CN112526124A - 一种花生免疫荧光检测试纸条及应用和检测方法 - Google Patents
一种花生免疫荧光检测试纸条及应用和检测方法 Download PDFInfo
- Publication number
- CN112526124A CN112526124A CN202011336762.0A CN202011336762A CN112526124A CN 112526124 A CN112526124 A CN 112526124A CN 202011336762 A CN202011336762 A CN 202011336762A CN 112526124 A CN112526124 A CN 112526124A
- Authority
- CN
- China
- Prior art keywords
- antibody
- ara
- peanut
- test strip
- line
- 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 57
- 241001553178 Arachis glabrata Species 0.000 title claims abstract description 46
- 235000020232 peanut Nutrition 0.000 title claims abstract description 46
- 235000017060 Arachis glabrata Nutrition 0.000 title claims abstract description 45
- 235000010777 Arachis hypogaea Nutrition 0.000 title claims abstract description 45
- 235000018262 Arachis monticola Nutrition 0.000 title claims abstract description 45
- 238000010166 immunofluorescence Methods 0.000 title claims abstract description 25
- 239000004816 latex Substances 0.000 claims abstract description 43
- 229920000126 latex Polymers 0.000 claims abstract description 43
- 239000004005 microsphere Substances 0.000 claims abstract description 43
- 239000012528 membrane Substances 0.000 claims abstract description 33
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 27
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 27
- 239000013566 allergen Substances 0.000 claims abstract description 23
- 238000003908 quality control method Methods 0.000 claims abstract description 19
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 13
- 239000002250 absorbent Substances 0.000 claims abstract description 11
- 230000002745 absorbent Effects 0.000 claims abstract description 11
- 235000013305 food Nutrition 0.000 claims description 18
- 239000011248 coating agent Substances 0.000 claims description 12
- 238000000576 coating method Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 9
- 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 description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 6
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 5
