CN109212218A - 基于apoa4蛋白胃癌检测试剂盒、及其使用方法 - Google Patents
基于apoa4蛋白胃癌检测试剂盒、及其使用方法 Download PDFInfo
- Publication number
- CN109212218A CN109212218A CN201811319452.0A CN201811319452A CN109212218A CN 109212218 A CN109212218 A CN 109212218A CN 201811319452 A CN201811319452 A CN 201811319452A CN 109212218 A CN109212218 A CN 109212218A
- Authority
- CN
- China
- Prior art keywords
- solution
- apoa4
- albumen
- gastric cancer
- detection kit
- 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
- 102100037320 Apolipoprotein A-IV Human genes 0.000 title claims abstract description 61
- 208000005718 Stomach Neoplasms Diseases 0.000 title claims abstract description 50
- 238000001514 detection method Methods 0.000 title claims abstract description 49
- 206010017758 gastric cancer Diseases 0.000 title claims abstract description 43
- 201000011549 stomach cancer Diseases 0.000 title claims abstract description 43
- 101000806793 Homo sapiens Apolipoprotein A-IV Proteins 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000000243 solution Substances 0.000 claims abstract description 44
- 239000012224 working solution Substances 0.000 claims abstract description 33
- 108010073614 apolipoprotein A-IV Proteins 0.000 claims abstract description 29
- 239000003085 diluting agent Substances 0.000 claims abstract description 18
- 102100033879 Alpha-amylase 1A Human genes 0.000 claims abstract description 11
- 101000779871 Homo sapiens Alpha-amylase 1A Proteins 0.000 claims abstract description 11
- 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 10
- 239000011534 wash buffer Substances 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 210000002966 serum Anatomy 0.000 claims abstract description 6
- 238000002965 ELISA Methods 0.000 claims abstract description 5
- 229960002685 biotin Drugs 0.000 claims abstract description 5
- 235000020958 biotin Nutrition 0.000 claims abstract description 5
- 239000011616 biotin Substances 0.000 claims abstract description 5
- 230000021615 conjugation Effects 0.000 claims abstract description 4
- 239000000203 mixture Substances 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims description 10
- 238000011534 incubation Methods 0.000 claims description 9
- 238000002835 absorbance Methods 0.000 claims description 8
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 210000005259 peripheral blood Anatomy 0.000 claims description 6
- 239000011886 peripheral blood Substances 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 3
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 13
