CN111272997A - Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit - Google Patents
Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit Download PDFInfo
- Publication number
- CN111272997A CN111272997A CN202010072100.0A CN202010072100A CN111272997A CN 111272997 A CN111272997 A CN 111272997A CN 202010072100 A CN202010072100 A CN 202010072100A CN 111272997 A CN111272997 A CN 111272997A
- Authority
- CN
- China
- Prior art keywords
- detected
- sample
- antigen
- reagent
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000003018 immunoassay Methods 0.000 title claims abstract description 21
- 239000000427 antigen Substances 0.000 claims abstract description 127
- 102000036639 antigens Human genes 0.000 claims abstract description 127
- 108091007433 antigens Proteins 0.000 claims abstract description 127
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 93
- 238000001514 detection method Methods 0.000 claims abstract description 53
- 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 44
- 238000009739 binding Methods 0.000 claims abstract description 29
- 230000027455 binding Effects 0.000 claims abstract description 28
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229960002685 biotin Drugs 0.000 claims abstract description 22
- 235000020958 biotin Nutrition 0.000 claims abstract description 22
- 239000011616 biotin Substances 0.000 claims abstract description 22
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 16
- 238000012360 testing method Methods 0.000 claims abstract description 15
- 230000005281 excited state Effects 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 37
- 239000000203 mixture Substances 0.000 claims description 25
- 150000001875 compounds Chemical class 0.000 claims description 15
- 239000012736 aqueous medium Substances 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 229910052751 metal Inorganic materials 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- -1 olefin compound Chemical class 0.000 claims description 5
- 230000003612 virological effect Effects 0.000 claims description 4
- 239000013522 chelant Substances 0.000 claims description 3
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 239000002994 raw material Substances 0.000 abstract description 9
- 238000012216 screening Methods 0.000 abstract description 2
- 239000000370 acceptor Substances 0.000 description 16
- 239000004005 microsphere Substances 0.000 description 16
- 239000003085 diluting agent Substances 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 12
- 239000000126 substance Substances 0.000 description 11
- 241000700605 Viruses Species 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 108700039791 Hepatitis C virus nucleocapsid Proteins 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000009870 specific binding Effects 0.000 description 6
- 239000012895 dilution Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000012898 sample dilution Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 101710132601 Capsid protein Proteins 0.000 description 4
- 208000005176 Hepatitis C Diseases 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 239000003504 photosensitizing agent Substances 0.000 description 3
- 150000003839 salts Chemical group 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- BVTJGGGYKAMDBN-UHFFFAOYSA-N Dioxetane Chemical compound C1COO1 BVTJGGGYKAMDBN-UHFFFAOYSA-N 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- WZELXJBMMZFDDU-UHFFFAOYSA-N Imidazol-2-one Chemical group O=C1N=CC=N1 WZELXJBMMZFDDU-UHFFFAOYSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical group C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 2
- 150000001491 aromatic compounds Chemical class 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 241000206761 Bacillariophyta Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010008177 Fd immunoglobulins Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- QYKIQEUNHZKYBP-UHFFFAOYSA-N Vinyl ether Chemical compound C=COC=C QYKIQEUNHZKYBP-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000002009 alkene group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001615 biotins Chemical class 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000012482 calibration solution Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- 239000001752 chlorophylls and chlorophyllins Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 125000005594 diketone group Chemical group 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 150000002081 enamines Chemical class 0.000 description 1
- 150000002084 enol ethers Chemical class 0.000 description 1
- 125000002534 ethynyl group Chemical class [H]C#C* 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 229940042795 hydrazides for tuberculosis treatment Drugs 0.000 description 1
- 150000007857 hydrazones Chemical class 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229920000592 inorganic polymer Polymers 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- KNJDBYZZKAZQNG-UHFFFAOYSA-N lucigenin Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.C12=CC=CC=C2[N+](C)=C(C=CC=C2)C2=C1C1=C(C=CC=C2)C2=[N+](C)C2=CC=CC=C12 KNJDBYZZKAZQNG-UHFFFAOYSA-N 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000006386 neutralization reaction 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
- 210000003463 organelle Anatomy 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000012048 reactive intermediate Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229930187593 rose bengal Natural products 0.000 description 1
- 229940081623 rose bengal Drugs 0.000 description 1
- STRXNPAVPKGJQR-UHFFFAOYSA-N rose bengal A Natural products O1C(=O)C(C(=CC=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 STRXNPAVPKGJQR-UHFFFAOYSA-N 0.000 description 1
- VDNLFJGJEQUWRB-UHFFFAOYSA-N rose bengal free acid Chemical compound OC(=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C(O)=C(I)C=C21 VDNLFJGJEQUWRB-UHFFFAOYSA-N 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229930192474 thiophene Natural products 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 239000000230 xanthan gum Substances 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
-
- 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
- G01N33/68—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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/186—Hepatitis C; Hepatitis NANB
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a homogeneous immunoassay kit and application thereof in the technical field of biology. The kit comprises a reagent I which comprises a first counterpart; the first counterpart is a known antigen or a known antibody capable of specifically binding to a test antigen in a test sample; a reagent II comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second counterpart capable of binding specifically to the first counterpart; a reagent III comprising a third counterpart capable of specifically binding to the first counterpart; a reagent IV comprising a donor capable of generating singlet oxygen in an excited state. The third counterpart is surface coated with biotin and the donor surface is coated with streptavidin. The homogeneous phase immunoassay method for detecting the antigen and the antibody by using the kit in combination reduces the difficulty of raw material screening and improves the detection sensitivity.
Description
The application is a divisional application of Chinese patent application with the application date of 2017, 11 and 27, and the application number of 201711203146.6, and the invention name of the application is 'a homogeneous immunoassay kit, a homogeneous immunoassay method and application thereof'.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a homogeneous immunoassay kit and application thereof in combined detection of a same virus antigen and a same virus antibody.
