CN117890575A - 一种用于检测神经因子的无微球均相化学发光检测方法 - Google Patents
一种用于检测神经因子的无微球均相化学发光检测方法 Download PDFInfo
- Publication number
- CN117890575A CN117890575A CN202311838660.2A CN202311838660A CN117890575A CN 117890575 A CN117890575 A CN 117890575A CN 202311838660 A CN202311838660 A CN 202311838660A CN 117890575 A CN117890575 A CN 117890575A
- Authority
- CN
- China
- Prior art keywords
- detection method
- nerve
- sample
- detection
- microsphere
- 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
- 210000005036 nerve Anatomy 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000002038 chemiluminescence detection Methods 0.000 title abstract description 3
- 238000001514 detection method Methods 0.000 claims abstract description 39
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 21
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims abstract description 5
- -1 acridine ester Chemical class 0.000 claims abstract description 4
- 239000003112 inhibitor Substances 0.000 claims abstract 2
- 108010025020 Nerve Growth Factor Proteins 0.000 claims description 7
- 102000015336 Nerve Growth Factor Human genes 0.000 claims description 7
- 229940053128 nerve growth factor Drugs 0.000 claims description 7
- 238000012545 processing Methods 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 3
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 238000000504 luminescence detection Methods 0.000 claims 1
- 210000002966 serum Anatomy 0.000 claims 1
- 238000004020 luminiscence type Methods 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 7
- 238000003018 immunoassay Methods 0.000 description 7
- 239000006249 magnetic particle Substances 0.000 description 6
- 239000004005 microsphere Substances 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 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 description 4
- 238000002965 ELISA Methods 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 2
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical compound NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 2
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 150000002016 disaccharides Chemical class 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 239000012780 transparent material Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- YQVOBDWTHOKQSV-UHFFFAOYSA-N Cl.Cl.Cl.Cl.ONC.ONC.ONC Chemical compound Cl.Cl.Cl.Cl.ONC.ONC.ONC YQVOBDWTHOKQSV-UHFFFAOYSA-N 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-O acridine;hydron Chemical compound C1=CC=CC2=CC3=CC=CC=C3[NH+]=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-O 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000010256 biochemical assay Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000000969 carrier 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