- 201000000498 stomach carcinoma Diseases 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 235000012000 cholesterol Nutrition 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 102000004895 Lipoproteins Human genes 0.000 description 5
- 108090001030 Lipoproteins Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 101150020529 APOA4 gene Proteins 0.000 description 4
- 108010004103 Chylomicrons Proteins 0.000 description 4
- 108010010234 HDL Lipoproteins Proteins 0.000 description 4
- 102000015779 HDL Lipoproteins Human genes 0.000 description 4
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 4
- 102000043296 Lipoprotein lipases Human genes 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 102000014190 Phosphatidylcholine-sterol O-acyltransferase Human genes 0.000 description 3
- 108010011964 Phosphatidylcholine-sterol O-acyltransferase Proteins 0.000 description 3
- 108010062497 VLDL Lipoproteins Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000004262 dental pulp cavity Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000004141 reverse cholesterol transport Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 150000003626 triacylglycerols Chemical class 0.000 description 2
- 239000000439 tumor marker Substances 0.000 description 2
- KEWSCDNULKOKTG-UHFFFAOYSA-N 4-cyano-4-ethylsulfanylcarbothioylsulfanylpentanoic acid Chemical compound CCSC(=S)SC(C)(C#N)CCC(O)=O KEWSCDNULKOKTG-UHFFFAOYSA-N 0.000 description 1
- 101150071783 APOA1 gene Proteins 0.000 description 1
- 101150001527 APOC3 gene Proteins 0.000 description 1
- 101150037123 APOE gene Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 102100033715 Apolipoprotein A-I Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 102100037637 Cholesteryl ester transfer protein Human genes 0.000 description 1
- 101100216294 Danio rerio apoeb gene Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 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 description 1
- 101000880514 Homo sapiens Cholesteryl ester transfer protein Proteins 0.000 description 1
- 102000016267 Leptin Human genes 0.000 description 1
- 108010092277 Leptin Proteins 0.000 description 1
- 108020005198 Long Noncoding RNA Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- -1 cholesteryl ester Chemical class 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 235000009200 high fat diet Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 1
- 229940039781 leptin Drugs 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000008604 lipoprotein metabolism Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003360 nephrocyte Anatomy 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000005748 tumor development Effects 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明公开了基于APOA4蛋白胃癌检测试剂盒，所述检测试剂盒的组成包括：溶液A：采用抗体稀释液将Human APOA4蛋白稀释成工作溶液，作为标准品；溶液B：采用生物素检测抗体稀释液将浓缩生物素检测抗体稀释成工作溶液，所述浓缩生物素检测抗体采用鼠抗人AMY1A单克隆抗体；溶液C：采用HPR共轭稀释剂将浓缩HRP结合物稀释成工作溶液；溶液D：采用抗体稀释液将兔抗人AMY1A多克隆抗体稀释成工作溶液；溶液E：Wash Buffer；溶液F：显色液；溶液G：终止液。本发明采用ELISA法检测将所述的基于APOA4蛋白胃癌检测试剂盒用于检测血清中APOA4蛋白表达水平，成本较低，操作方便，利于基层推广普及应用。