Background
In some in vitro diagnostic items using chemiluminescence detection, it is desirable to simultaneously detect both antigen and antibody in a sample. For example, the detection of Human Immunodeficiency Virus (HIV) has been developed from the former three generations of reagents for detecting only Virus antibodies to the current four generations of aids detection reagents for the combined detection of antigen and antibody. This avoids the risk of missed detection in patients with a window period who have only viral antigens and no corresponding antibodies or who have insufficient corresponding antibody titers to be detected. Therefore, establishing an efficient and reliable antigen-antibody combined detection method has important significance in the field of diagnostic reagent development and even in the medical and health industries.
The current chemiluminescence antigen-antibody joint detection method detects through the formation of a sandwich complex of a labeled antigen and a labeled antibody and an antibody or an antigen in a sample to be detected to finally generate a signal. The presence of either the antigen or antibody to be detected in the sample is reflected as a rise in signal. The disadvantage is that it is impossible to distinguish whether the positive signal value is caused by an antigen or an antibody, and therefore the course of the disease cannot be accurately judged, and reliable information cannot be provided for clinical treatment. In addition, the labeled raw materials in the detection reagent need to be strictly screened, and labeled antigens and antibodies cannot have cross reaction, so that the difficulty of reagent development is increased, and the development period is prolonged. Meanwhile, because some sites are avoided artificially, the sensitivity of detecting a part of antigens and/or antibodies is inevitably sacrificed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a homogeneous immunoassay kit and a homogeneous immunoassay method for jointly detecting the same virus antigen and antibody by using the kit. The method integrates a competition method and a sandwich method, only the antigen or the antibody needs to be marked, and the antigen and the antibody do not need to be marked simultaneously, so that potential cross reaction is avoided, the difficulty of raw material screening is reduced, and the detection sensitivity is improved. In addition, the method provided by the invention has more clinical significance in result interpretation, and the sample to be detected can be judged to be antigen positive or antibody positive through the result, so that a basis is provided for epidemic disease diagnosis and treatment scheme formulation.
To this end, the present invention provides in a first aspect a homogeneous immunoassay kit comprising:
a reagent i comprising a first counterpart; the first counterpart is a known antigen or a known antibody capable of specifically binding to a test antigen in a test sample; the reagent I can be used for diluting a sample to be detected;
a reagent II comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second counterpart capable of binding specifically to the first counterpart;
a reagent III comprising a third counterpart capable of specifically binding to the first counterpart;
a reagent IV comprising a donor capable of generating singlet oxygen in an excited state.
In some embodiments of the invention, the first counterpart is a known antibody capable of specifically binding to an antigen to be detected in a sample to be detected, and the second and third counterparts are both known antigens.
In some embodiments of the invention, the known antibody is a monoclonal antibody or an antibody positive serum.
In other embodiments of the invention, the first counterpart is a known antigen and the second and third counterparts are each a second antibody capable of specifically binding to the known antigen.
In some embodiments of the invention, the second antibody is a monoclonal antibody and/or a polyclonal antibody.
According to the invention, the third counterpart is surface coated with biotin and the donor surface is coated with streptavidin.
In some embodiments of the invention, the acceptor comprises an olefinic compound and a metal chelate and is soluble in an aqueous medium.
In other embodiments of the invention, all of the reagents are non-particulated and are soluble in aqueous media.
In a second aspect of the present invention, there is provided a homogeneous immunoassay method for the combined detection of the same viral antigen and antibody using the kit according to the first aspect of the present invention, comprising the following steps: firstly, detecting a negative sample without an antigen to be detected and an antibody to be detected, and taking an obtained detection result as a background signal value; and then detecting the chemiluminescence signal value of the sample to be detected, and comparing the chemiluminescence signal value of the sample to be detected with the background signal value, thereby judging whether the antigen to be detected and/or the antibody to be detected exist in the sample to be detected.
In some embodiments of the present invention, the first counterpart contained in reagent i is a known antibody capable of specifically binding to the antigen to be detected in the sample to be detected, and when:
if the detected chemiluminescence signal value of the sample to be detected is equal to the background signal value, the sample to be detected does not contain the antigen to be detected and the antibody to be detected;
if the detected chemiluminescence signal value of the sample to be detected is larger than the background signal value, the sample to be detected contains the antibody to be detected;
and if the detected chemiluminescence signal value of the sample to be detected is smaller than the background signal value, the sample to be detected contains the antigen to be detected.
In other embodiments of the invention, the first counterpart comprised in reagent i is a known antigen, when:
if the detected chemiluminescence signal value of the sample to be detected is equal to the background signal value, the sample to be detected does not contain the antigen to be detected and the antibody to be detected;
if the detected chemiluminescence signal value of the sample to be detected is larger than the background signal value, the sample to be detected contains the antigen to be detected;
and if the chemiluminescence signal value of the sample to be detected obtained by detection is smaller than the background signal value, the sample to be detected contains the antibody to be detected.
According to the invention, the method for detecting the chemiluminescence signal value of the sample to be detected comprises the following steps:
s1, mixing the sample to be detected with the reagent I to obtain a first mixture;
s2, adding a reagent II, a reagent III and a reagent IV into the first mixture to obtain a second mixture;
s3, treating the second mixture with energy or active compound, and detecting the chemiluminescence signal value of the second mixture;
and S4, analyzing the chemiluminescence signal value, and judging whether the sample to be detected contains the antigen to be detected and/or the antibody to be detected.
In some embodiments of the invention, reagent II and reagent III are added first, followed by reagent IV in step S2.
In other embodiments of the present invention, in step S1, a sample to be tested is added to a solution containing reagent I.
