CN112198313A - 一种pea检测用的试剂盒及其使用方法 - Google Patents
一种pea检测用的试剂盒及其使用方法 Download PDFInfo
- Publication number
- CN112198313A CN112198313A CN202011034892.9A CN202011034892A CN112198313A CN 112198313 A CN112198313 A CN 112198313A CN 202011034892 A CN202011034892 A CN 202011034892A CN 112198313 A CN112198313 A CN 112198313A
- Authority
- CN
- China
- Prior art keywords
- pea
- antibody
- kit
- solution
- detecting
- 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 35
- 238000000034 method Methods 0.000 title claims abstract description 15
- 239000000243 solution Substances 0.000 claims abstract description 27
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 claims abstract description 24
- 238000005406 washing Methods 0.000 claims abstract description 21
- 108010084695 Pea Proteins Proteins 0.000 claims abstract description 18
- 235000019702 pea protein Nutrition 0.000 claims abstract description 18
- 238000002965 ELISA Methods 0.000 claims abstract description 12
- 229960002685 biotin Drugs 0.000 claims abstract description 12
- 235000020958 biotin Nutrition 0.000 claims abstract description 12
- 239000011616 biotin Substances 0.000 claims abstract description 12
- 239000007790 solid phase Substances 0.000 claims abstract description 11
- 239000000758 substrate Substances 0.000 claims abstract description 9
- 239000003085 diluting agent Substances 0.000 claims abstract description 6
- 239000003550 marker Substances 0.000 claims abstract description 6
- 239000012089 stop solution Substances 0.000 claims abstract description 6
- 239000008055 phosphate buffer solution Substances 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 7
- 235000018102 proteins Nutrition 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 6
- 238000009739 binding Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 230000027455 binding Effects 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 238000006911 enzymatic reaction Methods 0.000 claims description 4
- 230000003053 immunization Effects 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 238000011161 development Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 241000589516 Pseudomonas Species 0.000 description 7
- 230000000694 effects Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 206010011409 Cross infection Diseases 0.000 description 2
- 206010029803 Nosocomial infection Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 239000000304 virulence factor Substances 0.000 description 2
- 230000007923 virulence factor Effects 0.000 description 2
- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 description 1
- 102000009062 ADP Ribose Transferases Human genes 0.000 description 1
- 108010049290 ADP Ribose Transferases Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 208000032536 Pseudomonas Infections Diseases 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
- 231100000776 exotoxin Toxicity 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
Images
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- 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
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
-
- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提出了一种PEA检测用的试剂盒及其使用方法,包括PEA蛋白、抗PEA抗体、SABC、酶标板、酶底物溶液、稀释液、洗涤液和终止液,PEA蛋白作为生化指标,抗PEA抗体用生物素标记作为检测抗体标记物,抗PEA抗体包被的酶标板作为固相载体,SABC用于放大检测信号;本发明以生物素标记抗体作为检测抗体,采用双抗体夹心法提高ELISA检测的特异性和精确性,再利用SABC放大了检测信号,从而大大提高了检测的灵敏度,相比现有的实验室检测手段,高效迅速,操作流程短难度低,成本低廉。
Description
技术领域
本发明涉及抗原检测技术领域,尤其涉及一种PEA检测用的试剂盒及其使用方法。
背景技术
假单孢菌又称绿脓杆菌,是一种条件致病菌,几乎可以感染人所有的体表组织和器官,由于假单胞菌在人体和自然界广泛存在,并且对多种抗生素不敏感,加之多种传播途径,它引起的医院内感染已成为控制院内交叉感染的棘手问题。
假单孢菌主要的毒力因子为外毒素A,即PEA,其为细菌感染过程中细菌体内产生的一种胞外酶。PEA合成后为71kd的前体蛋白。去掉25个氨基酸的信号序列后,以66kd的成熟蛋白形式分泌到上清。它有613个氨基酸残基组成,分3个结构功能区:I区为氨基酸末端区,与靶细胞识别有关,能与相应的表面受体结合;II区为跨膜区,与毒素的迁移定位有关;III区为羧基末端区,具有 ADP-核糖基转移酶活性,为毒素分子的活性部分。PEA与靶细胞表面受体结合,通过膜运动聚集于膜特化区。胞膜内陷形成胞内泡,小泡与溶酶体融合。PEA在转移定位至靶细胞的胞质溶胶后,通过抑制蛋白质合成导致细胞病变。研究证实,PEA在非毒性剂量下会影响免疫系统,从而抑制抗原抗体反应,并且PEA 在体外实验中还能抑制淋巴细胞的转化。经过长期实验发现,抗PEA抗体在假单孢菌感染的小鼠中表现出保护性作用。
PEA作为假单胞菌的主要毒力因子,成为评价假单孢菌感染的一个重要指标。目前,针对于PEA的检测主要采用荧光定量检测,检测用的RT-PCR设备成本高,且检测流程时间长,操作难度大,普遍仅适用于实验室研究。因此,开发一种特异性强,灵敏度高,使用方便,快速检测PEA的试剂盒对于研究假单胞菌的作用机理具有重要意义。
发明内容
有鉴于此,本发明提出了一种检测迅速,特异性强,灵敏度高,使用方便的PEA检测用的试剂盒及其使用方法。
本发明的技术方案是这样实现的:本发明提供了一种PEA检测用的试剂盒,包括PEA蛋白、抗PEA抗体、SABC、酶标板、酶底物溶液、稀释液、洗涤液和终止液,PEA蛋白作为生化指标,抗PEA抗体用生物素标记作为检测抗体标记物,抗PEA抗体包被的酶标板作为固相载体,SABC用于放大检测信号。
在以上技术方案的基础上,优选的,PEA蛋白为大肠杆菌表达的重组蛋白,其氨基酸序列为 GPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEER GYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQ DQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLP LRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKE QAIS。
在以上技术方案的基础上,优选的,抗PEA抗体为抗PEA的多克隆抗体,其制备方法包括,用PEA蛋白免疫兔子获得含有抗PEA抗体的血清,再将血清经过PEA亲和柱纯化,并浓缩和透析后获得纯化的多克隆抗体。
在以上技术方案的基础上,优选的,酶标板为96孔酶标板。
在以上技术方案的基础上,优选的,酶底物溶液为TMB应用液。
在以上技术方案的基础上,优选的,稀释液为10mmol/L,pH7.2-7.4的磷酸盐缓冲液,其中含有1%的牛血清白蛋白和0.1%的proclin 300。
在以上技术方案的基础上,优选的,洗涤液为10mol/L,pH7.2-7.4的磷酸盐缓冲液,其中含有0.05%Tween-20和0.1%的proclin 300。
在以上技术方案的基础上,优选的,终止液为1.0mol/LH2SO4溶液。
另一方面,提供了一种PEA检测用的试剂盒的使用方法,包括以下步骤,
S1将待检样品用稀释液稀释后,投入酶标板的微孔中,使待检样品中的PEA 与固相载体上的抗PEA抗体结合,用洗涤液洗去未结合的杂蛋白和其他物质;
S2使步骤S1中的获得物与检测抗体标记物结合,用洗涤液洗去多余的生物素标记抗体;
S3向步骤S2中的获得物加入SABC,使SABC和生物素标记抗体形成复合物,用洗涤液洗去多余的SABC;