- 108010090804 Streptavidin Proteins 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 235000020958 biotin Nutrition 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 claims description 3
- 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 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000011084 recovery Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 10
- 229920000915 polyvinyl chloride Polymers 0.000 description 8
- 239000004800 polyvinyl chloride Substances 0.000 description 8
- 206010016946 Food allergy Diseases 0.000 description 4
- 208000010668 atopic eczema Diseases 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000002572 peristaltic effect Effects 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 208000004262 Food Hypersensitivity Diseases 0.000 description 3
- 230000000172 allergic effect Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 235000020932 food allergy Nutrition 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 208000026935 allergic disease Diseases 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000012482 calibration solution Substances 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000013568 food allergen Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000007974 sodium acetate buffer Substances 0.000 description 2
- 239000012536 storage buffer Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 206010002199 Anaphylactic shock Diseases 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 206010012434 Dermatitis allergic Diseases 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 208000008267 Peanut Hypersensitivity Diseases 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000002052 anaphylactic effect Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 201000010853 peanut allergy Diseases 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 208000024891 symptom Diseases 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
-
- 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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
-
- 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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
-
- 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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
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)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Nanotechnology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
本发明提供了一种花生免疫荧光检测试纸条及应用和检测方法,涉及免疫荧光检测技术领域。本发明所述试纸条包括由左到右首尾相连且依次固定在PVC底板上的样品垫、结合垫、硝酸纤维素膜和吸水纸;所述结合垫上包被有荧光胶乳微球标记的混合抗体;在硝酸纤维素膜上包被抗Ara h1抗体(T1线)、抗Ara h2抗体(T2线)、抗Ara h3抗体(T3线)、抗花生总抗体(T4线)和兔抗鼠IgG抗体(C线),其中T1线、T2线、T3线、T4线为检测线,C线为质控线。本发明所述试纸条能够快速定量检测花生过敏原的种类和含量,并且操作简便、准确性高,回收率应在90%~110%、灵敏度高(≤0.84ng/ml)。
Description
技术领域
本发明属于免疫荧光检测技术领域,具体涉及一种花生免疫荧光检测试纸条及应用和检测方法。
背景技术
花生是世界粮农组织(FOA)1995年报告中提出的八大食物过敏原之一,据报道,花生是一种营养丰富、深受人们喜爱的常见食物,花生过敏占食物过敏的10%~47%,属于八大类容易引起过敏原的食品之首。花生相对于其他食品过敏而言,它的过敏发生率更高、临床症状更为严重,在公共卫生和食品安全领域备受人们的关注。
花生食物过敏可引起过敏性肠炎、过敏性皮炎等过敏性疾病,严重的甚至会发生过敏性休克和过敏性死亡。国内外均有报道食用花生食物引起过敏性死亡的病例。鉴于目前尚无根治这类疾病的方法,为此,美国及欧盟等国家要求在食品标签上标识花生等过敏原成分,以防止过敏患者误食。同时,美国及欧盟国家开始对部分进口食品进行花生过敏原抽检,而我国食品出口企业常因食物过敏原标识不正确而遭遇退货。因此,有必要开发一种灵敏度高、操作简单的检测花生过敏原成分及含量的检测方法。
发明内容
有鉴于此,本发明的目的在于提供一种花生免疫荧光检测试纸条及应用和检测方法,可快速定性和定量检测食品中的花生过敏原成分,操作简便、准确度高、灵敏度高。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种花生免疫荧光检测试纸条,所述试纸条上包括由左到右首尾相连且依次固定在PVC底板上的样品垫、结合垫、硝酸纤维素膜和吸水纸;所述结合垫上包被有荧光胶乳微球标记的混合抗体;所述混合抗体包括:抗Arah1、抗Arah2、抗Arah3和抗花生总抗体;
所述硝酸纤维素膜上包括平行的4条检测线和1条质控线;所述检测线上分别包被抗Arah1抗体、抗Arah2抗体、抗Arah3抗体和抗花生总抗体;所述质控线上包被兔抗鼠IgG抗体;
所述检测线上的包被抗体与所述荧光胶乳微球标记的混合抗体为配对抗体。
优选的,所述荧光胶乳微球的粒径为50~500nm。
优选的,所述包被有荧光胶乳微球标记的混合抗体的结合垫的制备方法,包括:a.将荧光标记的链霉亲和素和胶乳微球吸附结合,得到荧光胶乳微球;
b.将生物素与所述混合抗体结合,得到生物素化混合抗体;
c.将所述荧光胶乳微球和所述生物素化混合抗体混合,得荧光胶乳微球标记的混合抗体;
d.将所述荧光胶乳微球标记的混合抗体喷涂在结合垫上;
步骤a和b之间不具有时间上的先后关系。
优选的,步骤a所述荧光标记包括异硫氰酸荧光素、四乙基罗丹明、四甲基异硫氰酸罗丹明或荧光素CY5。
优选的,步骤d所述喷涂时,将所述荧光胶乳微球标记的混合抗体按照2~10μl/cm的用量喷涂在结合垫上。
优选的,所述混合抗体中抗Ara h1、抗Arah2、抗Ara h3和抗花生总抗体的质量比为1:1:1:1。
优选的,4条所述检测线上包被抗Arah1抗体、抗Ara h2抗体、抗Ara h3抗体和抗花生总抗体的包被浓度均为0.5~5.0μl/cm;
所述质控线上包被兔抗鼠IgG抗体的包被浓度为0.5~5μl/cm。
本发明提供了上述免疫荧光检测试纸条在检测食品中花生过敏原成分中的应用。
优选的,所述花生过敏原成分包括Arah1、Ara h2、Ara h3和总花生蛋白。
本发明还提供了一种检测食品中花生过敏原成分的方法,包括以下步骤:滴加100~120μl的食品溶液于上述免疫荧光检测试纸条的样品垫上,15min后读取所述检测试纸条的荧光信号,根据标准曲线测定花生过敏原的成分和含量;
其中x为过敏原浓度,单位IU/ml,y为荧光信号比值。