Here, the above-mentioned method is particularly a method for the purpose of non-disease diagnosis.
In a third aspect, the present invention provides a kit according to the first aspect of the present invention or a method according to the second aspect of the present invention for use in the combined detection of HCV antigens and antibodies.
The term "known antigen" in the present invention may be a standard substance of an antigen to be detected in a sample to be detected.
The term "known antibody specifically binding to the antigen to be detected in the sample to be detected" in the present invention may be a standard substance of the antibody to be detected in the sample to be detected.
The antigen to be detected and the antibody to be detected in the sample to be detected in the present invention may be antigens and antibodies against the same virus.
The phrase "the chemiluminescent signal value of the sample to be tested is equal to the background signal value" means that the chemiluminescent signal value of the sample to be tested is close to the local signal value by more than 90%, preferably more than 95%, and more preferably more than 99%.
The invention has the beneficial effects that:
1. the reagent components are simple, and only the labeled antigen or the labeled antibody needs to be added in the detection process, and the two kinds of the labeled antigen or the labeled antibody do not need to be added simultaneously.
2. The raw materials are easy to screen, the cross reaction among the raw materials is not needed to be considered, the raw materials have higher selectivity, and the proper raw materials are easier to find.
3. The detection sensitivity is high, partial epitopes do not need to be sacrificed in the raw material selection process to avoid cross reaction, so that more epitopes can be combined with the substance to be detected, and the detection sensitivity is correspondingly improved.
4. The result is accurately judged, and the comparison between the signal value read during detection and the background signal value can judge whether the sample is positive or not, can distinguish whether the positive result is antigen positive or antibody positive, and is more meaningful than the only common positive result.
5. The hook effect resistance is improved, and a sample which generates a hook due to strong yang can be detected because the signal value read during detection is lower than the background signal value.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings briefly described below are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of the homogeneous immunization method for combined detection of the same virus antigen and antibody. The reference numerals in the figures have the following meanings: 1, a known antibody contained in a reagent I and capable of specifically binding to an antigen to be detected in a sample to be detected; 2, the antibody to be detected in the sample to be detected; 3 antigen to be detected in the sample to be detected; 4, the luminescent microsphere coated by the known antigen; 5 Biotin labeled known antigens.
FIG. 2 is a flow chart of the homogeneous immunization method for combined detection of antigen and antibody.
FIG. 3 is a diagram showing the detection results of hepatitis C core antigen in the examples.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Term (I)
The term "homogeneous" as used herein is defined in english as "homogeneous" and means that the bound antigen-antibody complex and the remaining free antigen or antibody are detected without separation.
The term "test sample" as used herein refers to a mixture that may contain analytes, including but not limited to proteins, hormones, antibodies, antigens, etc. Typical test samples that can be used in the disclosed methods include body fluids such as blood, plasma, serum, urine, semen, saliva, and the like.
The term "antibody" as used herein is used in the broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any case desired, the antibody may be further conjugated to other moieties, such as specific binding pair members, e.g., biotin or streptavidin, and the like.
The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted from a monoclonal B lymphocyte, which can be prepared by methods known to those skilled in the art.
The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins produced by more than one B lymphocyte clone, which may be prepared by methods well known to those skilled in the art.
The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect.
The term "binding" as used herein refers to direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.
The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure.
The term "specific binding pair member" as used herein refers to a pair of molecules that are capable of specifically binding to each other, e.g., enzyme-substrate, antigen-antibody, ligand-receptor. An example of a specific binding pair member pair is the biotin-streptavidin system, where "biotin" is widely present in animal and plant tissues and has two cyclic structures on the molecule, an imidazolone ring and a thiophene ring, respectively, where the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be conjugated to almost any biological macromolecule known, including proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by a protein cross-linking agent; "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65 kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby creating a "tentacle effect" that increases assay sensitivity.
Any reagent used in the present invention, including antigens, antibodies, acceptors or donors, can be conjugated to biotin or streptavidin as desired, for example: the reagent III comprises a biotin-coated third counterpart; another example is: reagent iv contains a streptavidin-coated donor.
The term "donor" as used herein refers to a sensitizer capable of generating a reactive intermediate such as singlet oxygen that reacts with an acceptor upon activation by energy or an active compound. The donor may be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.).
In some embodiments of the invention, the donor is a photosensitizer which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. Pat. No. 5,5709994, which is incorporated herein by reference in its entirety, as well as derivatives of these compounds having 1 to 50 atom substituents that serve to render these compounds more lipophilic or more hydrophilic, and/or as a linker group to a member of a specific binding pair. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US patent No. US6406913, which is incorporated herein by reference.
In other embodiments of the invention, the donor is a chemically activated other sensitizer, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which are heated or directly absorb light to release singlet oxygen.
The term "acceptor" as used herein refers to a compound capable of reacting with singlet oxygen to produce a detectable signal. The donor is induced by energy or an active compound to activate and release singlet oxygen in a high energy state that is trapped by a close proximity acceptor, thereby transferring energy to activate the acceptor.
In some embodiments of the invention, the receptor is a substance that: it undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose with or following luminescence. Typical examples of such substances include, but are not limited to: enol ether, enamine, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridin, aromatic vinyl ether, diepoxy ethylene, dimethyl thiophene, aromatic imidazole or lucigenin.
In other embodiments of the invention, the acceptor is an alkene capable of reacting with singlet oxygen to form a hydroperoxide or dioxetane that can be decomposed into ketones or carboxylic acid derivatives; a stable dioxetane which can be decomposed by the action of light; acetylenes which can react with singlet oxygen to form diketones; hydrazones or hydrazides which can form azo compounds or azocarbonyl compounds, such as luminol; and aromatic compounds that can form endoperoxides. Specific, non-limiting examples of receptors that can be utilized in accordance with the disclosed and claimed invention are described in U.S. patent No. US5340716, which is incorporated herein by reference in its entirety.