S4向步骤S3中的的获得物加入酶底物溶液,37℃下进行孵育反应,用终止液终止酶反应后检测显色强度。
本发明的一种PEA检测用的试剂盒及其使用方法相对于现有技术具有以下有益效果:
(1)本发明设计了一种新型的试剂盒,以生物素标记抗体作为检测抗体,采用双抗体夹心法提高检测的特异性和精确性,再利用SABC放大了检测信号,从而大大提高了ELISA检测的灵敏度,相比现有的实验室检测手段,高效迅速,操作流程短难度低,成本低廉。
(2)本发明中采用的蛋白为大肠杆菌表达的重组蛋白,特异性强,且兔免疫获得的抗体为多克隆体抗体,相较于现有的检测手段普遍使用的单抗或单抗和多抗组合,在保证了检测的快速、精确和灵敏的基础上,其获取手段更方便简单,成本更低。
附图说明
图1为本发明的实施例1至3的PEA剂量(浓度)-反应(吸光度)曲线。
具体实施方式
下面将结合本发明实施方式,对本发明实施方式中的技术方案进行清楚、完整地描述,显然,所描述的实施方式仅仅是本发明一部分实施方式,而不是全部的实施方式。基于本发明中的实施方式,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式,都属于本发明保护的范围。
实施例1
本发明的一种PEA检测用的试剂盒,包括PEA蛋白、抗PEA抗体、SABC、酶标板、TMB应用液、含有1%的牛血清白蛋白和0.1%的proclin 300的 10mmol/L,pH7.2-7.4的磷酸盐缓冲液、含有0.05%Tween-20和0.1%的proclin 300 的0.25mol/L,pH7.2-7.4的磷酸盐缓冲液和1.0mol/LH2SO4溶液,PEA蛋白作为生化指标,抗PEA抗体用生物素标记作为检测抗体标记物,抗PEA抗体包被 96孔酶标板作为固相载体,SABC用于放大检测信号。
其中,PEA蛋白为大肠杆菌表达的重组蛋白,其氨基酸序列为 GPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEER GYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQ DQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLP LRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKE QAIS。
具体的,抗PEA抗体为抗PEA的多克隆抗体,其制备方法包括,用PEA 蛋白免疫兔子获得含有抗PEA抗体的血清,再将血清经过PEA亲和柱纯化,并浓缩和透析后获得纯化的多克隆抗体。
另一方面,其使用方法,包括以下步骤,
S1将待检样品用含有1%的牛血清白蛋白和0.1%的proclin 300的10mol/L,pH7.2-7.4的磷酸盐缓冲液稀释后,投入酶标板的微孔中,使待检样品中的PEA 与固相载体上的抗PEA抗体结合,用含有0.05%Tween-20和0.1%的proclin 300 的10mol/L,pH7.2-7.4的磷酸盐缓冲液,洗去未结合的杂蛋白和其他物质;
S2使步骤S1中的获得物与检测抗体标记物结合,用10mol/L,pH7.2-7.4 的磷酸盐缓冲液洗去多余的生物素标记抗体;
S3向步骤S2中的获得物加入SABC,使SABC和生物素标记抗体形成复合物,用10mol/L,pH7.2-7.4的磷酸盐缓冲液洗去多余的SABC;
S4向步骤S3中的的获得物加入TMB应用液,37℃下进行孵育反应,用 1.0mol/LH2SO4溶液终止酶反应后检测显色强度。
需要说明的是,ELISA检测,即酶联免疫吸附测定,是指将可溶性的抗原或抗体结合到聚苯乙烯等固相载体上,利用抗原抗体特异性结合进行免疫反应的定性和定量检测方法。
还需要说明的是,双抗体夹心法,是将抗PEA抗体结合到作为固相载体的酶标板表面,并保持其免疫活性,并使抗PEA抗体与酶连接成酶标抗体。这种酶标抗体既保留其免疫活性,又保留酶的活性。在测定时,使待检样品内的PEA 蛋白和酶标抗体进行抗原抗体结合反应,然后用洗涤的方法使固相载体上形成的抗原抗体复合物与其他物质分开,最后结合在固相载体上的酶量与标本中受检物质的量成一定的比例。在加入酶反应的底物后,底物被酶催化变为有色产物,产物的量与标本中受检物质的量直接相关,故可根据颜色反应的深浅进行定性或定量分析。由于酶的催化效率很高,故可极大的放大反应效果,从而使测定方法具有很高的敏感度。
在本发明中,通过步骤S1使待检样品中的PEA与固相载体上的抗PEA抗体结合,以及通过步骤S2中使获得物与检测抗体标记物结合,实现了双抗体夹心,然后用SABC放大反应效果,进而达到了提高检测灵敏度和精确度的目的。
实施例2和3为实施例1的相同条件下的复孔试验,试验结果如下表和图1 所示。其中,图1为PEA剂量(浓度)-反应(吸光度)曲线。
剂量-反应 | 实施例1 | 实施例2 | 实施例3 |
0ng/ml | 0.078 | 0.078 | 0.078 |
0.078ng/ml | 0.135 | 0.134 | 0.135 |
0.156ng/ml | 0.206 | 0.192 | 0.200 |
0.3125ng/ml | 0.324 | 0.322 | 0.331 |
0.625ng/ml | 0.537 | 0.538 | 0.535 |
1.25ng/ml | 0.996 | 0.954 | 0.974 |
2.5ng/ml | 1.673 | 1.655 | 1.663 |
5ng/ml | 2.262 | 2.265 | 2.273 |
由此可知,本发明检测灵敏度小于46.9pg/ml,检测范围为78-5000pg/ml,剂量-反应曲线的线性相关系数大于0.990,具有简便,灵敏,准确,快速的优点,为快速定量检测PEA提供了有效的工具。
