微粒试剂、,分别孵育并洗涤后加入发光底物;
(2)检测所述发光底物的相对发光强度,通过检测得到的相对发光强度,计算ST2蛋白的浓度;其中,所述裸ST2蛋白抗体与所述酶标记的ST2蛋白抗体用以竞争性地结合待测抗原。
实施例1:
反应体系参数的优化
(1)取9支离心管,分别加入10mg粒径为200、260和300nm带有羧基的聚苯乙烯微球,每三个离心管加入相同粒径的聚苯乙烯微球,装有相同粒径的羧基的聚苯乙烯微球的离心管分别加入pH5.5、6.5、7.5的MES缓冲液至1mL,每个pH条件下3支,分别标记为A1、A2、A3、B1、B2、B3、C1、C2和C3(微球活化PH选择);
(2)分别对A1、B1、C1的离心管中加入10μL20mg/mLEDC,分别对A2、B2、C2的离心管中加入20μL20mg/mLEDC、分别对A3、B3、C3的离心管中加入30μL的20mg/mLEDC,活化25min,离心,取沉淀分别重悬于对应1mL pH5.5、6.5、7.5的MES缓冲液中(EDC用量);
(3)加入0.1mg抗体,偶联1H;
(4)离心,取沉淀重悬于对应1mL PH5.5-9.5MES缓冲液中,再加入0.2mL10%BSA(260nm组平行加入0.2mL 0.1酪蛋白),封闭1h;
(5)以PB缓冲液定容为5mL,制得试剂R2;
(6)按照实施例1的方法(6.1-6.7)制备试剂R1;
(7)按照实施例1的方法(7.1-7.13)制备校准品、质控品;
(8)按照实施例1步骤(8)的方法获得相对标识浓度的偏差,根据测定质控品的相对偏差得到最佳反应体系参数。
实施例2:
随机取本发明检测试剂盒的三批试剂(5.0ng/ml磁微粒偶联的ST2蛋白抗体),分别对ST2蛋白浓度为30ng/ml和100ng/ml的样品进行检测,来验证本发明检测试剂盒的精密度。两个ST2蛋白样本各做10个平行测试,共获得30个检测结果。分别对30个检测结果的批内差及批间差进行计算。第1批试剂检测30ng/ml样品的批内差的变异系数为5.32%,检测100ng/ml样品的批内差的变异系数为3.11%;第2批试剂检测30ng/ml样品的批内差的变异系数为3.33%,检测100ng/ml样品的批内差的变异系数为3.11%;第3批试剂检测30ng/ml样品的批内差的变异系数为2.96%,检测100ng/ml样品的批内差的变异系数为2.31%;三批试剂的批内差的变异系数均<4%,符合检测标准。三批试剂检测30ng/ml样品的批间差的变异系数为6.40%,检测100ng/ml样品的批间差的变异系数为6.29%;变异系数均<7%,符合检测标准。以上结果说明,本发明检测试剂盒的精密度好。
以上对本发明及其实施方式进行了描述,这种描述没有限制性,总而言之如果本领域的普通技术人员受其启示,在不脱离本发明创造宗旨的情况下,不经创造性的设计出与该技术方案相似的结构方式及实施例,均应属于本发明的保护范围。
Claims (5)
1.可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒,其特征在于,包括所述试剂盒包含第一试剂,第二试剂,校准品,抗试剂,磁微粒试剂,发光底物。
其中所述第一试剂包含磁微粒偶联的ST2蛋白抗体、30g/L的电解质、20-70g/L的促进剂、0.5-10g/L防腐剂、1-20g/L表面活性剂和pH为6.5-9.5的1-100mmol/L的缓冲液;
所述第二试剂包含酶标记的ST2蛋白抗体或其抗原结合片段,按w/v计0.05%-5%的带有羧基的聚苯乙烯微球,和pH为5.5~9.5的1-200mmol/L的缓冲液。
所述校准品是通过在1L的新生牛血清中加入0.03g~0.06g的四环素和0.5g~0.8g的硫酸新霉素,完全溶解后经过0.3μm滤膜处理制备而成。
2.根据权利要求1所述的可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒,其特征在于:所述酶标记的ST2蛋白抗体的浓度为大于或等于2.5ng/ml,且小于或等于4.5ng/ml。
3.根据权利要求1所述的可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒,其特征在于:所述抗试剂缓冲液的制备:5~10Na 2PO 3H·12H 2O、2~4的NaPO 3H 2·12H 2O、2~56的绵羊血清、35~15的新生牛血清、1g~3g的马血清加入1L纯化水中,充分搅拌至完全溶解,用4M HCl调节pH至5~6,制得所述抗试剂缓冲液。
4.根据权利要求1所述的可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒,其特征在于:所述第二试剂的终浓度为0.1~3μg/ml;优选为1.25~2μg/ml;更优选为1.5~1μg/ml。
5.根据权利要求1-4任一项所述的可溶性生长刺激表达基因2蛋白(ST2)检测试剂盒的应用:包括以下步骤:
(1)依次混合待测样品、第一试剂、第二试剂,依次混合校准品、抗试剂、磁微粒试剂、,分别孵育并洗涤后加入发光底物;