In other embodiments of the invention, the receptor comprises an olefinic compound and a metal chelate, which is non-particulated and soluble in an aqueous medium, and the method of making such receptor can be found in patent PCT/US2010/025433 (which is incorporated herein by reference in its entirety).
In other embodiments of the invention, the "donor" and/or "acceptor" may be coated onto the substrate via a functional group to form "donor microspheres" and/or "acceptor microspheres". The "matrix" according to the present invention is microspheres or microparticles known to the skilled person, of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which have any density, but preferably have a density close to that of water, preferably are capable of floating in water, and which are made of a transparent, partially transparent or opaque material. The substrate may or may not have a charge, and when charged, is preferably negatively charged. The matrix may be a solid (e.g., polymers, metals, glass, organic and inorganic substances such as minerals, salts and diatoms), oil droplets (e.g., hydrocarbons, fluorocarbons, siliceous fluids), vesicles (e.g., synthetic such as phospholipids, or natural such as cells, and organelles). The matrix may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes. The matrix is generally multifunctional or capable of binding to a donor or recipient by specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxy, mercapto, and the like. One non-limiting example of a matrix suitable for use in the present invention is a carboxyl modified latex particle. Details of such substrates can be found in U.S. patent nos. US5709994 and US5780646 (both of which are incorporated herein by reference in their entirety).
Example II
As mentioned above, when the homogeneous immunoassay method is adopted to carry out the combined detection of the same virus antigen and antibody at present, the disease course can not be accurately judged because the positive signal value can not be distinguished to be caused by the antigen or the antibody, and reliable information can not be provided for clinical treatment. In addition, the marking raw materials in the detection reagent adopted in the detection process need to be strictly screened, and the marked antigen and the marked antibody can not have cross reaction, so that the difficulty of reagent research and development is increased, and the detection sensitivity is reduced. The inventor of the application finds that a certain amount of known antibody or known antigen which is specifically combined with the antigen to be detected in the sample to be detected is added into the reagent I (sample diluent), then the same virus antigen and antibody in the sample to be detected are detected by adopting a competition method and a sandwich method, only the marked antigen or the marked antibody is needed to be added, and finally the sample to be detected is judged to be antibody positive or antigen positive through the change of a signal value, so that the clinical diagnosis significance is achieved.
The detection principle of the present invention will be described in detail below by taking an example of adding a certain amount of known antibody that specifically binds to a test antigen in a test sample to reagent I (sample diluent), and a specific detection principle diagram is shown in FIG. 1.
Since a known antibody that specifically binds to an antigen to be detected in a sample to be detected is added to reagent i (sample dilution), reagent ii includes a receptor coated with a known antigen, and reagent iii includes a known antigen labeled with biotin.
When the sample to be detected does not contain the antigen to be detected and the antibody to be detected, the sample to be detected does not react with the known antibody in the sample diluent, and after the receptor coated with the known antigen and the biotin-labeled known antigen are added subsequently, the known antigen coated on the receptor and the biotin-labeled known antigen react with the known antibody in the sample diluent to form the sandwich complex. After a streptavidin-labeled donor contained in the reagent IV is added, the acceptor in the sandwich complexes can react with singlet oxygen emitted by a nearby donor through 680nm light excitation, the generated energy is emitted in the form of light with the wavelength of 610nm, and the signal value with the wavelength of 610nm is detected to serve as a detected background signal value.
When the sample to be detected contains the antibody to be detected, the total antibody amount is increased after the sample to be detected is mixed with the sample diluent, and the final reaction signal value is higher than the background signal value. The detection method is sandwich detection, sandwich reaction is enhanced, and reaction signal value is enhanced.
When the sample to be detected contains the antigen to be detected, the antigen to be detected in the sample to be detected reacts with the known antibody in the sample diluent after the sample to be detected is mixed with the sample diluent, and part of the known antibody is consumed by neutralization, so that the amount of the antibody which can react with the known antigen coated on the receptor and the known antigen marked by biotin in the subsequent process is reduced, and the signal value after the reaction is lower than the local signal value finally. The detection method in this case is a competitive detection method.
Accordingly, if a known amount of antigen is added to reagent I (sample dilution), reagent II comprises a receptor coated with a second antibody that specifically binds to the known antigen, and reagent III comprises a biotin-labeled second antibody that specifically binds to the known antigen.
At this time, when the antigen to be detected is contained in the sample to be detected, the total antigen amount is increased after the sample to be detected is mixed with the sample diluent, and the signal value after the final reaction is higher than the local signal value. The detection method in this case is a sandwich method detection, and the stronger the sandwich reaction, the stronger the reaction signal value.
When the sample to be detected contains the antibody to be detected, the antibody to be detected in the sample to be detected reacts with the known antigen in the sample diluent after the sample to be detected is mixed with the sample diluent, part of the known antigen is neutralized and consumed, so that the subsequent amount of the antigen capable of reacting with the second antibody coated on the receptor and the biotin-labeled second antibody is reduced, the signal value after the final reaction is lower than the local signal value, and the detection method is competition detection.
Accordingly, the present invention relates in a first aspect to a homogeneous immunoassay kit comprising:
a reagent i comprising a first counterpart; the first counterpart is a known antigen or a known antibody capable of specifically binding to a test antigen in a test sample;
a reagent II comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second counterpart capable of binding specifically to the first counterpart;
a reagent III comprising a third counterpart capable of specifically binding to the first counterpart.
A reagent IV comprising a donor capable of generating singlet oxygen in an excited state.
According to the invention, the third counterpart is surface coated with biotin and the donor surface is coated with streptavidin.