以上所述仅为本发明的较佳实施方式而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种PEA检测用的试剂盒,包括PEA蛋白、抗PEA抗体、SABC、酶标板、酶底物溶液、稀释液、洗涤液和终止液,其特征在于:所述PEA蛋白作为生化指标,所述抗PEA抗体用生物素标记作为检测抗体标记物,所述抗PEA抗体包被的酶标板作为固相载体,所述SABC用于放大检测信号。
2.根据权利要求1所述的一种PEA检测用的试剂盒,其特征在于:所述PEA蛋白为大肠杆菌表达的重组蛋白,其氨基酸序列为GPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAIS。
3.根据权利要求1所述的一种PEA检测用的试剂盒,其特征在于:所述抗PEA抗体为抗PEA的多克隆抗体,其制备方法包括,用PEA蛋白免疫兔子获得含有抗PEA抗体的血清,再将所述血清经过PEA亲和柱纯化,并浓缩和透析后获得纯化的所述多克隆抗体。
4.根据权利要求1所述的一种PEA检测用的试剂盒,其特征在于:所述酶标板为96孔酶标板。
5.根据权利要求1所述的一种PEA检测用的试剂盒,其特征在于:所述酶底物溶液为TMB应用液。
6.根据权利要求1所述的一种PEA检测用的试剂盒,其特征在于:所述稀释液为10mmol/L,pH7.2-7.4的磷酸盐缓冲液,其中含有1%的牛血清白蛋白和0.1%的proclin 300。
7.根据权利要求1所述的一种PEA检测用的试剂盒,其特征在于:所述洗涤液为10mol/L,pH7.2-7.4的磷酸盐缓冲液,其中含有0.05%Tween-20和0.1%的proclin 300。
8.根据权利要求1所述的一种PEA检测用的试剂盒,其特征在于:所述终止液为1.0mol/LH2SO4溶液。
9.一种PEA检测用的试剂盒的使用方法,其特征在于:包括以下步骤,
S1将待检样品用稀释液稀释后,投入所述酶标板的微孔中,使待检样品中的PEA与所述固相载体上的抗PEA抗体结合,用所述洗涤液洗去未结合的杂蛋白和其他物质;
S2使步骤S1中的获得物与所述检测抗体标记物结合,用所述洗涤液洗去多余的生物素标记抗体;
S3向步骤S2中的获得物加入所述SABC,使所述SABC和生物素标记抗体形成复合物,用所述洗涤液洗去多余的SABC;
S4向步骤S3中的的获得物加入酶底物溶液,37℃下进行孵育反应,用终止液终止酶反应后检测显色强度。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011034892.9A CN112198313A (zh) | 2020-09-27 | 2020-09-27 | 一种pea检测用的试剂盒及其使用方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011034892.9A CN112198313A (zh) | 2020-09-27 | 2020-09-27 | 一种pea检测用的试剂盒及其使用方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112198313A true CN112198313A (zh) | 2021-01-08 |
Family
ID=74007414
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011034892.9A Pending CN112198313A (zh) | 2020-09-27 | 2020-09-27 | 一种pea检测用的试剂盒及其使用方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112198313A (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101495516A (zh) * | 2005-07-29 | 2009-07-29 | 埃朗根-纽伦堡大学 | Cd-33特异性的单链免疫毒素及其使用方法 |
CN102590515A (zh) * | 2012-01-13 | 2012-07-18 | 张春华 | 艰难梭菌外毒素a检测试剂盒及组成该试剂盒的单克隆抗体 |
US20160025719A1 (en) * | 2014-07-25 | 2016-01-28 | Serum Institute Of India Ltd. | Highly sensitive immunoassay for rapid quantification of meningococcal capsular polysaccharide antigens |
CN111172301A (zh) * | 2019-12-27 | 2020-05-19 | 深圳市儿童医院 | 一种艰难梭菌毒素b的pcr荧光检测试剂盒及其应用 |
US20200249228A1 (en) * | 2019-01-31 | 2020-08-06 | Fresenius Medical Care Holdings, Inc. | Rapid diagnosis of peritonitis in peritoneal dialysis patients |
-
2020
- 2020-09-27 CN CN202011034892.9A patent/CN112198313A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101495516A (zh) * | 2005-07-29 | 2009-07-29 | 埃朗根-纽伦堡大学 | Cd-33特异性的单链免疫毒素及其使用方法 |
CN102590515A (zh) * | 2012-01-13 | 2012-07-18 | 张春华 | 艰难梭菌外毒素a检测试剂盒及组成该试剂盒的单克隆抗体 |
