CN113514636A - New crown neutralizing antibody fluorescence immunochromatographic assay test strip and preparation method thereof - Google Patents
New crown neutralizing antibody fluorescence immunochromatographic assay test strip and preparation method thereof Download PDFInfo
- Publication number
- CN113514636A CN113514636A CN202110234840.4A CN202110234840A CN113514636A CN 113514636 A CN113514636 A CN 113514636A CN 202110234840 A CN202110234840 A CN 202110234840A CN 113514636 A CN113514636 A CN 113514636A
- Authority
- CN
- China
- Prior art keywords
- pad
- test strip
- neutralizing antibody
- immunochromatographic assay
- detection
- 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
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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- 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
- G01N33/54346—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nanotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a new crown neutralization antibody fluorescence immunochromatographic assay test strip, which comprises a sample pad, a combination pad, a reaction membrane and an absorption pad, wherein the sample pad, the combination pad, the reaction membrane and the absorption pad are sequentially jointed on a bottom plate; wherein, the combination pad is coated with ACE2 and goat anti-chicken IgG coupled with time-resolved fluorescent microspheres; the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with RBD, and the quality control line is coated with chicken IgY antibody. The invention provides a novel corona neutralizing antibody fluorescence immunochromatographic assay test strip which has the characteristics of low cost, rapid and sensitive detection, instantaneity, no need of professional operation and the like, and can be completed only in a secondary biosafety laboratory. The test strip can effectively monitor the vaccination reaction in clinical research and be used as the evaluation index of the later vaccine effect; the detection rate of new coronas is improved, and the detection omission caused by false negative of nucleic acid detection is compensated; detecting the content of the neutralizing antibody in the body of the new coronary rehabilitation patient to judge whether the new coronary rehabilitation patient can be subjected to repeated work and production and whether the re-infection risk exists.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to a fluorescent immunochromatographic test strip for a new crown neutralizing antibody and a preparation method thereof.
Background
The novel coronavirus (SARS-CoV-2) is a novel beta coronavirus (beta-coronavirus), has a diameter of 60-140 nm, and is in a crown-like shape under an electron microscope. The S protein (spike protein) Receptor Binding Domain (RBD) of SARS-CoV-2 can bind to human ACE2 protein, so that the virus enters cells and replicates. Meanwhile, RBD is also the main target of neutralizing antibody. The novel coronavirus neutralizing antibody (NAb) is an antibody generated in novel coronavirus pneumonia patients and novel coronavirus vaccine injection patients, can be combined with RBD of S protein so as to block the combination of the virus and human ACE2, and shows direct neutralizing activity. Therefore, the detection of the neutralizing antibody can judge whether the human body has the capability of resisting the virus infection, and can effectively monitor the vaccination response and the evaluation index of the vaccine effect in clinical research.
Antibody detection mainly comprises virus neutralization test (cVNT), enzyme-linked immunosorbent assay (ELISA) and Lateral Flow Assay (LFA) rapid test. Virus neutralization tests (cvnts) detect neutralizing antibodies in the blood of patients, but require testing in tertiary biosafety laboratories, which are cumbersome and time consuming.
The fluorescence immunochromatographic test strip is a detection and analysis technology for a sample to be detected by using a microporous chromatographic membrane as a solid phase carrier, marking an antigen or an antibody by a fluorescent particle signal substance and carrying out antigen-antibody reaction based on capillary action.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a novel crown neutralization antibody fluorescence immunochromatographic assay test strip and a preparation method thereof. .
The invention aims to provide a novel crown neutralization antibody fluorescence immunochromatographic assay test strip.
The second purpose of the invention is to provide the application of any one of the novel crown neutralizing antibody fluorescence immunochromatographic assay test strips in the preparation of COVID-19 neutralizing antibody fluorescence immunochromatographic assay kits.
The third purpose of the invention is to provide a preparation method of any one of the novel crown neutralizing antibody fluorescence immunochromatographic assay test strip.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention discloses a novel corona neutralizing antibody fluorescence immunochromatographic assay test strip (as shown in figure 1), which sequentially comprises a PVC (polyvinyl chloride) base plate, a sample pad, a combination pad, a reaction membrane and a water absorption pad. Spraying ACE2 marked by fluorescent nano microspheres on a bonding pad by using a competitive bonding method; RBD was coated on the reaction membrane at the position of the Test line (T line).