Description
技术领域
本发明涉及生物医学技术领域，具体涉及一种基于APOA4蛋白胃癌检测试剂盒、及其使用方法。
背景技术
胃癌(Gastric Cancer)是世界上发病率最高的肿瘤之一，如何提高胃癌患者的早诊早治是目前国内研究的热点问题。因为早期胃癌没有明显的临床表现，胃癌的早期临床表现也常常与胃炎相混淆，所以大多数胃癌患者即使肿瘤发展至进展期或者晚期，也没有特异性临床症状出现，因而依靠临床表现诊断胃癌显然无法实现。虽然胃镜筛查已经成为中年以上人群的体检项目，但由于是侵入性检查方法，普通民众接受度不高，另检查设备成本较高在基层普及具有一定的困难。一些常规的检测指标诸如CEA、CA199和CA724等用于消化道肿瘤检测的肿瘤标志性蛋白质，也由于在普通人群中筛查的敏感性和特异性均不高的原因只能用于部分胃癌患者的术后复发与转移的监测。目前寻找特异性肿瘤标记物，尤其是在外周血中寻找对于胃癌有明确特异性的标志性蛋白质分子具有很高的临床价值。
APOA4基因编码载脂蛋白A-IV，该蛋白经水解、糖基化后生成酸性糖蛋白，主要存在于乳糜微粒(CM)、极低密度脂蛋白(VLDL)和高密度脂蛋白(HDL)中。在血脂运输和代谢，特别是在胆固醇逆转运过程中起重要作用。
国内外的众多研究表明，APOA4基因上的多态现象与血糖、血浆脂蛋白、胆固醇以及甘油三酯等的水平相关。APOA4基因的缺陷可直接导致血浆中的载脂蛋白A-IV水平的下降或活性的改变，其他一些脂蛋白和胆固醇的水平相应升高，从而大大提高罹患高脂蛋白血症和冠心病的风险。另外，也有研究发现APOA4基因和阿尔茨海默氏症也存在着一定的关系。
基因结构：APOA4全称Apolipoprotein A-IV(载脂蛋白A-IV)，位于第11号染色体11q23位置，与APOA1及APOC3基因连锁。该基因全长2,603bp,共有3个外显子和2个内含子。mRNA全长1,460nt，编码由396个氨基酸残基组成的蛋白。
生物学功能：载脂蛋白A-IV(apoA-IV)是一种酸性糖蛋白，分子量46kD。它是血浆脂蛋白家族中最常见的成员，具有多种调节脂蛋白代谢的功能。apoA-IV在人类和啮齿类动物肠道中广泛存在，主要由小肠和肝脏分泌。在哺乳动物中，apoA-IV主要是由小肠绒毛上皮细胞在吸收脂肪时合成，然后结合在乳糜微粒表面，随之进入循环系统。因此apoA-IV合成和分泌受乳糜微粒合成、瘦素、高脂饮食等多种因素的影响。
虽然有大量研究揭示了apoA-IV众多生理功能，但其确切作用机制尚不清楚，目前公认的生理生化功能有：
1)激活卵磷脂胆固醇酰基转移酶(LCAT)，催化游离胆固醇转变为胆固醇酯，参与HDL的成熟过程。Steinmetz等人的体外实验证明了apoA-IV同apoA-I类似都能激活LCAT，其激活效率与底物有关；
2)辅助apoC-Ⅱ激活脂蛋白脂酶(LPL)，LPL能水解血浆CM和VLDL中的甘油三酯，因此也参与外源性脂肪的吸收。Goldbery等人的实验证明只有在apoA-Ⅱ存在的条件下，apoA-IV能提高LPL活性，而apoA-I、apoE、CETP则无此作用；
3)apoA-IV能促进细胞内胆固醇外流，将胆固醇从外周细胞转运至肝细胞，此过程被称为胆固醇的逆转运(RCT)。apoA-IV几乎参与了此过程的每个步骤。Stein等人用体外细胞培养实验直接证明apoA-IV是转运胆固醇的极好接受体；
4)apoA-IV能与细胞结合，如肝细胞、肾细胞等。与其他脂蛋白相比，apoA-IV介导HDL与细胞结合的作用更强。Dallinga-Thie等人的实验证明了apoA-IV能与肝、肾细胞膜结合并进入细胞内。
由此可见，apoA-IV可能在脂质代谢，特别是在胆固醇逆转运过程中起重要作用。
5)最近的研究显示apoA-IV蛋白与前列腺癌的发生、发展有密切的关系。但是在胃癌中的作用并未见报道。
专利CN201510569987.3公开了一种与胃癌相关的血清/血浆lncRNA标志物试剂盒，其通过非编码RNA对胃癌进行标志物检测，但只能采用QPCR方法进行检测，QPCR的英文全名是Real-time Quantitative PCR Detecting System，即实时荧光定量核酸扩增检测系统，也叫实时定量基因扩增荧光检测系统，简称QPCR，采用基因技术进行检测，成本较高、操作复杂，不利于基层推广普及。
发明内容
本发明所要解决的技术问题是：现有胃癌检测技术成本较高、操作复杂，不利于基层推广普及，本发明提供了解决上述问题的基于APOA4蛋白胃癌检测试剂盒。
本发明通过下述技术方案实现：
基于APOA4蛋白胃癌检测试剂盒，所述检测试剂盒的组成包括：
溶液A：采用抗体稀释液将Human APOA4蛋白稀释成工作溶液，作为标准品；
溶液B：采用生物素检测抗体稀释液将浓缩生物素检测抗体稀释成工作溶液，所述浓缩生物素检测抗体采用鼠抗人AMY1A单克隆抗体；
溶液C：采用HPR共轭稀释剂将浓缩HRP结合物稀释成工作溶液；
溶液D：采用抗体稀释液将兔抗人AMY1A多克隆抗体稀释成工作溶液；
溶液E：Wash Buffer；
溶液F：显色液；
溶液G：终止液。
进一步地，所述溶液A中，Human APOA4蛋白浓度为300ng/mL。
进一步地，所述溶液B中，将浓缩生物素检测抗体100倍稀释成工作溶液。
进一步地，所述溶液C中，将浓缩HRP结合物100倍稀释成工作溶液。
进一步地，所述溶液D中，AMY1A抗体浓度为0.1ug/mL～5ug/mL。
进一步地，所述溶液E中，用ddH2O将Wash Buffer 25倍稀释成工作溶液。