In some embodiments of the invention, the kit specifically comprises:
a reagent I comprising a known antibody capable of specifically binding to an antigen to be detected in a sample to be detected;
reagent II, which comprises a receptor coated with a known antigen;
reagent iii comprising a biotin-coated known antigen;
reagent IV comprising a streptavidin coated donor.
In other embodiments of the present invention, the kit specifically comprises:
a reagent I comprising a known antigen;
reagent II comprising a receptor coated with a second antibody capable of specifically binding to a known antigen;
reagent iii comprising a biotin-coated secondary antibody that specifically binds to a known antigen;
reagent IV comprising a streptavidin coated donor.
The donor and acceptor of the above reagents may be coated onto a substrate to form particulate donor and acceptor microspheres, which may also be non-particulate reagents, soluble in an aqueous medium.
In some embodiments of the invention, the kit specifically comprises:
a reagent I comprising a known antibody capable of specifically binding to an antigen to be detected in a sample to be detected;
reagent II comprising receptor microspheres that bind to a known antigen;
reagent iii comprising a biotin-coated known antigen;
reagent iv comprising streptavidin coated donor microspheres.
In other embodiments of the invention, the kit specifically comprises:
a reagent I comprising a known antigen;
reagent II comprising a receptor microsphere coated with a second antibody capable of specifically binding to a known antigen;
reagent iii comprising a biotin-coated secondary antibody that specifically binds to a known antigen;
reagent iv comprising streptavidin coated donor microspheres.
In a second aspect, the present invention relates to a homogeneous immunoassay method for jointly detecting the same viral antigen and antibody by using the kit according to the first aspect of the present invention, which comprises the following steps: firstly, detecting a negative sample without an antigen to be detected and an antibody to be detected, and taking an obtained detection result as a background signal value; and then detecting the chemiluminescence signal value of the sample to be detected, and comparing the chemiluminescence signal value of the sample to be detected with the background signal value, thereby judging whether the antigen to be detected and/or the antibody to be detected exist in the sample to be detected.
In some embodiments of the present invention, the first counterpart contained in reagent i is a known antibody capable of specifically binding to the antigen to be detected in the sample to be detected, and when:
if the detected chemiluminescence signal value of the sample to be detected is equal to the background signal value, the sample to be detected does not contain the antigen to be detected and the antibody to be detected;
if the detected chemiluminescence signal value of the sample to be detected is larger than the background signal value, the sample to be detected contains the antibody to be detected;
and if the detected chemiluminescence signal value of the sample to be detected is smaller than the background signal value, the sample to be detected contains the antigen to be detected.
In other embodiments of the invention, the first counterpart comprised in reagent i is a known antigen, when:
if the detected chemiluminescence signal value of the sample to be detected is equal to the background signal value, the sample to be detected does not contain the antigen to be detected and the antibody to be detected;
if the detected chemiluminescence signal value of the sample to be detected is larger than the background signal value, the sample to be detected contains the antigen to be detected;
and if the chemiluminescence signal value of the sample to be detected obtained by detection is smaller than the background signal value, the sample to be detected contains the antibody to be detected.
According to the invention, the method for detecting the chemiluminescence signal value of the sample to be detected comprises the following steps:
s1, mixing the sample to be detected with the reagent I to obtain a first mixture;
s2, adding a reagent II, a reagent III and a reagent IV into the first mixture to obtain a second mixture;
s3, treating the second mixture with energy or active compound, and detecting the chemiluminescence signal value of the second mixture;
and S4, analyzing the chemiluminescence signal value, and judging whether the sample to be detected contains the antigen to be detected and/or the antibody to be detected. The specific reaction scheme is shown in FIG. 2.
In some embodiments of the invention, reagent II and reagent III are added first, followed by reagent IV in step S2.
In other embodiments of the present invention, in step S1, a sample to be tested is added to a solution containing reagent I.
In the methods of the invention, all reagents may be combined and mixed and/or incubated as desired. Specifically, the temperature of the incubation can be 35-45 ℃ and the time can be 10-20 min; preferably, the temperature of the incubation may be selected from 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃ or 44 ℃; the incubation time may be selected from 12min, 15min, 16min or 18 min.
In some embodiments of the invention, light having a wavelength of 680nm is used to excite the donor and/or donor microspheres to generate singlet oxygen, and the acceptor is capable of reacting with the singlet oxygen to generate a chemiluminescent signal having a wavelength of 615 nm.
Specifically, in some embodiments of the present invention, the method for detecting a chemiluminescent signal value of a sample to be detected includes the following steps:
s1, adding the reagent I into the pre-dilution plate, then adding a sample to be detected, oscillating, uniformly mixing, and then incubating for 10-15min at 35-40 ℃ to obtain a first mixture;
s2, adding the first mixture into the detection area, adding a reagent II and a reagent III, reacting at 35-40 ℃ for 10-15min, adding a reagent IV, and incubating at 35-40 ℃ for 10-15min to obtain a second mixture;
s3, treating the second mixture by using light with the wavelength of 680nm, exciting the donor and/or the donor microsphere to generate singlet oxygen, and combining the generated singlet oxygen with the acceptor and/or the acceptor microsphere to generate a chemiluminescent signal with the wavelength of 615 nm;
and S4, detecting the chemical invention signal, and judging whether the antigen and/or the antibody to be detected exist in the sample to be detected according to the obtained chemiluminescence signal value.
According to the invention, the detection sensitivity of the method is less than 10 ng/mL.
A third aspect of the invention relates to the use of a kit according to the first aspect of the invention or a method according to the second aspect of the invention for the combined detection of HCV antigens and antibodies.
In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
The light-activated chemiluminescence analyzer used in the following examples was a fully automatic light-activated chemiluminescence analyzer.