If the sample does not contain the neutralizing antibody of COVID-19, the fluorescence-ACE 2 complex is dissolved when the sample flows to the combination pad in the detection, ACE2 on the combination pad flows through the reaction membrane under the driving of the sample and is combined with RBD on the T line of the reaction membrane, and the fluorescence microspheres on ACE2 are gathered on the T line and show fluorescence when irradiated by exciting light; if the blood contains neutralizing antibodies to COVID-19, the neutralizing antibodies in the sample can flow to the T line on the reaction membrane synchronously with ACE2 on the binding pad, compete for binding to the coated RBD, the higher the content of the neutralizing antibodies, the less fluorescence ACE2 is captured by the RBD, and the fluorescence value in the detection zone is inversely related to the content of the neutralizing antibodies.
In the detection method, the goat anti-chicken IgG and the goat anti-chicken IgY which are fluorescently labeled are used as a quality control system (C line) to play a role in quality control and contrast. The fluorescence intensity (F) of the T lineT) Fluorescence intensity of C line (F)C) Making a comparison, establishing a test standard curve, and comparing F of an unknown sampleT/FCAnd (5) carrying out a curve to obtain the content of the neutralizing antibody in the sample. If fluorescence does not appear at the C line or the fluorescence does not reach the lowest predicted value during the test, the measured value is invalid, and the test is repeated.
The invention claims a novel corona neutralizing antibody fluorescence immunochromatographic assay test strip, which comprises a sample pad, a combination pad, a reaction membrane and an absorption pad, wherein the sample pad, the combination pad, the reaction membrane and the absorption pad are sequentially jointed on a bottom plate;
wherein, the combination pad is coated with ACE2 and goat anti-chicken IgG coupled with time-resolved fluorescent microspheres;
the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with RBD, and the quality control line is coated with chicken IgY antibody.
Preferably, the excitation wavelength of the time-resolved fluorescent microspheres is 300-400 nm, and the emission wavelength is 500-700 nm.
Preferably, the conjugate pad is coated with ACE2 and goat anti-chicken IgG coupled to europium-labeled time-resolved fluorescent microspheres.
Preferably, the time-resolved fluorescent microspheres are fluorescent microspheres with europium as a marker, and the diameter of the fluorescent microspheres is 100 nm-500 nm.
Preferably, characterised in that the conjugate pad is a glass fibre or polyester material.
Preferably, the reaction membrane is a nitrocellulose membrane or a cellulose acetate membrane.
Preferably, the sample pad is dried after being soaked in the buffer solution of the sample pad, and the buffer solution formula of the sample pad is as follows: 0.5% BSA, 2.5% sucrose, 1.5% Tween-20 were dissolved in 0.015M pH 9PB buffer.
Preferably, the combination pad is soaked and dried by 0.02-0.05 mol/L Tris-HCl buffer solution containing 1% of bovine serum albumin (w/w), 0.1-5% of trehalose (w/w) and 0.02-0.1% of Tween-20(v/v) and pH7.4, and then coated with ACE2 and goat anti-chicken IgG which are coupled with the time-resolved fluorescent microspheres.
The application of any test strip for detecting the fluorescent immunochromatography of the COVID-19 neutralizing antibody in the preparation of a kit for detecting the fluorescent immunochromatography of the COVID-19 neutralizing antibody also belongs to the protection scope of the invention.