上述基于APOA4蛋白胃癌检测试剂盒的使用方法，采用ELISA法检测将所述的基于APOA4蛋白胃癌检测试剂盒用于检测血清中APOA4蛋白表达水平。
上述基于APOA4蛋白胃癌检测试剂盒的使用方法，包括以下步骤：
步骤1，取兔抗人AMY1A多克隆抗体用抗体稀释液稀释成工作溶液D，以50uL/孔加入96孔板中，保持4℃温度条件下静置；
步骤2，去掉上清液，以350uL/孔加入Wash Buffer，放置1～2min，去上清液，反复清洗，去上清液；
步骤3，每孔加入100uL标准品或者样品，于37℃孵育90min；
步骤4，采用步骤2的方法清洗，以100μL/孔加入工作溶液B，于37℃孵育1h；
步骤5，采用步骤2的方法清洗，以100μL/孔加入工作溶液C，于37℃孵育30min；
步骤6，采用步骤2的方法清洗，以90μL/孔加入显色液，于37℃显色15min；
步骤7，以50μL/孔加入终止液；
步骤8，用酶标仪测量450nm处的吸光度值。
进一步地，所述步骤3中使用的样品为采集的外周血清采用PBS稀释100倍～150倍制备获得。
本发明具有如下的优点和有益效果：
本发明提供一种基于APOA4蛋白胃癌检测试剂，且采用ELISA法检测血清中APOA4蛋白表达水平差异，进而判断待检者患胃癌的风险：若APOA4蛋白的表达水平高，则患胃癌的风险高，若APOA4蛋白的表达水平低，则患胃癌的风险低，可用于临床胃癌的辅助诊断，临床应用前景良好。且ELISA法，操作简单方便，血清分泌性蛋白更容易检测到，在基层推广应用比较容易，相对于现有的APOA4蛋白检测试剂盒，亲和力高、灵敏度好、准确度好。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解，构成本申请的一部分，并不构成对本发明实施例的限定。在附图中：
图1为本发明的不同浓度标准样品和吸光度关系的标准曲线。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例和附图，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。
实施例
一、临床试验案例资料
选取胃癌患者外周血血样27例，正常人对照13例(指正常不患胃癌者的外周血血样)，基本信息见表1。
表1临床试验基本信息
| 基本信息 | 胃癌患者血样 | 正常人对照血样 |
| 人数/例 | 27 | 13 |
| 年龄/岁 | 30～60 | 30～60 |
| 男女比例 | 2:1 | 2:1 |
二、基于APOA4蛋白胃癌检测试剂盒的组成
给出基于APOA4蛋白胃癌检测试剂盒，如表2所示。
表2基于APOA4蛋白胃癌检测试剂盒组成
三、基于APOA4蛋白胃癌检测试剂盒的制备
步骤1，血清样品制备：用一次性无非内毒素采血管采集外周血，于室温下凝结2h或在4℃下过夜，然后在15℃离心15min在2～8℃下1000×g，收集上清液以进行测定。检测前需用PBS稀释样品至100倍～150倍获得待测样品；
步骤2、Human APOA4蛋白(标准品)制备：将标准物以10,000×g离心1min。加入1.0mL抗体稀释液，静置10min，轻轻颠倒几次。充分溶解后，轻轻涡旋。此时标准品为300ng/mL的工作溶液。然后根据需要进行连续稀释。具体稀释操作的稀释梯度如下：300,150,75,37.5,18.75,9.38,4.69,0ng mL。稀释方法：取7个EP管，每管加入500uL参比标准品和样品稀释液。移液器500uL将100ng/mL工作溶液加入第一管中并混合以产生150ng/mL工作溶液。根据这些步骤，将500uL的前一种管的溶液加入后一种溶液中。依次稀释。注意：最后一根管被视为空白。不要从前一根管中吸取溶液；
步骤3、溶液B的制备：在实验前计算所需量(100μL/孔)。
用生物素检测抗体稀释液将浓缩生物素检测抗体100倍稀释成工作溶液，浓度为0.01-0.5μg/mL；
步骤4、溶液C的制备：在实验前计算所需量(100μL/孔)。用HPR共轭稀释剂将浓缩HRP结合物100倍稀释成工作溶液；
步骤5、Wash Buffer：用ddH2O将Wash Buffer 25倍稀释成工作溶液，0.01％～0.1％Tween20的PBS。
四、基于APOA4蛋白胃癌检测试剂盒表达盒的使用
步骤1、取AMY1A抗体用抗体稀释液稀释成工作溶液(浓度为0.1ug/mL～5ug/mL)，以50uL/孔加入96孔板中，4℃包被过夜；
步骤2、第二天，倒扣去掉上清液，以350uL/孔加入Wash Buffer，放置1～2min，倒扣去掉上清液，反复清洗3次。于吸水纸上尽量除去上清液；
步骤3、每孔加入100uL标准品或者样品，于37℃孵育90min；
步骤4、采用步骤2的方法清洗3次，以100μL/孔加入工作溶液B，于37℃孵育1h；
步骤5、采用步骤2的方法清洗3次，以100μL/孔加入工作溶液C，于37℃孵育30min；
步骤6、采用步骤2的方法清洗3次，以90μL/孔加入显色液，于37℃显色15min。(不超过30min)；
步骤7、以50μL/孔加入终止液；
步骤8、用酶标仪测量450nm处的吸光度值。
五、检测结果
在用酶标仪测量450nm处的吸光度值后，获得标准品Human APOA4蛋白浓度和吸光度(OD)关系的标准曲线，如图1所示；基于临床试验案例制备的样品中Human APOA4蛋白表达水平如表3所示：
表3临床试验中Human APOA4蛋白表达水平
| 吸光度 | 吸光度 | ||||
| 病人编号 | 稀释100倍 | 浓度ng/mL | 正常人编号 | 稀释100倍 | 浓度ng/mL |
| 217516 | 0.6073 | 6768.00 | S-20171107-269015 | 0.0552 | -593.33 |
| 216523 | 0.4976 | 5305.33 | S-20171107-269025 | 0.1871 | 1165.33 |
| 216502 | 0.8952 | 10606.67 | S-20171107-269054 | 0.8552 | 10073.33 |
| 215523 | 0.6416 | 7225.33 | S-20171107-269018 | 0.0685 | -416.00 |