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 239000000852 hydrogen donor Substances 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000000979 retarding effect Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000002699 waste material Substances 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Plasma & Fusion (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明涉及一种用于检测神经因子的无微球均相化学发光检测方法,其包括如下步骤:首先HRP偶联标记特制抗体分子A,吖啶酯偶联标记特制抗体分子B。然后将样本与特定的均相化学发光试剂混合进行反应,且无需添加本底抑制液。最后将上述检测体系于37℃孵育15分钟,加入含有H2O2的激发液,立即检测发光信号。
Description
技术领域
本发明涉及化学发光体系技术领域,具体涉及一种快速无微球均相化学发光免疫分析方法及其系统。
背景技术
在体外诊断领域,尤其是在神经科学和临床神经病学中,神经因子的准确检测对于疾病的诊断和治疗至关重要。神经因子,如神经生长因子(NGF)、脑源性神经营养因子(BDNF)等,是在神经系统发育、维持和病理状态中发挥作用的一类生物活性物质。它们的异常表达与多种神经退行性疾病有关,传统的神经因子检测方法依赖于复杂的实验过程和耗时的步骤,如酶联免疫吸附测定(ELISA)、磁微粒化学发光免疫分析法等。这些方法虽然在灵敏度和特异性上有一定的优势,但它们往往需要昂贵的仪器设备,且操作过程繁琐,耗时较长。此外,固定化微粒或固相载体在这些方法中的使用往往导致样品处理复杂化,且易受到非特异性结合的干扰,影响检测结果的准确性。因此,开发一种快速、简便、高灵敏度且成本效益高的神经因子检测方法是该领域的迫切需求。
磁微粒化学发光免疫分析法是目前较为常用的检测方法,该方法是将生物素标记的抗体、待测抗原与碱性磷酸酶标记的抗体形成“三明治”结构的复合物。随后加入连有链霉亲和素的磁性微粒,通过链霉亲和素与生物素的特异性结合使抗原抗体复合物连接在磁微粒上,在外加磁场中将免疫反应形成的复合物与未结合的其他物质分离。去上清后清洗磁微粒复合物,加入发光底物,通过发光仪检测反应的发光强度,发光强度与待测抗原含量呈正比,使用相应的计算方法即可计算出样本中待测抗原的浓度。该方法使用磁微粒作为固相支持物,检测过程中需要外加磁场,并且需要洗涤步骤,因此存在操作繁琐,检测用时长的缺陷。而本发明使用的无微球均相化学发光免疫分析技术,它摒弃了传统检测方法中的固定化介质,大大简化了样品处理流程,缩短了整个检测周期,为神经因子的快速和高效检测提供了新的可能性。此外,这种方法通过简化检测步骤,减少了非特异性结合的可能性,从而提高了检测结果的准确性和可靠性。
发明内容
为了更加充分地利用均相化学发光分析的优势,开发出快速准确的免疫分析检测平台,本发明提供了一种基于无微球均相化学发光技术的神经因子检测方法。该方法不仅提高了检测效率和准确性,而且操作简便。本发明的核心在于利用无微球均相化学发光技术进行神经因子的检测。不同于传统的基于微粒或固相载体的方法,本发明不需要以微孔板、磁珠或微球等作为载体包被抗体,检测抗体能够与待测分析物充分进行免疫反应,没有洗涤操作,因此检测过程中无洗涤废液,是一种真正意义上的均相化学发光技术。本发明采用的无微球均相化学发光免疫分析的检测流程如下(结合图1无微球均相发光即时检测原理图):
首先通过HRP偶联标记特制抗体分子A,吖啶酯偶联标记特制抗体分子B。然后加入待测样本进行反应,且无需添加本底抑制液。最后将上述检测体系于(37±0 .5)℃孵育(15±1)分钟;然后加入含有H2O2的激发液,立即检测发光信号。
检测流程中所述的均相发光试剂是包含HRP、H2O2、吖啶酯、抗体A、抗体B以及缓冲液的混合物。
所述的HRP(辣根过氧化物酶,Horseradish Peroxidase)是一种常用的酶标记物,在生物化学和分子生物学的许多应用中都非常重要。这种酶主要用于酶联免疫吸附试验(ELISA)、西方印迹(Western Blot)以及组织化学染色等技术中。HRP的主要优势是其高灵敏度和稳定性,以及对多种底物的广泛催化能力。HRP易与过氧化氢(H2O2)结合,而所得的[HRP-H2O2]络合物可氧化各种氢供体。HRP可以通过几种不同的方法与抗体结合,包括戊二醛,高碘酸盐氧化,通过二硫键,以及通过氨基和巯基定向交联剂。它比酶标记β-半乳糖苷酶和碱性磷酸酶更小且更稳定,因此是最理想的标记。
所述的吖啶酯(Acridinium ester)是一种化学发光底物,广泛用于生物化学检测中,特别是在化学发光免疫分析(CLIAs)中。这类化合物在特定条件下能够产生强烈的化学发光,使其成为检测生物标记物如蛋白质、核酸和小分子的理想选择。吖啶酯在某些化学反应中,如暴露于过氧化氢和适当的催化剂(如过氧化物酶)时,能发生化学发光反应。
所述的本底抑制液是浓度为10mM抗坏血酸水溶液与1mM 2-氨基苯酚水溶液的复合溶液,主要作用是阻滞或降低化学反应速度,在此化学反应中主要是防止其他物质与本底结合,造成信号干扰。