The invention also claims a preparation method of any COVID-19 neutralizing antibody fluorescence immunochromatography detection test strip, wherein the coupled time-resolved fluorescent microsphere ACE2 protein is sprayed on a conjugate release pad to obtain a conjugate pad coated with the coupled time-resolved fluorescent microsphere ACE2 protein; soaking and drying the combination pad by using 0.02-0.05 mol/L Tris-HCl buffer solution containing 1% of bovine serum albumin (w/w), 0.1-5% of trehalose (w/w), 0.02-0.1% of Tween-20(v/v) and pH7.4, and respectively spraying RBD and chicken IgY on a reaction membrane as a detection line and a quality control line; the sample pad is obtained by soaking the sample pad in a buffer solution of the sample pad and drying the sample pad, wherein the formula of the buffer solution of the sample pad is as follows: 0.5% (mass fraction) BSA, 2.5% (mass fraction) sucrose, 1.5% (volume fraction) Tween-20 were dissolved in 0.015M pH 9PB buffer.
Preferably, the time-resolved fluorescent microspheres are activated, mixed with ACE2 protein, and covalently coupled. After sufficient reaction, blocking buffer was added for blocking.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a novel corona neutralizing antibody fluorescence immunochromatographic assay test strip which has the characteristics of low cost, rapid and sensitive detection, instantaneity, no need of professional operation and the like, and can be completed only in a secondary biosafety laboratory. The test strip can effectively monitor the vaccination reaction in clinical research and be used as the evaluation index of the later vaccine effect; the detection rate of new coronas is improved, and the detection omission caused by false negative of nucleic acid detection is compensated; detecting the content of the neutralizing antibody in the body of the new coronary rehabilitation patient to judge whether the new coronary rehabilitation patient can be subjected to repeated work and production and whether the re-infection risk exists.
Drawings
FIG. 1 is a schematic diagram of a fluorescent immunochromatographic assay test strip for detecting a novel crown neutralizing antibody
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Embodiment 1A novel corona neutralizing antibody fluorescence immunochromatographic assay test strip and a preparation method thereof
First, experiment method
1. Preparation of sample pad
And soaking the glass fiber with the specification of 25cm multiplied by 30cm in the buffer solution of the sample pad, taking out after soaking for 3-5 min, drying for 16-18 h at room temperature, and cutting into the sample pad with the specification of 2cm multiplied by 30 cm.
The buffer formulation for the sample pad was as follows: 0.5% (mass fraction) BSA, 2.5% (mass fraction) sucrose, 1.5% (volume fraction) Tween-20 were dissolved in 0.015M pH 9PB buffer.
2. Preparation of fluorescent marker and preparation method of combined pad
1) Preparation of fluorescently labeled ACE 2: adding 200 μ L of europium-labeled time-resolved fluorescent microspheres into a 2mL EP tube, adding 800 μ L of 0.1M MES with pH6.0, and mixing in dark; then 200 mu L of 1mg/mL NHS (N-hydroxysuccinimide) and 200 mu L of 50mg/mL EDC (carbodiimide) are added, after uniform mixing, the mixture is activated and reacted for 30 minutes at room temperature, after the activation and reaction, the mixture is centrifuged at 15000rpm for 25 minutes, the supernatant is discarded, 1mL MES is added, the latex microspheres are resuspended by ultrasonic, 100 mu L of 2mg/mL ACE2 is added, and after uniform mixing, the mixture is reacted for 2 hours at room temperature in a dark place; adding 10% BSA (bovine serum albumin) to make the BSA at the final level of 1%, mixing uniformly, and reacting at room temperature for 1 hour; centrifugation at 15000rpm for 15 minutes, followed by collection of the pellet and reconstitution with a reconstitution fluid (0.015M PBS, pH7.4: 10% BSA: Tween-20 in a volume ratio of 90: 10: 1) to twice the original volume, yielded fluorescently labeled ACE 2.
2) Preparing fluorescence labeling sheep anti-chicken IgG: adding 200 μ L of time-resolved fluorescent microspheres into a 2mL EP tube, adding 800 μ L of 0.1M MES with pH6.0, and mixing in dark; then adding 200 mu L of NHS (N-hydroxysuccinimide) with the concentration of 1mg/mL and 200 mu L of EDC (carbodiimide) with the concentration of 50mg/mL, uniformly mixing, carrying out activation reaction at room temperature for 30 minutes, after the activation reaction, centrifuging at 15000rpm for 25 minutes, discarding supernatant, adding 1mL of MES, carrying out ultrasonic heavy suspension on latex microspheres, adding 20 mu L of goat anti-chicken IgG with the concentration of 2mg/mL, uniformly mixing, and carrying out reaction at room temperature in a dark place for 2 hours; adding 10% BSA (bovine serum albumin) to make the BSA at the final level of 1%, mixing uniformly, and reacting at room temperature for 1 hour; the mixture was centrifuged at 15000rpm for 15 minutes, and the pellet was collected after centrifugation and reconstituted with a reconstitution solution (0.015M PBS, pH7.4: 10% BSA: Tween-20 in a volume ratio of 90: 10: 1) to twice the original volume to give fluorescently labeled goat anti-chicken IgG.
3) The fluorescently-labeled ACE2 was mixed with goat anti-chicken IgG at a volume ratio of 9: 1.
4) Soaking the glass fiber in 0.02-0.05 mol/L Tris-HCl (containing 0.1-5% of trehalose and 0.02-0.1% of Tween-20) buffer solution containing 1% of bovine serum albumin (mass fraction) and pH7.4 for 2h, drying at 37 ℃ for 2h, and storing in a dry environment for later use.
5) And spraying the mixed marker onto glass fiber with the thickness of 10mm multiplied by 30cm by using a gold spraying and film scratching instrument, and drying at 37 ℃ for 18-24 hours to obtain the bonding pad for later use.
3. Preparation of the reaction film
1) Coating the reaction film.
Quality control line (line C): marking on a nitrocellulose membrane, wherein the distance between a line C and the lower edge of the nitrocellulose membrane is 0.5cm, the distance between the line C and the upper edge of the nitrocellulose membrane is 2cm, diluting the chicken IgY antibody to 1mg/mL by using a coating buffer solution, and coating, wherein the marking parameter is 1 mu L/cm;
detection line (T line): the distance between the T line and the upper edge of the nitrocellulose membrane is 2cm, the distance between the T line and the upper edge is 0.5cm, RBD is diluted to 1.0mg/mL by using a coating buffer solution for coating, and the marking parameter is 1 mu L/cm;
wherein, the coating buffer solution: 0.01M PBS pH7.4+ 1% dextran.
2) Drying
And (5) putting the mixture into a 37 ℃ oven, and drying for 6-12 hours.
4. Assembly
The arrangement sequence of the components on the bottom plate is sequentially absorption pad-reaction membrane (nitrocellulose membrane) -combination pad-sample pad,
1) pasting the absorption pad: the bottom plate is flatly paved on a working table top; the protective film at the position where the absorption pad is adhered on the upper edge of the bottom plate is uncovered, the absorption pad is adhered on the protective film, and the absorption pad covers the reaction film (nitrocellulose film) for 2 mm.
2) Bonding the bonding pad: the protective film at the joint of the reaction membrane (nitrocellulose membrane) and the bonding pad is removed, and the bonding pad is adhered to the protective film by the same method as the absorption pad, and the bonding pad is covered on the reaction membrane (nitrocellulose membrane) for 2 mm.
3) Pasting the sample pad: the sample pad is adhered to the lower portion of the conjugate pad in the same manner as the absorbent pad. The sample pad was covered with 2mm on the conjugate pad.
4) Cutting and clamping the test strip: and (3) placing the adhered base plate into a slitter, cutting the base plate into test strips with the width of 4mm, and then placing the test strips into a plastic card shell.
II, composition
Comprises a sample pad, a combination pad, a reaction membrane (nitrocellulose membrane) and a test paper of an absorption pad which are sequentially jointed on a bottom plate,
wherein, the bonding pad is coated with ACE2 coupled with europium-labeled time-resolved fluorescent microspheres and goat anti-chicken IgG,
sample pad: the glass fiber with the specification of 25cm multiplied by 30cm is soaked in the buffer solution of the sample pad and then dried, and the formula of the buffer solution of the sample pad is as follows: 0.5% BSA (mass fraction), 2.5% sucrose (mass fraction), 1.5% Tween-20 (volume fraction) was dissolved in 0.015M pH 9PB buffer;
the reaction membrane (nitrocellulose membrane) is 25mm in width and is provided with a detection line and a quality control line, the detection line is coated with RBD, and the quality control line is coated with chicken IgY antibody.