| 235675 | 0.6256 | 7012.00 | S-20171107-269036 | 0.2448 | 1934.67 |
| 237009 | 0.0835 | -216.00 | S-20171107-269051 | 1.0205 | 12277.33 |
| 190002 | 0.1414 | 556.00 | S-20171107-269021 | 0.0577 | -560.00 |
| 179502 | 0.1056 | 78.67 | S-20171107-269060 | 0.1155 | 210.67 |
| 202009 | 0.5149 | 5536.00 | S-20171107-269045 | 0.1018 | 28.00 |
| 198002 | 1.645 | 20604.00 | S-20171107-269042 | 0.2066 | 1425.33 |
| 224082 | 0.5625 | 6170.67 | S-20171107-269039 | 0.2426 | 1905.33 |
| 309509 | 0.3662 | 3553.33 | S-20171107-269024 | 0.3222 | 2966.67 |
| 206590 | 0.8822 | 10433.33 | S-20171107-269063 | 0.0734 | -350.67 |
| 227175 | 0.0719 | -370.67 | |||
| 205623 | 0.1183 | 248.00 | |||
| 236509 | 0.0557 | -586.67 | |||
| 227168 | 0.0982 | -20.00 | |||
| 227182 | 0.1273 | 368.00 | |||
| 178015 | 0.0831 | -221.33 | |||
| 220502 | 0.1225 | 304.00 | |||
| 178523 | 0.0934 | -84.00 | |||
| 204661 | 0.0783 | -285.33 | |||
| 178001 | 0.2687 | 2253.33 | |||
| 308502 | 0.4149 | 4202.67 | |||
| 平均值 | 0.38 | 3726.72 | 0.27 | 2312.82 |
根据不同浓度标准品吸光度得到相应标准曲线(图1所示)。以分光光度计测定结果以及标准曲线计算各样品APOA4蛋白浓度，对比胃癌病人与正常人APOA4蛋白浓度含量的差异。如表1所示，由结果显示胃癌病人APOA4蛋白浓度明显高于正常人。
综上，本发明试剂盒通过检测APOA4蛋白的表达水平，可以判断APOA4蛋白表达过高者有患胃癌的风险：若APOA4蛋白的表达水平高，则患胃癌的风险高，若APOA4蛋白的表达水平低，则患胃癌的风险低，可用于临床胃癌的辅助诊断，为患者采取相关的治疗措施或者决策提供有效的依据，临床应用前景良好。
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、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.基于APOA4蛋白胃癌检测试剂盒，其特征在于，所述检测试剂盒的组成包括：
溶液A：采用抗体稀释液将Human APOA4蛋白稀释成工作溶液，作为标准品；
溶液B：采用生物素检测抗体稀释液将浓缩生物素检测抗体稀释成工作溶液，所述浓缩生物素检测抗体采用鼠抗人AMY1A单克隆抗体；
溶液C：采用HPR共轭稀释剂将浓缩HRP结合物稀释成工作溶液；
溶液D：采用抗体稀释液将兔抗人AMY1A多克隆抗体稀释成工作溶液；
溶液E：Wash Buffer；
溶液F：显色液；
溶液G：终止液。
2.根据权利要求1所述的基于APOA4蛋白胃癌检测试剂盒，其特征在于，所述溶液A中，Human APOA4蛋白浓度为300ng/mL。
3.根据权利要求1所述的基于APOA4蛋白胃癌检测试剂盒，其特征在于，所述溶液B中，将浓缩生物素检测抗体100倍稀释成工作溶液。
4.根据权利要求1所述的基于APOA4蛋白胃癌检测试剂盒，其特征在于，所述溶液C中，将浓缩HRP结合物100倍稀释成工作溶液。
5.根据权利要求1所述的基于APOA4蛋白胃癌检测试剂盒，其特征在于，所述溶液D中，AMY1A抗体浓度为0.1ug/mL～5ug/mL。
6.根据权利要求1所述的基于APOA4蛋白胃癌检测试剂盒，其特征在于，所述溶液E中，用ddH2O将Wash Buffer 25倍稀释成工作溶液。
7.根据权利要求1-6任一项所述的基于APOA4蛋白胃癌检测试剂盒的使用方法，其特征在于，采用ELISA法检测将所述的基于APOA4蛋白胃癌检测试剂盒用于检测血清中APOA4蛋白表达水平。
8.根据权利要求7所述的基于APOA4蛋白胃癌检测试剂盒的使用方法，其特征在于，所述使用方法包括以下步骤：
步骤1，取兔抗人AMY1A多克隆抗体用抗体稀释液稀释成工作溶液D，以50uL/孔加入96孔板中，保持4℃温度条件下静置；
步骤2，去上清液，以350uL/孔加入Wash Buffer，放置1～2min，去上清液，反复清洗，去上清液；
步骤3，每孔加入100uL标准品或者样品，于37℃孵育90min；
步骤4，采用步骤2的方法清洗，以100μL/孔加入工作溶液B，于37℃孵育1h；
步骤5，采用步骤2的方法清洗，以100μL/孔加入工作溶液C，于37℃孵育30min；
步骤6，采用步骤2的方法清洗，以90μL/孔加入显色液，于37℃显色15min；
步骤7，以50μL/孔加入终止液；
步骤8，用酶标仪测量450nm处的吸光度值。
9.根据权利要求8所述的基于APOA4蛋白胃癌检测试剂盒的使用方法，其特征在于，所述步骤3中使用的样品为采集的外周血清采用PBS稀释100倍～150倍制备获得。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811319452.0A CN109212218A (zh) | 2018-11-07 | 2018-11-07 | 基于apoa4蛋白胃癌检测试剂盒、及其使用方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811319452.0A CN109212218A (zh) | 2018-11-07 | 2018-11-07 | 基于apoa4蛋白胃癌检测试剂盒、及其使用方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109212218A true CN109212218A (zh) | 2019-01-15 |