所述的抗体A、B为特制的鼠抗人单克隆抗体A和鼠抗人单克隆抗体B,委托CRO公司进行定制。
所述的缓冲液包括缓冲体系、表面活性剂、无机盐、二糖稳定剂、蛋白保护剂和防腐剂组成。所述缓冲体系可以是三羟基氨基甲烷盐酸(Tris-HCl)缓冲体系、2-吗啉乙磺酸(MES)缓冲液体系中的任一种,缓冲体系浓度范围为10-100mmol/L,缓冲体系pH范围为5.5-7.5;所述表面活性剂可以是0.1%-1%的Tween-20、Tween-60、Tween-80、Triton X-100中的任一种;所述无机盐可以是10-100mmol/L的氯化钠或氯化钾;所述二糖稳定剂可以是0.5-4%的蔗糖或海藻糖;所述蛋白保护剂可以是牛血清蛋白(BSA)、酪蛋白中的任意一种;防腐剂可以是0.1-0.4% Proclin-300。
配合上述检测方法,本发明也设计了一种应用该检测方法的检测系统,包括光电控制系统、孵育系统、反应杯、光信号采集系统、数据处理系统。
所述的光电控制系统为电路控制系统,可以控制光源、搅拌、加热、供氧、信号采集等其他子系统。
所述的孵育系统为主要用于样本的孵育和反应。控制系统可以检测系统环境温度,启动或者停止电热板加热,保持反应体系温度在37℃左右。同时为确保反应充分进行,通常配备漩涡混匀器等设备,用于混匀反应液。
所述的反应杯为本系统的一种耗材,可以是一次性使用材料。反应杯需要至少在光路位置由透明材质制备。可以是各种常见的透明高分子材料,也可以是硅酸盐等各种硅基的无机材料,还可以是水晶等天然透明材料。优选地,可以采用聚丙烯材料。反应杯容积可以有300uL, 500uL, 1mL,2mL等多种选择。反应杯中可以预装反应试剂,也可以在进行检测时,再根据试剂要求装入试剂。
所述光信号采集系统包括滤光片、光电倍增管、选频放大器。
所述的数据收集处理系统用来光信号采集系统收集到的光电信号并根据预置软件的设定做出数学转换,提交数据给人机界面软件或接输出端口把数据送到其它外设设备。
附图说明
图1为无微球均相发光即时检测原理图。
图2为无微球和有微球的试剂检测发光值对比图。
实施例
本发明无微球均相化学发光试剂与市售有微球的化学发光试剂测试对比。
样品准备:准备不同浓度的神经因子抗原样品,具体浓度为0 pg/mL(对照组)、100pg/mL、391 pg/mL、782 pg/mL、1563 pg/mL、3125pg/mL、6250pg/mL、12500pg/mL、25000pg/mL、50000pg/mL和100000pg/mL。
神经因子(NGF)检测抗体溶液:1ug/mL HRP-A、1ug/mL 吖啶酯-B分别用50mMTris缓冲液(pH=7.2)稀释。
反应体系为:神经因子(NGF)检测抗体溶液1ug/mL、样品20uL。将上述反应体系于反应杯中37℃孵育10min后,加入100ul激发液进行检测。
测定发光值:使用本发明无微球均相化学发光试剂与市售有微球的化学发光试剂,同时测定上述样品进行对比。
数据记录:记录每个样品的发光值,检测数据见表1。根据表1数据绘制比对图(图2)。
表1 检测数据
待测样品浓度(pg/mL) | 无微球发光均值RLU | 有微球发光均值RLU |
0 | 462 | 581 |
100 | 815 | 693 |
391 | 929 | 785 |
782 | 1765 | 1327 |
1563 | 3391 | 2495 |
3125 | 6612 | 4797 |
6250 | 12695 | 8923 |
12500 | 24628 | 16865 |
25000 | 46547 | 33055 |
50000 | 91233 | 62144 |
100000 | 180641 | 117451 |
结论:图表数据表明,在神经因子(NGF)0~100000 pg/mL浓度范围内,本发明无微球均相化学发光试剂相关系数为R2=0.9999,高于市售有微球的化学发光试剂相关系数R2=0.9988。相同样本浓度下,本发明无微球均相化学发光试剂发光值高于市售有微球的化学发光试剂,体现了低端灵敏度和高端线性的优势。综合比较可得,本发明无微球均相化学发光试剂性能明显优于市售有微球的化学发光试剂。
Claims (10)
1.一种用于检测神经因子的无微球均相化学发光检测方法,其特征在于包括以下步骤:
A.准备含有目标神经因子的样本;
B.准备与HRP偶联的特制单克隆抗体A、与吖啶酯偶联的特制单克隆抗体B;
C.将样本与特定的均相化学发光试剂混合,且无需使用本底抑制剂。该试剂包括HRP标记的抗体、吖啶酯标记的抗体;
D.在37℃下孵育,使抗体与神经因子结合,无需清洗;
E.采用发光检测装置,检测所述溶液中的化学发光信号,其中该信号与所述神经因子的浓度成正比;
F.通过分析所述化学发光信号的强度,定量确定所述样本中的神经因子含量。
2.根据权利要求1所述的检测方法,其中所述神经因子选自神经生长因子(NGF),所述的单克隆抗体A、B均为特制。
3.根据权利要求1或2所述的检测方法,其中所述均相化学发光试剂包括对特定激光波长敏感的发光底物。
4.根据权利要求1至3中任一项所述的检测方法,其中所述激光的波长设定为适合激发所述发光底物的特定波长为470nm。
5.根据权利要求1至4中任一项所述的检测方法,其中所述发光检测设备包括光电倍增管、荧光光谱仪或光电二极管阵列。
6.根据权利要求1至5中任一项所述的检测方法,其中所述的孵育条件包括温度控制在37°C。
7.根据权利要求1至6中任一项所述的检测方法,其中所述的发光强度计算包括采用线性拟合标准曲线以计算所述神经因子的浓度。
8.根据权利要求1至7中任一项所述的检测方法,其中所述样本为血清、血浆。
9.根据权利要求1至8中任一项所述的检测方法,其中所述均相化学发光试剂的pH值调整至7.2至7.4,以优化反应条件。