本发明提供了一种花生免疫荧光检测试纸条,其结构如图1所示,包括由左到右首尾相连且依次固定在PVC底板上的样品垫、结合垫、硝酸纤维素膜和吸水纸;所述结合垫上包被有荧光胶乳微球标记的混合抗体;所述混合抗体包括:抗Ara h1、抗Arah2、抗Arah3和抗花生总抗体;在硝酸纤维素膜(NC膜)上包被抗Arah1抗体(T1线)、抗Ara h2抗体(T2线)、抗Arah3抗体(T3线)、抗花生总抗体(T4线)和兔抗鼠IgG抗体(C线),其中T1线、T2线、T3线、T4线为检测线,C线为质控线。本发明所述混合抗体和NC膜上包被的抗体分别配对,且混合抗体类似于二抗,NC膜上包被抗体类似于一抗。本发明所述试纸条能够快速定量检测花生过敏原的种类和含量,并且操作简便、准确性高,回收率应在90%~110%、灵敏度高(≤0.84ng/ml)。
附图说明
图1为本发明所述免疫荧光检测试纸条的结构图;
图2为荧光免疫层析法测定标准曲线。
具体实施方式
本发明提供了一种花生免疫荧光检测试纸条,其结构如图1所示,所述试纸条上包括由左到右首尾相连且依次固定在PVC底板上的样品垫、结合垫、硝酸纤维素膜和吸水纸;所述结合垫上包被有荧光胶乳微球标记的混合抗体;所述混合抗体包括:抗Arah1、抗Arah2、抗Arah3和抗花生总抗体;
所述硝酸纤维素膜上包括平行的4条检测线和1条质控线;所述检测线上分别包被抗Ara h1抗体、抗Ara h2抗体、抗Ara h3抗体和抗花生总抗体;所述质控线上包被兔抗鼠IgG抗体;
所述检测线上的包被抗体与所述荧光胶乳微球标记的混合抗体为配对抗体。
本发明所述结合垫上包被有荧光胶乳微球标记的混合抗体,所述包被有荧光胶乳微球标记的混合抗体的结合垫的制备方法,优选包括:a.将荧光标记的链霉亲和素和胶乳微球吸附结合,得到荧光胶乳微球;
b.将生物素与所述混合抗体结合,得到生物素化混合抗体;
c.将所述荧光胶乳微球和所述生物素化混合抗体混合,得荧光胶乳微球标记的混合抗体;
d.将所述荧光胶乳微球标记的混合抗体喷涂在结合垫上;步骤a和b之间不具有时间上的先后关系。
本发明步骤a中所述荧光标记的链霉亲和素和胶乳微球的质量比优选为1:40;步骤b中所述生物素和混合抗体的体积比优选为1:4;步骤c中荧光胶乳微球和生物素化混合抗体的体积比优选为10:1。本发明所述荧光胶乳微球的粒径优选为50~500nm。本发明步骤a所述荧光标记优选包括异硫氰酸荧光素、四乙基罗丹明、四甲基异硫氰酸罗丹明或荧光素CY5。本发明对所述混合抗体中抗Ara h1、抗Ara h2、抗Arah3和抗花生总抗体的质量比优选为1:1:1:1。本发明对所述抗Ara h1、抗Arah2、抗Ara h3和抗花生总抗体的来源并没有特殊限定,优选为本领域的常规市售抗体。本发明步骤d中所述结合垫上所述荧光胶乳微球标记的混合抗体的喷涂量优选为2~10μl/cm。在本发明中,所述荧光胶乳微球标记的混合抗体均为二抗。
本发明所述硝酸纤维素膜(NC膜)上包括平行的4条检测线和1条质控线;所述检测线上分别包被抗Arah1抗体、抗Ara h2抗体、抗Ara h3抗体和抗花生总抗体;所述质控线上包被兔抗鼠IgG抗体。本发明对所述NC膜的制备方法并没有特殊限定,优选的包括:用包被缓冲液分别稀释抗Ara h1抗体、抗Arah2抗体、抗Arah3抗体、抗花生总抗体和兔抗鼠IgG抗体,并将稀释后的五种抗体分别平行地划在硝酸纤维素膜上,待抗体渗入硝酸纤维素膜后分别形成包被抗Ara h1抗体、抗Ara h2抗体、抗Ara h3抗体、抗花生总抗体的检测线以及包被兔抗鼠IgG抗体的质控线。本发明4条所述检测线上包被抗Arah1抗体、抗Arah2抗体、抗Ara h3抗体和抗花生总抗体的包被浓度均为0.5~5.0μl/cm;所述质控线上包被兔抗鼠IgG抗体的包被浓度为0.5~5μl/cm。本发明在进行划线时,针对检测限和质控线的操作不同,在进行检测限的划线时,优选的将抗Ara h1抗体、抗Ara h2抗体、抗Ara h3抗体和抗花生总抗体分别依次按3mg/ml的浓度,蠕动泵授液量0.4ml/min,划线速度50m/20min,在干燥箱内20℃鼓风干燥12h;在质控线划线时,优选将兔抗鼠IgG抗体按8mg/ml的浓度,蠕动泵授液量0.4ml/min,划线速度50m/20min,在硝酸纤维素膜上划线,该线与检测线的线平行,放入干燥箱内20℃鼓风干燥12h。
本发明优选依次将样品垫、结合垫、硝酸纤维素膜和吸水滤纸组装粘贴在PVC底板上,按照要求(4mm)用切条机将其切割成如图1所示的试纸条。本发明所示试纸条可以批量生产,适用于临床快速诊断和现场快速检测,易于保存。
本发明提供了上述免疫荧光检测试纸条在检测食品中花生过敏原成分中的应用。
本发明所述花生过敏原成分优选包括Ara h1、Ara h2、Ara h3和总花生蛋白,其中总花生蛋白中不涉及Arah1、Ara h2和Ara h3。
本发明还提供了一种检测食品中花生过敏原成分的方法,包括以下步骤:滴加100~120μl的食品溶液于上述免疫荧光检测试纸条的样品垫上,15min后读取所述检测试纸条的荧光信号,根据标准曲线测定花生过敏原的成分和含量;所述标准曲线为R2=0.9997;
其中x为过敏原浓度,单位IU/ml,y为荧光信号比值。
本发明将所述待测样本滴加与样品垫上后,样品经过结合垫上的混合抗体进行反应结合,再依次经过检测区的抗Ara h1抗体、抗Ara h2抗体、抗Arah3抗体和抗总花生蛋白抗体进行反应,最后达到质控区反应结束。