Family
ID=64994668
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811319452.0A Pending CN109212218A (zh) | 2018-11-07 | 2018-11-07 | 基于apoa4蛋白胃癌检测试剂盒、及其使用方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109212218A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118290562A (zh) * | 2024-04-11 | 2024-07-05 | 复旦大学 | 一种用于前列腺癌评估的分子指标ApoA4表位肽，试剂盒 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100136584A1 (en) * | 2008-09-22 | 2010-06-03 | Icb International, Inc. | Methods for using antibodies and analogs thereof |
| CN102844661A (zh) * | 2010-02-11 | 2012-12-26 | 香港理工大学 | 胃癌的生物标志物及其应用 |
| CN105899953A (zh) * | 2013-11-05 | 2016-08-24 | 新加坡科技研究局 | 膀胱癌生物标志物 |
| CN108700593A (zh) * | 2015-11-02 | 2018-10-23 | 拜奥克罗斯公司 | 载脂蛋白检测方法 |
-
2018
- 2018-11-07 CN CN201811319452.0A patent/CN109212218A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100136584A1 (en) * | 2008-09-22 | 2010-06-03 | Icb International, Inc. | Methods for using antibodies and analogs thereof |
| CN102844661A (zh) * | 2010-02-11 | 2012-12-26 | 香港理工大学 | 胃癌的生物标志物及其应用 |
| CN105899953A (zh) * | 2013-11-05 | 2016-08-24 | 新加坡科技研究局 | 膀胱癌生物标志物 |
| CN108700593A (zh) * | 2015-11-02 | 2018-10-23 | 拜奥克罗斯公司 | 载脂蛋白检测方法 |
Non-Patent Citations (3)
| Title |
|---|
| CAO MENG 等: "Monodisperse magnetic mesoporous silica microspheres facilitate the studies of gastric cancer-specific peptides in sera", 《RSC ADVANCES》 * |
| USCN生命科学有限公司: "载脂蛋白A4(APOA4)检测试剂盒-酶联免疫吸附试验法-SEB967Hu", 《CLOUD-CLONE CORP.》 * |
| 范衡: "糖脂代谢紊乱与慢性萎缩性胃炎的相关性", 《中国优秀硕士学位论文全文数据库-医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118290562A (zh) * | 2024-04-11 | 2024-07-05 | 复旦大学 | 一种用于前列腺癌评估的分子指标ApoA4表位肽，试剂盒 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lin et al. | Nationwide epidemiological survey of chronic pancreatitis in Japan | |
| Yasui et al. | Characteristics of patients with nonalcoholic steatohepatitis who develop hepatocellular carcinoma | |
| Cheung et al. | Relationship between HB sAg, HB crAg and hepatocellular carcinoma in patients with undetectable HBV DNA under nucleos (t) ide therapy | |
| Jia et al. | Transient elastography compared to serum markers to predict liver fibrosis in a cohort of C hinese patients with chronic hepatitis B | |
| Umehara et al. | A novel clinical entity, IgG4-related disease (IgG4RD): general concept and details | |
| Shorbagi et al. | Experience of a single center with congenital hepatic fibrosis: a review of the literature | |