10.一种使用权利要求1至9中任一项所述方法的检测系统,其特征在于所述系统进一步包括自动化样品处理装置、自动化试剂分配系统及与光电检测设备相连接的数据处理及分析软件。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311838660.2A CN117890575A (zh) | 2023-12-28 | 2023-12-28 | 一种用于检测神经因子的无微球均相化学发光检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311838660.2A CN117890575A (zh) | 2023-12-28 | 2023-12-28 | 一种用于检测神经因子的无微球均相化学发光检测方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117890575A true CN117890575A (zh) | 2024-04-16 |
Family
ID=90645146
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311838660.2A Pending CN117890575A (zh) | 2023-12-28 | 2023-12-28 | 一种用于检测神经因子的无微球均相化学发光检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117890575A (zh) |
-
2023
- 2023-12-28 CN CN202311838660.2A patent/CN117890575A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10732111B2 (en) | Automated immunoanalyzer system for performing diagnostic assays for allergies and autoimmune diseases | |
Hayrapetyan et al. | Enzyme-linked immunosorbent assay: types and applications | |
RU2608656C2 (ru) | Связанные со стрептавидином магнитные частицы и способ их изготовления | |
Bock | The new era of automated immunoassay | |
US9383354B2 (en) | Anti-antibody reagent | |
US6087188A (en) | Two-site immunoassay for an antibody with chemiluminescent label and biotin bound ligand | |
AU2013222444B2 (en) | Methods and systems for signal amplification of bioassays | |
US10914732B2 (en) | Method and device for determining biological analytes | |
CN112698041A (zh) | 一种复合物及其生长分化因子15检测试剂盒与应用 | |
US20120021944A1 (en) | Co-Coupling To Control Reactivity Of Reagents In Immunoassays | |
JP3194585B2 (ja) | 化学発光ラベル及びビオチン結合リガンドによる抗体の2サイトイムノアッセイ | |
EP0201211A1 (en) | Method and compositions for visual solid phase immunoassays based on luminescent microspheric particles | |
CN117554615A (zh) | 一种adamts13酶活性发光免疫检测方法及adamts13酶活性检测试剂盒 | |
Zhu et al. | An automated and highly sensitive chemiluminescence immunoassay for diagnosing mushroom poisoning | |
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 | |
JP2014228385A (ja) | 免疫試験方法および免疫試験キット | |
CN110988325A (zh) | 封闭剂及包含该封闭剂的试剂盒 | |
CN117890575A (zh) | 一种用于检测神经因子的无微球均相化学发光检测方法 | |
US20240044893A1 (en) | Ultrasensitive sensing method for detection of biomolecules | |
JPH07270415A (ja) | 酵素免疫測定法 | |
Schwickart et al. | Immunoassays | |
CN117269503A (zh) | 可溶性生长刺激表达基因2蛋白(st2) 检测试剂盒 | |
CN115902204A (zh) | 一种外泌体形式半乳糖凝集素9的检测方法与试剂盒 | |
JP2024524928A (ja) | 標識固定化及び増幅戦略を使用した単一分子に対するデジタル免疫センシングのための方法 | |
CN117871871A (zh) | 一种基于亚微米磁力化学发光法检测人岩藻糖基化蛋白lcn2的方法及检测试剂盒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication |