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CN110456073B - Method and kit for detecting antibody by double antigen sandwiches - Google Patents

Method and kit for detecting antibody by double antigen sandwiches Download PDF

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Publication number
CN110456073B
CN110456073B CN201910772077.3A CN201910772077A CN110456073B CN 110456073 B CN110456073 B CN 110456073B CN 201910772077 A CN201910772077 A CN 201910772077A CN 110456073 B CN110456073 B CN 110456073B
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antigen
kit
antibody
biotin
parasite
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CN110456073A (en
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周峻
黄记有
潘少丽
程珍珠
池朗山
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Guangdong Fapon Biotech Co Ltd
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Guangdong Fapon Biotech Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention relates to the technical field of biology, in particular to a method and a kit for detecting an antibody by double antigen sandwiches. The method is improved on the basis of the existing double antigen sandwich of a one-step two-step combination method, and the method can simultaneously improve the hook problem and the low-concentration sample omission problem by forming a marker-anti-marker compound to amplify signals.

Description

Method and kit for detecting antibody by double antigen sandwiches
Technical Field
The invention relates to the technical field of biology, in particular to a method and a kit for detecting an antibody by double antigen sandwiches.
Background
The double antigen sandwich method uses the labeled antigen to replace the labeled secondary antibody in the indirect method, solves the defect of the indirect method in methodology, and has the characteristics of high sensitivity, high specificity and further shortened window period. Conventional detection modes of the double antigen sandwich method can be classified into "one-step method", "two-step method" and "three-step method". Because of the characteristics of simplicity and short time consumption, the one-step method kit is favored by the clinical detection market. However, the "one-step method" has one disadvantage: "hook effect", false negative results are likely to occur when the antibody concentration is too high.
In the conventional one-step method, the sample to be tested and the labeled antigen are added simultaneously, i.e., the free labeled antigen and the test antibody are present in the reaction system simultaneously, and the test antibody partially forms a "coated antigen-antibody" complex with the coated antigen and partially forms a "labeled antigen-antibody" complex with the labeled antigen. Particularly, when the content of the detected antibody in the reaction system is high, the formation of a sandwich structure of the coated antigen-antibody-labeled antigen is seriously influenced by a labeled antigen-antibody complex formed by the detected antibody and the free labeled antigen, so that a hook effect is formed, and even false negative detection omission is caused.
To solve the hook effect, the applicant has previously invented a "one-step two-step binding method" (CN 109444434 a), i.e. a first step of contacting the coated antigen, the labelled antigen and the test substance under conditions sufficient for an antibody/antigen binding reaction to take place, forming an immune complex; washing away unbound antibody to be detected; the second step is to add a labeled antigen again and bind it to the remaining antigen binding sites in the immunocomplexes, which is effective in improving the HOOK problem, but still has a missed detection problem when the concentration of the antibody to be detected is too low.
In view of this, the present invention has been made.
Disclosure of Invention
The invention relates to a method for detecting an antibody by double antigen sandwich, which is used for forming a first antigen Ag 1 -antibody to be detected-second antigen Ag 2 In the form of (2) complete the test, wherein Ag 1 Coupled to a solid support, and the second antigen Ag 2 Comprising two forms, the method comprising the steps of:
a) Ag with 1 First form Ag 2 Contacting the test substance under conditions sufficient for an antibody/antigen binding reaction to occur, to form an immune complex;
wherein in terms of mole, ag 1 More than the first form Ag 2 And the first form Ag 2 For Ag coupled with a marker 2 ；
b) Washing away unbound antibody to be detected;
c) Addition of the second form of Ag 2 And allowing it to bind to the remaining antigen binding sites in the immune complex; the second form Ag 2 For Ag coupled with signalling substances 2 ；
d) Adding an anti-tag coupled with a signal indicator, said anti-tag being capable of forming a specific tag-anti-tag complex with said tag and performing signal amplification;
step c) and d) are not sequential;
e) Detecting the signal indicator to indicate the presence and/or amount of the antibody to be detected.
The method can simultaneously improve the hook problem and the low-concentration sample omission problem.
The invention also relates to a kit for implementing the method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic diagram of an embodiment of the present invention (AE is taken as an example of a signal indicator).
Detailed Description
The invention relates to a method for detecting an antibody by double antigen sandwich, which is used for forming a first antigen Ag 1 -antibody to be detected-second antigen Ag 2 In the form of (2) complete the test, wherein Ag 1 Coupled to a solid support, and the second antigen Ag 2 Comprising two forms, the method comprising the steps of:
a) Ag with 1 First form Ag 2 Contacting the test substance under conditions sufficient for an antibody/antigen binding reaction to occur, to form an immune complex;
wherein in terms of mole, ag 1 More than the first form Ag 2 And the first form Ag 2 For Ag coupled with a marker 2 ；
b) Washing away unbound antibody to be detected;
c) Addition of the second form of Ag 2 And allowing it to bind to the remaining antigen binding sites in the immune complex; said firstTwo forms of Ag 2 For Ag coupled with signalling substances 2 ；
d) Adding an anti-tag coupled with a signal indicator, said anti-tag being capable of forming a specific tag-anti-tag complex with said tag and performing signal amplification;
step c) and d) are not sequential;
e) Detecting the signal indicator to indicate the presence and/or amount of the antibody to be detected.
In the present invention, the first antigen Ag 1 And a second antigen Ag 2 The antigens of (2) may be the same or different.
In some embodiments, in step a), ag in molar ratio 1 :Ag 2 =6:2 to 6:5; either 3:2 or 2:1 may also be selected.
Suitable Ag 1 With Ag 2 The addition ratio of the (C) can avoid the hook effect on the premise of ensuring the sensitivity. This ratio may vary somewhat depending on the specific antigen-antibody characteristics, but the inventors have verified that most antigens meet the above ratios.
In the present invention, the "molar ratio" may also be the activity ratio of an antigen, and a protein having an epitope to which an antibody to be detected binds may be regarded as an activity unit.
In some embodiments, the Ag added in step a) is in molar ratio 2 With Ag added in step c) 2 The ratio of (2) to (4) is 6:2-6:4.
In some embodiments, the Ag added in step a) is in molar ratio 2 With Ag added in step c) 2 The ratio of (2) to (1).
Ag in step c) 2 The addition in a proper ratio can effectively reduce the residue of the marker to reduce the background.
In the present invention, (anti-tag/tag) combination means two compounds that can bind in a specific manner.
In some embodiments, the label/anti-label combination in the label-anti-label complex is selected from biotin or a derivative thereof/streptavidin, biotin or a derivative thereof/avidin, biotin or a derivative thereof/NeutrAvidin, biotin or a derivative thereof/avidin or a derivative antibody thereof, hapten/antibody, antigen/antibody, peptide/antibody, receptor/ligand, digoxin/digoxigenin, carbohydrate/lectin, and polynucleotide/complementary polynucleotide.
These various combinations and others are known and are well known to those skilled in the art.
The invention is preferably a family of biotin/biotin-aggregated proteins.
The family of biotin-binding proteins includes streptavidin, avidin and NeutrAvidin proteins described above, each of which is capable of binding four biotin molecules with high affinity and specificity. Among them, streptavidin is most commonly used, which is not glycosylated and has a very low level of non-specific binding. Avidin is a highly cationic glycoprotein with isoelectric point around 10.5, whose positively charged residues and oligosaccharide components mediate nonspecific binding, resulting in excessive background in some applications. Neutravidin protein undergoes deglycosylation and reduces the isoelectric point, thereby reducing its background staining.
Furthermore, it is well known to those skilled in the art that biotin may be replaced with a homofunctional derivative thereof, such as D-biotin, activated biotin, biocytin, ethylenediamine biotin, cadaverine biotin or desthiobiotin.
In some embodiments, the second form of Ag 2 In which the signaling material is coupled to Ag 2 Is achieved indirectly through a bridge.
In some embodiments, the bridge is selected from one or more of a protein, a protein complex, or a bifunctional crosslinking agent.
In some embodiments, the bridge is selected from a marker-anti-marker complex, which is as defined in the above.
In some embodiments, the protein or protein complex comprises at least one of bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, immunoglobulin, thyroglobulin, polylysine.
In a preferred embodiment, the bridge is selected from bovine serum albumin. Bovine serum albumin is not only commonly used in the field, but also has a certain function of reducing nonspecific signals, and can increase the signal-to-noise ratio of experiments.
In some embodiments, the bifunctional crosslinking agent is selected from the group consisting of:
1) Any of MPBH (4- [ 4-N-maleimidopynyl ] butyric acid hydrazide hydrochloride), MPEG2A (1- [2- [2- (2-amino-methoxy) ethyl ] maleimide hydrochloride), BMPH (N- [ beta-maleimidopropionic acid ] hydrate, trifluoroacetic acid salt), EMCH (N- [ epsilon-Maleimidocaproic acid) hydrate, trifluoroacetic acid salt), KMUH (N- [ kappa-maleimidoundecanoic acid ] hydrate, trifluoroacetic acid salt);
2) Or 1) a derivative of a substance containing a maleimide group and an amino or hydrazide group;
3) Or a group consisting of 1) and 2).
In the present invention, the bridge will also typically have the effect of amplifying the signal, e.g., the bridge may be coupled to more signal indicators to amplify the signal.
In the present invention, the antigen Ag 1 Usually immobilized on a solid support in advance, the term "immobilized" means creating a covalent or non-covalent bond. In some embodiments, the solid support is a magnetic bead, a slide, a membrane substrate, or a plate provided with a loading well.
In some embodiments, the magnetic beads are γfe 2 O 3 Or Fe (Fe) 3 O 4 Magnetic nanoparticles, or their complexes with organic polymeric materials.
In the present invention, a signaling indicator refers to a substance capable of providing a detected signal, which in some embodiments, in steps c) and d) is independently selected from any one or more of a chromophore, a digoxin-labeled probe, an electron dense substance, colloidal gold, or an enzyme. These labels are listed in the following non-limiting section:
enzymes that produce detectable signals, such as by colorimetry, fluorescence and luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.
Chromophores such as fluorescence, quantum dots, fluorescent microspheres, luminescent compounds and dyes.
A group having an electron density which can be detected by an electron microscope or by its electrical characteristics such as conductivity, amperometry, voltage measurement, resistance, and the like.
A detectable group, such as a molecule of sufficient size to induce a detectable modification in its physical and/or chemical properties; such detection may be achieved by optical methods (e.g., diffraction, surface plasmon resonance, surface variation and angle of contact variation) or physical methods (e.g., atomic force spectroscopy and tunneling).
Electronically dense materials, e.g. radioactive molecules (e.g 32 P， 35 S or 125 I)。
In some embodiments, the signaling indicator is an Acridinium Ester (AE).
The invention also provides a kit comprising an anti-marker as defined above, a first antigen Ag 1 Second antigen Ag 2 。
In some embodiments, the kit further comprises one or more of a sample pretreatment solution, a buffer, and a color developer.
The pretreatment solution may include a lysate (lysing tissues or cells), a purification reagent for proteins, particularly antibodies, a protease inhibitor, etc. capable of releasing, purifying, protecting antibodies. The pretreatment liquid is preferably an agent that does not cause denaturation of the antibody.
In some embodiments, the first antigen Ag 1 Second antigen Ag 2 An antigen capable of binding to antibodies raised by a disease pathogen;
in some embodiments, the disease pathogen comprises one or more of a virus, a bacterium, a fungus, or a parasite;
in some embodiments, the virus comprises: one or more of adenoviridae (adenoviridae), arenaviridae (arenaviridae), astroviridae (astroviridae), bunyaviridae (bunyaviridae), calicividae (calicividae), flaviviridae (flavoviridae), hepaciviridae (hepeviridae), single-molecule negative-strand RNA viridae (mononegavirales), reticuloviridae (nidovirales), picornaviridae (picornaviridae), orthomyxoviridae (orthomyxoviridae), papillomaviridae (papuloviridae), parvoviridae (parvoviridae), polyomaviridae (polyomaviridae), poxviridae (poxviridae), reoviridae (reoviridae), retroviridae (retroviridae) and togaviridae (togaviridae);
in some embodiments, the bacteria comprise: one or more of staphylococcus, streptococcus, li Shi, erysipelas, renieratia, bacillus, clostridium, mycobacterium, actinomyces, nocardia, corynebacterium, rhodococcus, and/or one or more of bacillus anthracis, erysipelas, tetanus, listeria, mycobacterium tuberculosis, escherichia coli, proteus, shigella, pneumobacillus, brucella, pneumophila, haemophilus influenzae, haemophilus parainfluenza, moraxella catarrhalis, acinetobacter, yersinia, pneumophila, pertussis, bordetella parapertussis, shigella, pasteurella, vibrio and haemolytic bacteria;
in some embodiments, the fungus comprises: one or more of coccidioidomycosis, geotrichum puusii, histoplasma capsulatum, histoplasma duchenne, lobayensis, paracoccidiomycosis brazil, blastomyces dermatitidis, sporotrichum, penicillium marneffei, candida albicans, candida glabrata, candida tropicalis, candida vinosa, aspergillus, ectobottle mold, pei Shi chromomyces lanuginosus, phoma verrucosum, chromomyces dermatitis, geotrichum candidum, cryptococcus neoformans, candida, rhizopus oryzae, mucor indicus, colpitis umbellifera, copperus, frog manure, auricularia coronaria, auricularia ani, nose sporotriclopyr, myces transparent and myceliophthora obscurus;
in some embodiments, the parasite comprises: digestive tract endoparasites, hepatic endoparasites, pulmonary endoparasites, brain tissue parasites, vascular endoparasites, lymphatic endoparasites one or more of a muscle tissue parasite, an intracellular parasite, a bone tissue parasite, and an intraocular parasite.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
1. HCV antigen coating of magnetic bead (MP-HCVAg)
Activating carboxyl magnetic beads with EDC and NHS, adding HCV antigen to incubate and couple with the activated carboxyl magnetic beads, removing supernatant by magnetic separation, and storing the HCV antigen coated magnetic beads in a magnetic bead preservation solution.
HCV antigen biotin labeling (HCVAg-bio)
HCV antigen was reacted with activated biotin (Bio-NHS) in a well-mixed manner. And (3) performing overnight dialysis by using a dialysis bag, and recovering residues in the dialysis bag to obtain the purified biotin-labeled HCV antigen.
3. Streptavidin AE label (SA-AE)
Streptavidin was mixed with activated acridine ester (AE-NHS) and reacted well. And (3) performing overnight dialysis by using a dialysis bag, and recovering residues in the dialysis bag to obtain the purified streptavidin AE marker (SA-AE).
4. Bovine serum albumin AE marker (BSA-AE)
The BSA solid was weighed and dissolved in buffer. To the BSA solution was added an activated acridine ester (AE-NHS). The reaction is carried out at room temperature. Samples were dialyzed overnight with a dialysis bag. Recovering residues in the dialysis bag to obtain bovine serum albumin AE marker (BSA-AE).
5. Maleimide activated ester activated BSA-AE
Maleimide activated ester activation was added to BSA-AE samples. The samples were transferred into dialysis bags for dialysis. Recovering the residue from the dialysis bag to obtain maleimide activated BSA-AE.
Coupling of HCVAg-BSA-AE
The maleimide activated BSA-AE sample was reacted in admixture with the HCVAg sample. And (3) transferring the sample into a dialysis bag for dialysis, and recovering residues in the dialysis bag to obtain the HCV antigen AE cross-linked product (HCVAg-BSA-AE).