下面结合实施例对本发明提供的一种花生免疫荧光检测试纸条及应用和检测方法进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
本发明中所用试剂,如非特殊说明,均为常规购买产品,其中:
抗Arah 1抗体:IndoorBiotechnologies(货号:MA-2F7-1,MA-5C2-1);
抗Ara h 2抗体:IndoorBiotechnologies(货号:MA-2H3-1,MA-1C4-1);
抗Ara h 3抗体:IndoorBiotechnologies(货号:MA-1E8-1,MA-4G9-1);
抗总花生蛋白抗体:IndoorBiotechnologies(货号:MA-3B8-1)。
实施例1
1、结合垫的制备
1)荧光胶乳微球的制备
荧光胶乳微球的制备:用吸附缓冲液(50mM、pH5.8的柠檬酸盐缓冲液)稀释粒径为400nm胶乳微球至终浓度30mg/ml,体积为6ml,制得胶乳微球悬浮液;添加适当的红色荧光素罗丹明标记连霉亲和素在吸附缓冲液中,终体积为6ml;将上述胶乳微球悬浮液加入到上述含有红色荧光素罗丹明标记霉亲和素的吸附缓冲液中,制得混合液;将所得混合液在室温下温浴1~2h,并不断搅拌,然后离心,收集沉淀,沉淀用储存缓冲液(含有0.06%BSA的吸附缓冲液)溶解,放置4℃保存,备用。
2)生物素化抗花生混合抗体的制备
将抗Ara h 1抗体、抗Ara h 2抗体、抗Ara h 3抗体和抗总花生蛋白抗体按照1:1:1:1的质量比混合后(混合抗体,M1),用0.2M pH4.7醋酸钠缓冲液稀释至3ml,交互用0.2MpH4.7醋酸钠缓冲液对花生抗体M1充分透析;用1ml DMSO溶解1ml的NHSB,得到NHSB溶液;向上述3ml花生抗体M1加入25μLNHSB,搅拌2~4h,继续室温搅拌10min,然后用20mM、pH3.9PBS缓冲液透析,即得生物素化混合抗体M1。
3)荧光胶乳微球标记花生抗体的制备
将步骤1)制得的荧光胶乳微球和步骤2)制得的生物素化混合抗体按照10:1的体积比混合在一起,反应30min后离心,沉淀用储存缓冲液溶解,恢复至原体积。
4)将荧光胶乳微球标记花生混合抗体按照2~10μl/cm的用量喷涂在结合垫上。
2、硝酸纤维素膜的制备
1)膜处理取一张硝酸纤维素膜,做好标记后放在pH膜处理液(TBS)中浸泡5~10min;
2)组装点样装置,将浸泡过的硝酸纤维素膜放在平铺的垫子上,放上抗体点样板子,上面要留出贴标记纸的地方。
3)花生抗体检测区的制备:将抗Ara h1抗体、抗Ara h2抗体、抗Ara h3抗体、抗花生总抗体分别依次按3mg/ml的浓度,蠕动泵授液量0.4ml/min,划线速度50m/20min,在干燥箱内20℃鼓风干燥12h。
4)质控区的制备:将兔抗鼠IgG抗体按8mg/ml的浓度,蠕动泵授液量0.4ml/min,划线速度50m/20min,在硝酸纤维素膜4上划线,该线与检测区的线平行,放入干燥箱内20℃鼓风干燥12h。
5)用上述封闭液(100ml PBS和0.5g BSA配备)将的硝酸纤维素膜于37℃封闭60min,取出后置37℃下烘干处理2h,封袋备用。
6)试纸条组装
依次将样品垫、结合垫、硝酸纤维素膜和吸水滤纸组装粘贴在PVC底板上,按照要求(4mm)用切条机将其切割成如图1所示的试纸条。
3、待检抗原检测
将被检测样本滴加100~120μl于样品垫上,后经过结合垫上的抗花生混合抗体进行反应结合,再依次经过检测区的抗Ara h1抗体、抗Ara h2抗体、抗Arah3抗体、抗花生总抗体进行反应,最后达到质控区反应结束,后将试纸条放于专门的荧光信号读取仪器中,读取荧光信号的大小,进行定量测定。
剂量-反应曲线的线性:测定试剂盒中校准品配制的校准液系列,浓度分别为100.00、50.00、17.50、3.50、0.35IU/ml,用双对数或其它适当的数学模式进行拟合,模型拟合结果应符合分析内精密度(CV%)应≤10.0%;分析间精度(CV%)应≤15.0%。
剂量-反应曲线线性分析结果显示,0.35~100IU/ml范同内,各浓度与测试值呈平滑递增曲线,测量下限(CV<15%测试系统所能检测到的最低浓度)为0.35IU/ml,曲线方程如图2所示:
其中x为过敏原浓度,单位IU/ml,y为荧光信号比值。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种花生免疫荧光检测试纸条,其特征在于,所述试纸条上包括由左到右首尾相连且依次固定在PVC底板上的样品垫、结合垫、硝酸纤维素膜和吸水纸;所述结合垫上包被有荧光胶乳微球标记的混合抗体;所述混合抗体包括:抗Ara h1、抗Ara h2、抗Ara h3和抗花生总抗体;
所述硝酸纤维素膜上包括平行的4条检测线和1条质控线;所述检测线上分别包被抗Ara h1抗体、抗Ara h2抗体、抗Ara h3抗体和抗花生总抗体;所述质控线上包被兔抗鼠IgG抗体;
所述检测线上的包被抗体与所述荧光胶乳微球标记的混合抗体为配对抗体。
2.根据权利要求1所述免疫荧光检测试纸条,其特征在于,所述荧光胶乳微球的粒径为50~500nm。