The paper strip encapsulates and becomes the detection card in the casing, has set gradually sample application of sample hole and observation hole window on the casing, and wherein detection line and quality control line correspond with observation hole window position, and sample application of sample hole sets up in sample pad top.
Third, using method
1) Restoring the test strip and the sample to be tested to room temperature;
2) starting the immunofluorescence analyzer, and inserting a corresponding blank detection card to adjust the reading position; when in use, the outer package of the reagent strip is removed, the reagent is taken out and placed horizontally;
3) adding 100 μ L of serum of sample to be tested diluted 20 times with PBST into the sample adding hole of the prepared detection card, reacting for 15min, inserting the detection card into an immunofluorescence analyzer, and clicking the 'test' button on the analyzer
4) The time-resolved fluorescent microspheres trapped on the detection line and the quality control line of the test strip emit strong fluorescent strips under the optimal excitation light;
5) adopting an immunochromatography analyzer to read the fluorescence intensity of the detection line and the quality control line of the test strip, giving a T/C value, and taking the inhibition rate as 1- (T)Measuring/CMeasuring÷TYin (kidney)/CYin (kidney)) And calculating the content of the neutralizing antibody in the sample, wherein the positive results show that the inhibition rates are all more than 30%.
Example 2 detection of blood samples from Xinguan convalescent and vaccine injection patients
First, experiment method
Clinical samples were tested using the test strip of example 1. This example included 8 new crown convalescent, 8 vaccinees, and 16 healthy subjects as controls. To evaluate the test strip of example 1 for its detection effect on neutralizing antibodies against the novel coronavirus.
Second, experimental results
The detection results are shown in table 1, the inhibition rates of 8 Xinguan convalescent patients and 8 vaccinees are both more than 30%, and the test strip can detect neutralizing antibodies in serum.
Table 1:
it should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A new crown neutralization antibody fluorescence immunochromatographic assay test strip is characterized by comprising a sample pad, a combination pad, a reaction membrane and an absorption pad which are sequentially jointed on a bottom plate;
wherein, the combination pad is coated with ACE2 and goat anti-chicken IgG coupled with time-resolved fluorescent microspheres;
the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with RBD, and the quality control line is coated with chicken IgY antibody.
2. The COVID-19 neutralizing antibody fluorescent immunochromatographic assay test strip of claim 1, wherein the binding pad is coated with ACE2 and goat anti chicken IgG coupled to europium labeled time resolved fluorescent microspheres.
3. The COVID-19 neutralizing antibody fluorescence immunochromatographic assay test strip of claim 1, wherein the time-resolved fluorescent microspheres are fluorescent microspheres with europium as a label with a diameter of 100nm to 500 nm.
4. The COVID-19 neutralizing antibody fluorescent immunochromatographic assay test strip of claim 1, wherein said conjugate pad is a glass fiber or polyester material.
5. The COVID-19 neutralizing antibody fluorescence immunochromatographic assay test strip according to claim 1, wherein the reaction membrane is a nitrocellulose membrane or a cellulose acetate membrane.
6. The new crown neutralization antibody fluorescence immunochromatographic assay test strip according to claim 1, wherein the sample pad is dried after being soaked in the buffer solution of the sample pad, and the formula of the buffer solution of the sample pad is as follows: 0.5% BSA (w/w), 2.5% sucrose (w/w), 1.5% Tween-20(v/v) was dissolved in 0.015M pH 9PB buffer.
7. The test strip for the immunofluorometric detection of the neo-corona neutralizing antibody according to claim 1, wherein the conjugate pad is coated with ACE2 and goat anti-chicken IgG coupled with time-resolved fluorescent microspheres after being soaked and dried in 0.02-0.05 mol/L Tris-HCl buffer solution containing 1% bovine serum albumin (w/w), 0.1-5% trehalose (w/w) and 0.02-0.1% Tween-20(v/v) and pH 7.4.