Example 1: sensitivity experiment of homogeneous immunization method for single-hole combined detection of human hepatitis C Core antigen (HCV Core Ag)
1. Preparation of the marker
The HCV antibody detection kit produced by Shanghai Boyang is adopted, and the batch number is as follows: 1606L. Wherein the specific marking process of each reagent component is omitted.
2. Preparation of reagent I (sample dilution)
1 part of antibody positive serum is subjected to gradient dilution by using a sample diluent in the finished product kit, and is respectively diluted to 10 times, 100 times, 1000 times, 1 ten thousand times, 10 ten thousand times and 100 ten thousand times. The diluted solution is used as a sample to be detected by the kit, and the dilution with the signal value of about 20000 is found. The dilution was used as a ratio of adding the antibody to the sample dilution, and 20ml of the sample dilution was prepared in this ratio.
3. Detection of recombinant HCV core antigen concentration
Experiments were performed with recombinant HCV Core Ag. The antigen was diluted with Phosphate Buffered Saline (PBS) to 2-fold, 5-fold, and 10-fold, respectively, and then detected with a BCA protein concentration detection kit. For detection, Bovine Serum Albumin (BSA) calibration solutions (6 spots in total of 0mg/ml, 0.1mg/ml, 0.25mg/ml, 0.5mg/ml, 0.75mg/ml and 1 mg/ml) were first used for calibration, and after the operation according to the kit instructions, OD was measured on a microplate reader562nmA reading is taken. The detection result is that the original concentration of the antigen is less than 1 mg/ml.
4. Detection of sensitivity in homogeneous immunoassay combined detection of human Hepatitis C (HCV) core antigen
The antigen described in step 3 was diluted with PBS buffer in a gradient of 10-fold, 1000-fold, 1-thousand-fold and 10-thousand-fold, respectively.
Adding a sample diluent into a pre-dilution plate, wherein each well is 25ul, then adding the diluted HCV core antigen solution serving as a sample to be detected into each well of 25ul of the sample diluent respectively, uniformly mixing by oscillation, and incubating at 37 ℃ for 10min to obtain a first mixture.
25ul of the first mixture was pipetted per well and added to the assay well, then 25ul of each of the receptor microsphere solution bound to the HCV core antigen and the biotin-coated HCV core antigen solution was added, and after 15min of reaction at 37 ℃, 175ul of the streptavidin-coated donor microsphere solution was added, and after further incubation at 37 ℃ for 10min, a second mixture was obtained.
And (3) processing the second mixture by adopting light with the wavelength of 680nm, exciting the donor microsphere to generate singlet oxygen, and combining the generated singlet oxygen with the acceptor microsphere to generate a chemiluminescent signal with the wavelength of 615 nm. The chemical invention signal was detected and the value of the obtained chemical invention signal is shown in FIG. 3. As can be seen from FIG. 3, the change of the detection signal value is not obvious after the HCV core antigen is diluted by 10 ten thousand times, which indicates that the analysis sensitivity of the method for detecting the human hepatitis C core antigen is less than 10 ng/ml.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (12)
1. A homogeneous immunoassay kit comprising:
a reagent I comprising a first counterpart, said first counterpart being a known antigen;
a reagent II comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second counterpart capable of binding specifically to the first counterpart;
a reagent III comprising a third counterpart capable of specifically binding to the first counterpart;
a reagent IV comprising a donor capable of generating singlet oxygen in an excited state.
2. The kit of claim 1, wherein the second counterpart and the third counterpart are each a second antibody capable of specifically binding to a known antigen.
3. The kit according to claim 2, wherein the second antibody is a monoclonal antibody and/or a polyclonal antibody.
4. The kit of any one of claims 1 to 3, wherein the third counterpart is surface coated with biotin and the donor surface is surface coated with streptavidin.
5. The kit according to any one of claims 1 to 4, wherein the acceptor comprises an olefin compound and a metal chelate compound, and is soluble in an aqueous medium.
6. A kit as claimed in any one of claims 1 to 5, wherein all reagents are non-particulate and soluble in aqueous media.
7. A homogeneous immunoassay method for the combined detection of the same viral antigen, antibody for non-disease diagnostic purposes using the kit of any one of claims 1 to 6, comprising the steps of: firstly, detecting a negative sample without an antigen to be detected and an antibody to be detected, and taking an obtained detection result as a background signal value; and then detecting the chemiluminescence signal value of the sample to be detected, and comparing the chemiluminescence signal value of the sample to be detected with the background signal value, thereby judging whether the antigen to be detected and/or the antibody to be detected exist in the sample to be detected.
8. The method of claim 7, wherein if the detected chemiluminescent signal value of the test sample is equal to the background signal value, then the test sample is free of test antigen and test antibody;
if the detected chemiluminescence signal value of the sample to be detected is larger than the background signal value, the sample to be detected contains the antigen to be detected;
and if the chemiluminescence signal value of the sample to be detected obtained by detection is smaller than the background signal value, the sample to be detected contains the antibody to be detected.
9. The method according to claim 7 or 8, wherein the method for detecting the chemiluminescent signal value of the sample to be tested comprises the steps of:
s1, mixing the sample to be detected with the reagent I to obtain a first mixture;
s2, adding a reagent II, a reagent III and a reagent IV into the first mixture to obtain a second mixture;
s3, treating the second mixture with energy or active compound, and detecting the chemiluminescence signal value of the second mixture;
and S4, analyzing the chemiluminescence signal value, and judging whether the sample to be detected contains the antigen to be detected and/or the antibody to be detected.
10. The method of claim 9, wherein in step S2, reagent ii and reagent iii are added, followed by reagent iv.
11. The method according to claim 9 or 10, wherein in step S1, the sample to be tested is added to a solution containing the reagent i.