3.根据权利要求1所述免疫荧光检测试纸条,其特征在于,所述包被有荧光胶乳微球标记的混合抗体的结合垫的制备方法,包括:a.将荧光标记的链霉亲和素和胶乳微球吸附结合,得到荧光胶乳微球;
b.将生物素与所述混合抗体结合,得到生物素化混合抗体;
c.将所述荧光胶乳微球和所述生物素化混合抗体混合,得荧光胶乳微球标记的混合抗体;
d.将所述荧光胶乳微球标记的混合抗体喷涂在结合垫上;
步骤a和b之间不具有时间上的先后关系。
4.根据权利要求3所述免疫荧光检测试纸条,其特征在于,步骤a所述荧光标记包括异硫氰酸荧光素、四乙基罗丹明、四甲基异硫氰酸罗丹明或荧光素CY5。
5.根据权利要求3所述免疫荧光检测试纸条,其特征在于,步骤d所述喷涂时,将所述荧光胶乳微球标记的混合抗体按照2~10μl/cm的用量喷涂在结合垫上。
6.根据权利要求1或3所述免疫荧光检测试纸条,其特征在于,所述混合抗体中抗Arah1、抗Ara h2、抗Ara h3和抗花生总抗体的质量比为1:1:1:1。
7.根据权利要求1所述免疫荧光检测试纸条,其特征在于,4条所述检测线上包被抗Arah1抗体、抗Ara h2抗体、抗Ara h3抗体和抗花生总抗体的包被浓度均为0.5~5.0μl/cm;
所述质控线上包被兔抗鼠IgG抗体的包被浓度为0.5~5μl/cm。
8.权利要求1~7任一项所述免疫荧光检测试纸条在检测食品中花生过敏原成分中的应用。
9.根据权利要求8所述应用,其特征在于,所述花生过敏原成分包括Ara h1、Ara h2、Ara h3和总花生蛋白。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011336762.0A CN112526124A (zh) | 2020-11-25 | 2020-11-25 | 一种花生免疫荧光检测试纸条及应用和检测方法 |
AU2021100135A AU2021100135A4 (en) | 2020-11-25 | 2021-01-11 | Test Strip for Peanut Immunofluorescence Assay (IFA), Use Thereof and Detection Method |
US17/211,535 US20220163523A1 (en) | 2020-11-25 | 2021-03-24 | Test strip for peanut immunofluorescence assay (ifa), use thereof and detection method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011336762.0A CN112526124A (zh) | 2020-11-25 | 2020-11-25 | 一种花生免疫荧光检测试纸条及应用和检测方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112526124A true CN112526124A (zh) | 2021-03-19 |
Family
ID=74993701
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011336762.0A Pending CN112526124A (zh) | 2020-11-25 | 2020-11-25 | 一种花生免疫荧光检测试纸条及应用和检测方法 |
Country Status (3)
Country | Link |
---|---|
US (1) | US20220163523A1 (zh) |
CN (1) | CN112526124A (zh) |
AU (1) | AU2021100135A4 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115057927A (zh) * | 2022-05-30 | 2022-09-16 | 南开大学 | 一种花生过敏原Ara h1特异性纳米抗体及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112526142A (zh) * | 2020-11-25 | 2021-03-19 | 杭州福德敏生物技术有限公司 | 一种鸡蛋免疫荧光检测试纸条及应用和检测方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102879586A (zh) * | 2012-10-11 | 2013-01-16 | 南京基蛋生物科技有限公司 | 微量白蛋白荧光免疫定量检测试剂盒及其制备方法 |
CN104101581A (zh) * | 2013-04-11 | 2014-10-15 | 南昌大学 | 一种快速检测食品中花生致敏蛋白的装置及其制备方法 |
-
2020
- 2020-11-25 CN CN202011336762.0A patent/CN112526124A/zh active Pending
-
2021