8. Use of the novel corona neutralizing antibody fluorescent immunochromatographic assay test strip of any one of claims 1 to 7 in the preparation of a COVID-19 neutralizing antibody fluorescent immunochromatographic assay kit.
9. The method for preparing the test paper strip for the immunofluorometric detection of the neo-corona neutralizing antibody according to any one of claims 1 to 7, characterized in that the conjugated time-resolved fluorescent microsphere ACE2 protein is sprayed on the conjugate release pad to obtain a conjugate pad coated with the conjugated time-resolved fluorescent microsphere ACE2 protein; soaking and drying the combination pad by using 0.02-0.05 mol/L Tris-HCl buffer solution containing 1% of bovine serum albumin (w/w), 0.1-5% of trehalose (w/w), 0.02-0.1% of Tween-20(v/v) and pH7.4, and respectively spraying RBD and chicken IgY on a reaction membrane as a detection line and a quality control line; the sample pad is obtained by soaking the sample pad in a buffer solution of the sample pad and drying the sample pad, wherein the formula of the buffer solution of the sample pad is as follows: 0.5% BSA, 2.5% sucrose, 1.5% Tween-20 were dissolved in 0.015M pH 9PB buffer.
10. The method for preparing the novel corona neutralizing antibody fluorescence immunochromatographic assay test strip of any one of claims 1 to 7, wherein the time-resolved fluorescent microspheres are activated, mixed with ACE2 protein, and covalently coupled. After sufficient reaction, blocking buffer was added for blocking.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110234840.4A CN113514636A (en) | 2021-03-03 | 2021-03-03 | New crown neutralizing antibody fluorescence immunochromatographic assay test strip and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110234840.4A CN113514636A (en) | 2021-03-03 | 2021-03-03 | New crown neutralizing antibody fluorescence immunochromatographic assay test strip and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113514636A true CN113514636A (en) | 2021-10-19 |
Family
ID=78060179
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110234840.4A Pending CN113514636A (en) | 2021-03-03 | 2021-03-03 | New crown neutralizing antibody fluorescence immunochromatographic assay test strip and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113514636A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113985037A (en) * | 2021-12-23 | 2022-01-28 | 广东忠信生物科技有限公司 | Novel detection method, detection test strip and kit for coronavirus neutralizing antibody |
| CN114755416A (en) * | 2022-02-28 | 2022-07-15 | 广州中医药大学顺德医院(佛山市顺德区中医院) | New coronavirus neutralizing antibody latex method detection test strip tracing to new coronavirus neutralizing test titer |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111273016A (en) * | 2020-02-26 | 2020-06-12 | 浙江诺迦生物科技有限公司 | Kit for rapidly detecting coronavirus based on S protein ligand and ACE2 receptor competition chromatography |
| CN111537747A (en) * | 2020-06-10 | 2020-08-14 | 广州再生医学与健康广东省实验室 | Test strip and kit for detecting novel human coronavirus IgG antibody and preparation method thereof |
| CN111562368A (en) * | 2020-06-18 | 2020-08-21 | 威海威高生物科技有限公司 | SARS-CoV-2 neutralizing antibody detection kit |
| CN111562369A (en) * | 2020-06-18 | 2020-08-21 | 威海威高生物科技有限公司 | SARS-CoV-2 neutralizing antibody detection kit |
| CN211905393U (en) * | 2020-09-22 | 2020-11-10 | 正元盛邦(天津)生物科技有限公司 | Novel coronavirus specific antibody and neutralizing antibody combined detection test paper and device |
| CN111999507A (en) * | 2020-08-25 | 2020-11-27 | 中国人民解放军军事科学院军事医学研究院 | Fluorescence immunochromatography test paper for detecting novel coronavirus antibody |
| CN112394180A (en) * | 2021-01-19 | 2021-02-23 | 南京立顶医疗科技有限公司 | Detection method and detection kit for SARS-CoV-2 neutralizing antibody |
-
2021
- 2021-03-03 CN CN202110234840.4A patent/CN113514636A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111273016A (en) * | 2020-02-26 | 2020-06-12 | 浙江诺迦生物科技有限公司 | Kit for rapidly detecting coronavirus based on S protein ligand and ACE2 receptor competition chromatography |