7. Preparation of HCVAg-MPBH-AE
MPBH is mixed with AE-activated ester. The unreacted AE activated ester was removed by reaction with BSA. MPBH-AE conjugate was reacted in admixture with HCVAg sample. And (3) transferring the sample into a dialysis bag for dialysis, and recovering residues in the dialysis bag to obtain the HCV antigen AE marker (HCVAg-MPBH-AE).
Example 2
In this embodiment, each group is based on a one-step two-step combination method, and for technical details of this method reference is made to the prior art: chinese patent CN109444434a, publication date 2019, 03, 08.
Taking the B mode in (1) as an example:
the detection flow is as follows:
1) Taking 50uL of a sample to be detected (containing HCV antibody), adding 50uL of magnetic microspheres coated with HCV antigen, simultaneously adding 50uL of biotin-labeled antigen, and incubating for 15min at 37 ℃;
2) Adding a magnetic field to perform magnetic separation, and removing a supernatant;
3) Washing for 4 times, and repeating the operation of the step 2, wherein 250uL of washing liquid is washed each time;
4) Adding 100uL of SA-AE (Ag-BSA-AE) mixed reagent, and incubating for 10min at 37 ℃;
5) Adding a magnetic field to perform magnetic separation, and removing a supernatant;
6) Washing for 4 times, wherein 250uL of washing liquid is washed each time, and the operation of the step 4 is repeated;
7) The photo-activated A liquid (containing H) is added into each hole 2 O 2 ) 100uL, and adding photoexcitation liquid B (containing NaO)H) 100uL, relative Luminescence Units (RLU) of each well was measured with a scintillation photon counter at the same time as the addition, measuring time 1 second/well.
8) And (3) taking 4 times of the average value of the RLU of the negative control as a threshold value (cutoff value), comparing the RLU value of each well with the threshold value, and judging positive if the measured value of the sample is greater than or equal to the threshold value, otherwise, judging negative.
(1) Comparison of hook problem solving effects
The results of the 3 tests are shown below:
note that: the first column is positive quality control blood at different dilutions.
A mode: the first step of reaction, adding a coating antigen, serum and a certain amount of Ag-Bio, and initially forming a sandwich; the second step of the reaction adds an amount of SA-AE for providing a signal (RLU value).
B mode: the first step of reaction, adding a coating antigen, serum and a certain amount of Ag-Bio, and initially forming a sandwich; the second reaction step adds a certain amount of SA-AE and Ag-BSA-AE mixture for providing a signal (RLU value).
C mode: the first step of reaction, adding coating antigen, serum and a certain amount of Ag-BSA-AE, and initially forming a sandwich; the second reaction step adds a quantity of Ag-BSA-AE, and the two steps together provide a signal (RLU value).
Wherein, the Ag-Bio and the Ag-BSA-AE are the amounts which are enough to fully react with the antibody to be detected, and the SA-AE is also the amount which is enough to fully react with the Ag-Bio, namely, the addition amount is excessive. The other experiments below are the same.
Results: A. and B and C detection mode data are compared, when the concentration of the positive sample is increased to a certain value, the signal value of the A mode is reduced, and the concentrations of the B and C modes and the signal value still keep positive correlation. The results show that the "one-step two-step method" is an effective solution to the hook phenomenon.
( And (3) injection: coating antigen: magnetic beads coated with HCV antigen; ab: an antibody; ag-Bio: biotinylated HCV antigen; sA-AE: a streptavidin AE label; ag-BSA-AE: an HCV antigen bovine serum albumin AE marker; ag-MPBH-AE: HCV antigen-MPBH-AE marker; the following is the same as )
(2) Low value problem solving effect contrast
The results of the 3 tests are shown below:
note that: "1/24K" means dilution of 2.4X10 4 Positive quality control blood.
B mode: the first step of reaction, adding a coating antigen, serum and a certain amount of Ag-Bio, and initially forming a sandwich; the second reaction step adds a certain amount of SA-AE and Ag-BSA-AE mixture for providing a signal (RLU value).