| Kumar et al. | Characteristics of hepatocellular carcinoma in India: a retrospective analysis of 191 cases | |
| Canet et al. | Altered regulation of hepatic efflux transporters disrupts acetaminophen disposition in pediatric nonalcoholic steatohepatitis | |
| García‐Monzón et al. | Increased soluble CD 36 is linked to advanced steatosis in nonalcoholic fatty liver disease | |
| Chen et al. | Possible association between Helicobacter pylori infection and nonalcoholic fatty liver disease | |
| Abruzzi et al. | Coinfection of Schistosoma species with hepatitis B or hepatitis C viruses | |
| Song et al. | The combined measurement of total serum IgG and IgG4 may increase diagnostic sensitivity for autoimmune pancreatitis without sacrificing specificity, compared with IgG4 alone | |
| Yang et al. | The association between non‐alcoholic fatty liver disease and serum ferritin levels in American adults | |
| Cadranel et al. | Low incidence of spontaneous bacterial peritonitis in asymptomatic cirrhotic outpatients | |
| Duan et al. | Multicenter prospective study to validate a new transient elastography device for staging liver fibrosis in patients with chronic hepatitis B | |
| Lee et al. | Comparison of surrogate serum markers and transient elastography (Fibroscan) for assessing cirrhosis in patients with chronic viral hepatitis | |
| Goel et al. | Use of serum vitamin B12 level as a marker to differentiate idiopathic noncirrhotic intrahepatic portal hypertension from cryptogenic cirrhosis | |
| Liu et al. | The potential effects of diabetes mellitus on liver fibrosis in patients with primary biliary cholangitis | |
| Dong et al. | Duodenal microbiome and serum metabolites predict hepatocellular carcinoma in a multicenter cohort of patients with cirrhosis | |
| Lu et al. | Liver Cancer–Specific Serine Protease Inhibitor Kazal Is a Potentially Novel Biomarker for the Early Detection of Hepatocellular Carcinoma | |
| Shi et al. | Characteristics of gut microbiota and fecal metabolomes in patients with celiac disease in Northwest China | |
| Hendrickson et al. | Reduced infant rhesus macaque growth rates due to environmental enteric dysfunction and association with histopathology in the large intestine | |
| Kamisawa et al. | Management strategies for autoimmune pancreatitis | |
| CN109212218A (zh) | 基于apoa4蛋白胃癌检测试剂盒、及其使用方法 | |
| Hong et al. | Prevalence and risk factors of significant fibrosis in chronic hepatitis B patients with concurrent metabolic dysfunction-associated steatotic liver disease |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190115 |
|
| RJ01 | Rejection of invention patent application after publication |