| CN111537747A (en) * | 2020-06-10 | 2020-08-14 | 广州再生医学与健康广东省实验室 | Test strip and kit for detecting novel human coronavirus IgG antibody and preparation method thereof |
| CN111562368A (en) * | 2020-06-18 | 2020-08-21 | 威海威高生物科技有限公司 | SARS-CoV-2 neutralizing antibody detection kit |
| CN111562369A (en) * | 2020-06-18 | 2020-08-21 | 威海威高生物科技有限公司 | SARS-CoV-2 neutralizing antibody detection kit |
| CN111999507A (en) * | 2020-08-25 | 2020-11-27 | 中国人民解放军军事科学院军事医学研究院 | Fluorescence immunochromatography test paper for detecting novel coronavirus antibody |
| CN211905393U (en) * | 2020-09-22 | 2020-11-10 | 正元盛邦(天津)生物科技有限公司 | Novel coronavirus specific antibody and neutralizing antibody combined detection test paper and device |
| CN112394180A (en) * | 2021-01-19 | 2021-02-23 | 南京立顶医疗科技有限公司 | Detection method and detection kit for SARS-CoV-2 neutralizing antibody |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113985037A (en) * | 2021-12-23 | 2022-01-28 | 广东忠信生物科技有限公司 | Novel detection method, detection test strip and kit for coronavirus neutralizing antibody |
| CN114755416A (en) * | 2022-02-28 | 2022-07-15 | 广州中医药大学顺德医院(佛山市顺德区中医院) | New coronavirus neutralizing antibody latex method detection test strip tracing to new coronavirus neutralizing test titer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106872420B (en) | Kit and method for time-resolved fluorescence quantitative detection of microalbuminuria | |
| CN111562369A (en) | SARS-CoV-2 neutralizing antibody detection kit | |
| EP0636886B1 (en) | A calibrator or control composition for an IgM serology assay | |
| CN109765384B (en) | Canine coronavirus antibody fluorescence detection test strip and preparation method and application thereof | |
| CN111233985A (en) | Preparation method of novel coronavirus IgA antibody rapid detection test strip | |
| ES2339834T3 (en) | USE OF CONTROL SURFACES TO DETECT SAMPLES THAT INTERFER IN AN IDENTIFICATION PROCESS. | |
| US5989840A (en) | Estimation of active infection by heliobacter pylori | |
| CN113156129B (en) | High-sensitivity detection method and product of neutralizing antibody | |
| CN113514636A (en) | New crown neutralizing antibody fluorescence immunochromatographic assay test strip and preparation method thereof | |
| CN112798795A (en) | Assays for detecting blocking analytes | |
| JP7241696B2 (en) | Immunochromatographic device | |
| CN109856406B (en) | Canine parvovirus antibody fluorescence detection test strip and preparation method and application thereof | |
| CN111007257A (en) | Porcine pseudorabies virus gE protein antibody detection immune blocking chromatography kit and application thereof | |
| CN112379089A (en) | New coronavirus detection method based on quantum dot microsphere immunochromatographic test strip | |
| CN109856407B (en) | Canine distemper virus antibody fluorescence detection test strip and preparation method and application thereof | |
| JP3859027B2 (en) | Method for measuring specific substances in nipple discharge | |
| KR20210080410A (en) | Lateral flow analysis for differential isotype detection | |
| CN113985037A (en) | Novel detection method, detection test strip and kit for coronavirus neutralizing antibody | |
| US7067264B2 (en) | Test device for detecting human blood and method of use | |
| JP2007218593A (en) | Immuno-chromatography measuring method for bio-pilin detection, and its device | |
| CN220690951U (en) | Kit for detecting cardiac troponin I | |
| CN115943313A (en) | Immunochromatographic device for detecting neutralizing antibodies of coronaviruses | |
| CN109212203B (en) | Quantum dot immunochromatography test strip for rapidly detecting brucella antibody | |
| JP3493544B2 (en) | Antibody assay device | |
| CN114930171A (en) | Lateral flow test strip with competitive assay controls |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211019 |