- 2021-01-11 AU AU2021100135A patent/AU2021100135A4/en active Active
- 2021-03-24 US US17/211,535 patent/US20220163523A1/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112526142A (zh) * | 2020-11-25 | 2021-03-19 | 杭州福德敏生物技术有限公司 | 一种鸡蛋免疫荧光检测试纸条及应用和检测方法 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115057927A (zh) * | 2022-05-30 | 2022-09-16 | 南开大学 | 一种花生过敏原Ara h1特异性纳米抗体及其应用 |
CN115057927B (zh) * | 2022-05-30 | 2024-02-20 | 南开大学 | 一种花生过敏原Ara h1特异性纳米抗体及其应用 |
Also Published As
Publication number | Publication date |
---|---|
AU2021100135A4 (en) | 2021-04-15 |
US20220163523A1 (en) | 2022-05-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN202794178U (zh) | 降钙素原快速定量免疫层析检测试剂盒 | |
CN101769932B (zh) | 全量程c反应蛋白检测试剂盒 | |
CN104090248A (zh) | 铕螯合物乳胶时间分辨荧光免疫层析定量检测食品中β-受体激动剂试剂 | |
CN106248974A (zh) | 一种高敏感性免疫层析检测试纸 | |
US20140322724A1 (en) | Homogeneous competitive lateral flow assay | |
CN106053794A (zh) | 一种用于准确检测待测物的试剂卡、试剂盒和用途 | |
CN111308072A (zh) | 快速检测新型冠状病毒IgG抗体的胶体金免疫层析试剂盒及其制备方法 | |
CN112526124A (zh) | 一种花生免疫荧光检测试纸条及应用和检测方法 | |
CN114441766A (zh) | 一种定量检测抗pla2r抗体的荧光免疫层析试纸条及其制备方法 | |
CN109459570A (zh) | 一种快速检测人体血液中铅离子的胶体金免疫试纸条制备方法 | |
CN112526142A (zh) | 一种鸡蛋免疫荧光检测试纸条及应用和检测方法 | |
CN112630423A (zh) | 一种小麦免疫荧光检测试纸条及应用 | |
CN112595847A (zh) | 一种虾免疫荧光检测试纸条及应用 | |
CN115993451B (zh) | 一种甲型流感病毒和腺病毒抗原定量检测试剂盒、制备方法及定量检测方法 | |
AU2021103258A4 (en) | Fluorescence immunochromatography assay (fica) test strip for fish, use thereof, and detection method using same | |
CN110927386B (zh) | C-反应蛋白胶体金检测试剂卡及其制备方法 | |
AU2021103250A4 (en) | Immunofluorescent assay test strip for hazelnut, use thereof, and detection method using same | |
CN105223349B (zh) | 一种检测样本的装置 | |
CN110672862B (zh) | 一种血型检测卡及其制备方法 | |
AU2021103259A4 (en) | Fluorescence immunochromatography assay (fica) test strip for quantitatively detecting soybean allergens, use thereof, and detection method using same | |
CN101762693A (zh) | 一种检测tp抗体的磁性免疫层析试纸条及其制备方法 | |
CN112362865A (zh) | 一种牛奶免疫荧光检测试纸条及应用 | |
CN106872690B (zh) | 一种高灵敏度胶体金试纸及其制备方法与应用 | |
CN103995131B (zh) | 用于特异性杂草类花粉过敏原IgE筛查的试纸条及其制备方法 | |
JP2002214236A (ja) | 血中抗原検出方法及び装置 |
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 |