US20160025719A1 (en) * | 2014-07-25 | 2016-01-28 | Serum Institute Of India Ltd. | Highly sensitive immunoassay for rapid quantification of meningococcal capsular polysaccharide antigens |
US20200249228A1 (en) * | 2019-01-31 | 2020-08-06 | Fresenius Medical Care Holdings, Inc. | Rapid diagnosis of peritonitis in peritoneal dialysis patients |
CN111172301A (zh) * | 2019-12-27 | 2020-05-19 | 深圳市儿童医院 | 一种艰难梭菌毒素b的pcr荧光检测试剂盒及其应用 |
Non-Patent Citations (1)
Title |
---|
ALAIN R. OGAARD等: "Exotoxin a From Various Pseudomonas Aeruginosa Cultures: In Vitro Activity Measured by Enzyme-linked Immunosorbent", 《ACTA PATH. MICROBIOL. IMMUNOL. SCAND. SECT. B》, vol. 93, 31 December 1985 (1985-12-31), pages 218 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3037822B1 (en) | Mycoplasma pneumoniae immunological detection method and kit | |
CN112679605B (zh) | 针对新型冠状病毒核衣壳蛋白的抗体或其抗原结合片段及其应用 | |
JP2009185037A (ja) | EhrlichiacanisおよびEhrlichiachaffeensis抗体の検出のための組成物および方法 | |
Rennard et al. | Enzyme linked immunoassay (ELISA) for connective tissue proteins: type I collagen | |
WO2018208977A1 (en) | Optimizing diagnostics for galactofuranose containing antigens | |
CN114878833A (zh) | 一种检测抗过氧化物还原酶-1-IgG抗体的试剂盒 | |
US20120064543A1 (en) | Method for detecting substance in biological sample | |
CN112305230A (zh) | 一种定量检测降钙素原的免疫层析试纸条及其定量检测方法 | |
CN113024669A (zh) | 含氨基或羧基基团物质标记的rbp抗体、人rbp免疫层析检测试剂盒及其制备方法 | |
US8178344B2 (en) | System and method for antigen structure-independent detection of antigens captured on antibody arrays | |
CN112198313A (zh) | 一种pea检测用的试剂盒及其使用方法 | |
CN114720691B (zh) | 一种检测生物标志物的试剂盒及其制备方法和应用 | |
CN114702579B (zh) | 一种碱性磷酸酶标记抗体及其制备方法 | |
US11320427B2 (en) | Tandemly repeated antibody-binding protein and its applications | |
US6489148B1 (en) | Immunoassay for equine protozoal myeloencephalitis in horses | |
EP1504265B1 (en) | Sandwich assay | |
KR20060027404A (ko) | 사료 효소 검출 시약, 방법 및 키트 | |
KR20200015276A (ko) | 쯔쯔가무시균 유래 엑소좀을 이용한 쯔쯔가무시병의 진단방법 | |
KR101311736B1 (ko) | 샌드위치 면역분석 바이오칩 및 이를 사용한 면역분석법 | |
Castro et al. | Highly sensitive biotin-avidin sandwich ELISA for the rapid detection of pneumococcal capsular polysaccharide antigens | |
KR102404143B1 (ko) | 라싸 바이러스 핵단백질에 특이적인 항체, 이를 생산하는 하이브리도마 세포주 및 이를 이용한 라싸 바이러스 검출 키트 | |
RU2545909C2 (ru) | Способ иммунохроматографического определения специфических антител | |
KR20100101248A (ko) | 리스테리아 오염 탐지용 특이단백질 부위 및 그에 대한 항체제조법과 활용 | |
KR20170041956A (ko) | 유럽형 돼지생식기호흡기증후군 바이러스 특이항체 진단용 래피드 키트 | |
WO2021133328A1 (en) | Mannheimia haemolytica serotype a1 lateral flow immunochromatography diagnosis kit |
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 |