C mode: the first step of reaction, adding coating antigen, serum and a certain amount of Ag-BSA-AE, and initially forming a sandwich; the second step of the reaction adds a certain amount of Ag-BSA-AE mixture, and the two steps together provide a signal.
Results: the P/N value data indicates that the detection sensitivity B mode > C mode. The results show that the combination of two different bridging marker systems, especially the combination of HCVAg-bio-marker and HCVAg-BSA-marker, can simultaneously improve the hook problem and the low concentration sample omission problem, and has higher detection sensitivity than the detection sensitivity of a bridging marker system (such as Ag-BSA-AE) alone.
(3) Bridging system
Note that: "1/24K" means dilution of 2.4X10 4 Positive quality control blood.
A combination: the first step of reaction, adding a coating antigen, serum and a certain amount of Ag-Bio, and initially forming a sandwich; the second reaction step adds a certain amount of SA-AE and Ag-BSA-AE mixture for providing a signal (RLU value).
And B, combination: the first step of reaction, adding a coating antigen, serum and a certain amount of Ag-Bio, and initially forming a sandwich; the second reaction step adds a certain amount of SA-AE and Ag-MPBH-AE mixture for providing a signal.
And C, combination: the first step of reaction, adding a coating antigen, serum and a certain amount of Ag-Bio, and initially forming a sandwich; the second reaction step adds an amount of SA-AE and Ag-Bio/SA-AE mixture to provide a signal.
(4) Comparison of the effects of the mode of labeling antigen AE in the composition
Note that: "1/24K" means dilution of 2.4X10 4 Positive quality control blood.
Column 2 data testing step: the first step of reaction, adding a coating antigen, serum and a certain amount of Ag-Bio, and initially forming a sandwich; the second reaction step adds a certain amount of SA-AE and Ag-BSA-AE mixture for providing a signal (RLU value).
Column data testing step 3: the first step of reaction, adding a coating antigen, serum and a certain amount of Ag-Bio, and initially forming a sandwich; the second reaction step adds a certain amount of SA-AE and Ag-MPBH-AE mixture for providing a signal.
Column 4 data testing step: the first step of reaction, adding a coating antigen, serum and a certain amount of Ag-Bio, and initially forming a sandwich; the second reaction step adds a certain amount of SA-AE and Ag-AE mixture for providing signals.
Results: the order of the P/N values is Ag-BSA-AE > Ag-MPBH-AE > Ag-AE. The results show that when two bridging systems are used, the other bridging labelling system used in combination with Ag-Bio/SA-AE is better in an indirect bridging manner than in a direct labelling manner.
(5) High and low value sample detection
And selecting 20 negative samples and 20 positive samples, detecting by adopting several reaction modes at the same time, comparing the RLU value of each average value hole with CUTOFF, judging positive if the measured value of the sample is more than or equal to 20X negative, otherwise, judging negative, and using +to represent positive, -representing negative, wherein the result is as follows:
the results show that the method can solve the problem of hook missed detection of the one-step method, can further reduce the missed detection probability (such as sample P5) of the weak positive specimen on the basis of the one-step two-step method, and improves the detection sensitivity.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (12)

1. A method of double antigen sandwich detection of antibodies to form a first antigen Ag 1 -antibody to be detected-second antigen Ag 2 In the form of (2) complete the test, wherein Ag 1 Coupled to a solid support, and the second antigen Ag 2 Comprising two forms, the method comprising the steps of:
a) Ag with 1 First form Ag 2 Contacting the test substance under conditions sufficient for an antibody/antigen binding reaction to occur, to form an immune complex;
wherein in terms of mole, ag 1 More than the first form Ag 2 And the first form Ag 2 For Ag coupled with a marker 2 ；
b) Washing away unbound antibody to be detected;
c) Addition of the second form of Ag 2 And allowing it to bind to the remaining antigen binding sites in the immune complex; the second form Ag 2 For Ag coupled with signalling substances 2 ；
d) Adding an anti-tag coupled with a signal indicator, said anti-tag being capable of forming a specific tag-anti-tag complex with said tag and performing signal amplification;
step c) and d) are not sequential;
e) Detecting the labelling agent to indicate the presence and/or amount of the antibody to be detected;
the label/anti-label combination in the label-anti-label complex is selected from biotin or a derivative thereof/streptavidin, biotin or a derivative thereof/avidin;
wherein the derivative of biotin is any one of D-biotin, activated biotin, ethylenediamine biotin, cadaverine biotin or desthiobiotin;
the second form Ag 2 In (2) coupling a marking agent to Ag 2 Is indirectly realized through a bridge;
the bridge is selected from one or more of bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, polylysine or MPBH;
in steps c) and d), the signaling indicators are independently selected from any one or more of chromophores, digoxin-labeled probes, electron dense substances, colloidal gold, or enzymes.