(2)检测所述发光底物的相对发光强度,通过检测得到的相对发光强度,计算ST2蛋白的浓度;其中,所述裸ST2蛋白抗体与所述酶标记的ST2蛋白抗体用以竞争性地结合待测抗原。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311000592.2A CN117269503A (zh) | 2023-08-10 | 2023-08-10 | 可溶性生长刺激表达基因2蛋白(st2) 检测试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311000592.2A CN117269503A (zh) | 2023-08-10 | 2023-08-10 | 可溶性生长刺激表达基因2蛋白(st2) 检测试剂盒 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117269503A true CN117269503A (zh) | 2023-12-22 |
Family
ID=89207119
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311000592.2A Pending CN117269503A (zh) | 2023-08-10 | 2023-08-10 | 可溶性生长刺激表达基因2蛋白(st2) 检测试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117269503A (zh) |
-
2023
- 2023-08-10 CN CN202311000592.2A patent/CN117269503A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220155296A1 (en) | Peptides, Reagents And Methods For Detecting Food Allergy | |
WO2016127318A1 (zh) | 心肌肌钙蛋白i超敏检测试剂盒及其超敏检测方法 | |
CA2779191C (en) | Method and kit for measuring component to be assayed in specimen | |
CN109863406B (zh) | 用于测定维生素d的方法和成分 | |
JPH0562302B2 (zh) | ||
WO2016127320A1 (zh) | 一种用于检测胃泌素-17的试剂盒及其制备方法和应用 | |
CN112014566A (zh) | 一种氨基末端脑利钠肽前体检测试剂盒、制备方法及用途 | |
JP7503505B2 (ja) | 自己抗体の直接イムノアッセイ測定法 | |
CN114252592B (zh) | 一种可溶性fms样酪氨酸激酶-1检测试剂盒及其制备方法与应用 | |
CN114252594A (zh) | 一种胎盘生长因子检测试剂盒及其制备方法与应用 | |
WO2016175406A1 (ko) | 쇼그렌증후군 특이적 항체반응 검사를 이용한 쇼그렌증후군 진단방법 | |
US20040077027A1 (en) | Assays and kits for detecting and monitoring heart disease | |
JP2010091308A (ja) | レクチン吸収法による前立腺がんの診断方法及び判定キット | |
CN115651065A (zh) | 一种用于肝纤维化和肝硬化检测的试剂盒 | |
CN117269503A (zh) | 可溶性生长刺激表达基因2蛋白(st2) 检测试剂盒 | |
EP4286850A1 (en) | Immunological assay method | |
CN114371292A (zh) | 一种检测可溶性st2蛋白的试剂盒 | |
Yang et al. | Development of a novel parallel determination platform: a feasibility study tested on a chemiluminescence device | |
EP3978921A1 (en) | Method and reagent for measuring thyroglobulin | |
CN111505268A (zh) | 一种自身免疫抗体检测方法 | |
CN111289759B (zh) | Saa检测试剂盒及saa定量检测的方法 | |
WO2023229015A1 (ja) | 測定方法及び測定装置 | |
CA2246894C (en) | Ligand binding surfaces | |
TW202221323A (zh) | 用於檢測心肌肌鈣蛋白i的方法以及利用該方法之用於診斷心肌肌鈣蛋白i-相關疾病的方法與套組 | |
JP4013270B2 (ja) | 尿中生理活性ペプチドの測定法及び測定用試薬 |
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 |