12. Use of a kit according to any one of claims 1 to 6 or a method according to any one of claims 7 to 12 for the combined detection of HCV antigens and antibodies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010072100.0A CN111272997A (en) | 2017-11-27 | 2017-11-27 | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711203146.6A CN108051585B (en) | 2017-11-27 | 2017-11-27 | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit |
| CN202010072100.0A CN111272997A (en) | 2017-11-27 | 2017-11-27 | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711203146.6A Division CN108051585B (en) | 2017-11-27 | 2017-11-27 | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111272997A true CN111272997A (en) | 2020-06-12 |
Family
ID=62120569
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711203146.6A Active CN108051585B (en) | 2017-11-27 | 2017-11-27 | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit |
| CN202010072100.0A Pending CN111272997A (en) | 2017-11-27 | 2017-11-27 | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711203146.6A Active CN108051585B (en) | 2017-11-27 | 2017-11-27 | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN108051585B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112051402A (en) * | 2020-09-15 | 2020-12-08 | 郑州伊美诺生物技术有限公司 | Kit for quantitatively detecting thyroid peroxidase and non-diagnosis-purpose detection method |
| CN116429754A (en) * | 2023-02-13 | 2023-07-14 | 科美博阳诊断技术(上海)有限公司 | HCV detection kit and using method thereof |
Families Citing this family (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116413255A (en) * | 2018-07-18 | 2023-07-11 | 科美博阳诊断技术(上海)有限公司 | Homogeneous chemiluminescence detection kit and application thereof |
| CN116539597A (en) * | 2018-07-18 | 2023-08-04 | 科美博阳诊断技术(上海)有限公司 | cTnI homogeneous phase chemiluminescence detection kit, detection method and device |
| EP3839485B1 (en) * | 2018-08-13 | 2024-03-13 | Chemclin Diagnostics (Shanghai) Co., Ltd. | Microsphere composition for chemiluminescence analysis and use thereof |
| WO2020034939A1 (en) * | 2018-08-13 | 2020-02-20 | 博阳生物科技（上海）有限公司 | Chemiluminescence analysis method and application thereof |
| CN116559153A (en) * | 2018-08-13 | 2023-08-08 | 科美博阳诊断技术(上海)有限公司 | Homogeneous chemiluminescence detection kit and application thereof |
| CN111122871A (en) * | 2018-10-31 | 2020-05-08 | 博阳生物科技（上海）有限公司 | Homogeneous detection kit and method for triiodothyronine and application |
| CN118010974A (en) * | 2018-10-31 | 2024-05-10 | 科美博阳诊断技术(上海)有限公司 | Homogeneous phase chemiluminescence detection kit |
| CN111122844A (en) * | 2018-10-31 | 2020-05-08 | 博阳生物科技（上海）有限公司 | Chemiluminescence immunoassay method for anti-biotin interference and application thereof |
| CN111122852A (en) * | 2018-10-31 | 2020-05-08 | 博阳生物科技（上海）有限公司 | Homogeneous phase chemiluminescence analysis method and application thereof |
| CN111122845A (en) * | 2018-10-31 | 2020-05-08 | 博阳生物科技（上海）有限公司 | Chemiluminescence immunoassay kit for resisting biotin interference and application thereof |
| CN111521804B (en) * | 2019-02-01 | 2024-01-19 | 科美诊断技术股份有限公司 | Treating agent and application thereof |
| CN112240937B (en) * | 2019-07-19 | 2023-06-13 | 科美博阳诊断技术(上海)有限公司 | Application of donor reagent in diagnosis of bacterial inflammatory diseases of subject infection |
| CN112240928B (en) * | 2019-07-19 | 2023-06-13 | 科美博阳诊断技术(上海)有限公司 | Homogeneous chemiluminescence analysis method and application thereof |
| CN113125696B (en) * | 2019-12-31 | 2024-03-26 | 科美博阳诊断技术(上海)有限公司 | Estradiol homogeneous chemiluminescence detection kit and application thereof |
| CN113125711B (en) * | 2019-12-31 | 2023-07-28 | 科美博阳诊断技术(上海)有限公司 | Acceptor reagent and application thereof |
| CN113125701B (en) * | 2019-12-31 | 2024-01-30 | 科美博阳诊断技术(上海)有限公司 | Homogeneous chemiluminescence detection kit and application thereof |
| CN113125712B (en) * | 2019-12-31 | 2023-06-16 | 科美博阳诊断技术(上海)有限公司 | Homogeneous chemiluminescence detection kit for hepatitis C virus antibody and application thereof |
| CN113125730B (en) * | 2019-12-31 | 2023-07-07 | 科美博阳诊断技术(上海)有限公司 | Homogeneous detection kit for interleukin 6 and application thereof |
| CN113125703B (en) * | 2019-12-31 | 2023-08-04 | 科美诊断技术(苏州)有限公司 | Myoglobin homogeneous detection kit and application thereof |
| CN113391065A (en) * | 2020-03-13 | 2021-09-14 | 科美诊断技术股份有限公司 | Receptor reagent for detecting novel coronavirus and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1030300A (en) * | 1987-06-15 | 1989-01-11 | 科特公司 | Utilize the enzyme-labeled immunity detection method to measure antigen and/or antibody in the human body simultaneously |
| US4943525A (en) * | 1987-11-02 | 1990-07-24 | Bioventures, Inc. | Simultaneous immunoassay for the determination of antigens and antibodies |
| CN101281137A (en) * | 2008-04-24 | 2008-10-08 | 博阳生物科技（上海）有限公司 | Light activating chemical luminescence luminous immune detecting method |
| CN101363853A (en) * | 2007-08-06 | 2009-02-11 | 北京科美东雅生物技术有限公司 | Human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit and method for preparing same |
| CN101750493A (en) * | 2008-12-17 | 2010-06-23 | 北京科美东雅生物技术有限公司 | Enzymatic chemiluminescence immunoassay qualitative diagnostic reagent kit for simultaneously testing communicable disease projects |