2. The method of claim 1, wherein the solid support is a magnetic bead, a slide, a membrane substrate, or a plate provided with a loading well.
3. The method of claim 2, wherein the magnetic beads are γfe 2 O 3 Or Fe (Fe) 3 O 4 Magnetic nanoparticles, or their complexes with organic polymeric materials.
4. The method of double antigen sandwich detection antibody of claim 1 wherein in steps c) and d) the signal indicator is an acridinium ester.
5. A kit comprising an anti-marker as defined in any one of claims 1 to 4, a first antigen Ag 1 Second antigen Ag 2 。
6. The kit of claim 5, further comprising one or more of a sample pretreatment solution, a buffer solution, and a color developer.
7. The kit of claim 6, wherein the first antigen Ag 1 Second antigen Ag 2 Is an antigen capable of binding to antibodies raised by a disease pathogen.
8. The kit of claim 7, wherein the disease pathogen comprises one or more of a virus, a bacterium, a fungus, or a parasite.
9. The kit of claim 8, wherein the virus comprises: adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, flaviviridae, hepaciviridae, single-molecule negative-strand RNA viridae, reticuloviridae, picornaviridae, orthomyxoviridae, papillomaviridae, parvoviridae, polyomaviridae, poxviridae, reoviridae, retrovirus, and togaviridae.
10. The kit of claim 8, wherein the bacteria comprise: staphylococcus, streptococcus, li Shi, erysipelas, renieratia, bacillus, clostridium, mycobacterium, actinomyces, nocardia, corynebacterium, rhodococcus, and/or bacillus anthracis, erysipelas, tetanus, listeria, mycobacterium tuberculosis, escherichia coli, proteus, shigella, pneumobacillus, brucellosis, pneumophila, haemophilus influenzae, haemophilus parainfluenza, moraxella catarrhalis, acinetobacter, yersinia, pneumophila, pertussis, bordetella parapertussis, shigella, pasteurella, vibrio and haemolytic bacteria.
11. The kit of claim 8, wherein the fungus comprises: one or more of coccidioidomycosis, geotrichum puusii, histoplasma capsulatum, histoplasma dulcis, lobayensis, paracoccidiomycosis brazil, blastomyces dermatitidis, sporomyces lanuginosus, penicillium marneffei, candida albicans, candida glabrata, candida tropicalis, candida vitis, aspergillus, ectobottle mold, pei Shi chromomyces lanuginosus, phoma verrucosum, chromomyces dermatitis, geotrichum candidum, cryptococcus neoformans, candida, rhizopus oryzae, mucor indicus, colpitis umbellifera, coppernix, frog manure, auricularia coronal, auricularia, myceliophthora, and myceliophthora obscurca.
12. The kit of claim 8, wherein the parasite comprises: digestive tract endoparasites, hepatic endoparasites, pulmonary endoparasites, brain tissue parasites, vascular endoparasites, lymphatic endoparasites one or more of a muscle tissue parasite, an intracellular parasite, a bone tissue parasite, and an intraocular parasite.
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