| CN101806802A (en) * | 2010-04-29 | 2010-08-18 | 华中科技大学 | Fluorescence resonance energy transfer-based homogeneous immunoassay method for multi-component simultaneous detection |
| CN102348980A (en) * | 2009-03-12 | 2012-02-08 | 西门子医疗保健诊断公司 | Immunoassays employing non-particulate chemiluminescent reagent |
-
2017
- 2017-11-27 CN CN201711203146.6A patent/CN108051585B/en active Active
- 2017-11-27 CN CN202010072100.0A patent/CN111272997A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1030300A (en) * | 1987-06-15 | 1989-01-11 | 科特公司 | Utilize the enzyme-labeled immunity detection method to measure antigen and/or antibody in the human body simultaneously |
| US4943525A (en) * | 1987-11-02 | 1990-07-24 | Bioventures, Inc. | Simultaneous immunoassay for the determination of antigens and antibodies |
| CN101363853A (en) * | 2007-08-06 | 2009-02-11 | 北京科美东雅生物技术有限公司 | Human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit and method for preparing same |
| CN101281137A (en) * | 2008-04-24 | 2008-10-08 | 博阳生物科技（上海）有限公司 | Light activating chemical luminescence luminous immune detecting method |
| CN101750493A (en) * | 2008-12-17 | 2010-06-23 | 北京科美东雅生物技术有限公司 | Enzymatic chemiluminescence immunoassay qualitative diagnostic reagent kit for simultaneously testing communicable disease projects |
| CN102348980A (en) * | 2009-03-12 | 2012-02-08 | 西门子医疗保健诊断公司 | Immunoassays employing non-particulate chemiluminescent reagent |
| CN101806802A (en) * | 2010-04-29 | 2010-08-18 | 华中科技大学 | Fluorescence resonance energy transfer-based homogeneous immunoassay method for multi-component simultaneous detection |
Non-Patent Citations (4)
| Title |
|---|
| P.CROVARI ET AL: "Radioimmunoassay for the simultaneous detection and quantitation of hepatitis B surface antigen and of the homologous antibody", BOLLETTINO DELL\'ISTITUTO SIEROTERAPICO MILANESE, vol. 54, no. 2, pages 149 - 160 * |
| 戚中田: "医学微生物学实验指导", vol. 1, 30 June 2011, 第二军医大学出版社, pages: 172 * |
| 胡燕 等: "HIV-1/2抗体和P24抗原联合检测试剂盒的研制与评价", vol. 21, no. 4, pages 391 - 393 * |
| 胡远贵: "化学发光免疫分析技术在检验医学中的应用", vol. 1, 31 July 2009, 湖北科学技术出版社, pages: 114 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112051402A (en) * | 2020-09-15 | 2020-12-08 | 郑州伊美诺生物技术有限公司 | Kit for quantitatively detecting thyroid peroxidase and non-diagnosis-purpose detection method |
| CN116429754A (en) * | 2023-02-13 | 2023-07-14 | 科美博阳诊断技术(上海)有限公司 | HCV detection kit and using method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108051585A (en) | 2018-05-18 |
| CN108051585B (en) | 2021-08-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108051585B (en) | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit | |
| CN109709317B (en) | Homogeneous phase immunoassay kit without matrix effect and analysis method and application thereof | |
| CN109725153B (en) | Homogeneous phase immunoassay method and application thereof | |
| JP4163869B2 (en) | Detection method | |
| WO2019223692A1 (en) | Chemical luminescence analysis and measurement method, system using same, and kit | |
| CN107976535B (en) | Homogeneous immunoassay kit for detecting target IgM antibody in sample and use method and application thereof | |
| CN108445239B (en) | Homogeneous phase immunoassay reagent kit for detecting beta human chorionic gonadotropin, and preparation method and application thereof | |
| CN108445216B (en) | Human anti-mullerian hormone determination kit and preparation method and application thereof | |
| CN114217077A (en) | Homogeneous phase immunoassay reagent kit for detecting target anti-Carp antibody and application thereof | |
| CN111521779B (en) | Combined detection method and kit for hepatitis C virus antigen and antibody | |
| CN113156119A (en) | Method for detecting coronavirus by adopting angiotensin converting enzyme II (ACE2) | |
| WO2009084369A1 (en) | Reagent for detecting hiv-1 antigen and detection method | |
| CN114354919A (en) | Homogeneous immunoassay kit for detecting 14-3-3eta protein and application thereof | |
| CN111505302A (en) | Homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide, preparation method, detection method and device | |
| CN113125704A (en) | Homogeneous phase chemiluminescence assay kit and application thereof | |
| CN113125706A (en) | Competitive homogeneous phase chemiluminescence assay kit and application thereof | |
| JP7488812B2 (en) | Chemiluminescence immunoassay method and system and reagent kit using said method | |
| CN110514650B (en) | Chemiluminescence analysis determination method, system and kit using same | |
| CN110514646B (en) | Chemiluminescence analysis determination method, system and kit using same | |
| CN110514647B (en) | Chemiluminescence analysis determination method, system and kit using same | |
| CN110161232B (en) | Homogeneous phase immunoassay kit for detecting anti-CCP antibody and application thereof | |
| CN113125418A (en) | Homogeneous phase chemiluminescence detection method and application thereof | |
| JP2002189027A (en) | Immunity detection method | |
| CN110579602A (en) | Method for assessing whether rheumatoid arthritis exists in vitro through biomarker-associated sample | |
| CN113125416A (en) | Competitive homogeneous phase chemiluminescence detection method and application thereof |
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 |