CN112904021A - Test strip for double-line detection of antibody after vaccination of new coronavirus - Google Patents
Test strip for double-line detection of antibody after vaccination of new coronavirus Download PDFInfo
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- G—PHYSICS
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- 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
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- 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
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- 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
Abstract
The invention discloses a test strip for detecting antibodies after vaccination of new corona viruses, which comprises a bottom plate, a sample pad, a combination pad, a reaction pad and a water absorption pad, wherein the sample pad, the combination pad, the reaction pad and the water absorption pad are sequentially lapped on the bottom plate; the other detection zone detects whether the neutralizing antibody generated by the organism can reach a certain titer, so that the protective effect is formed. The test strip has good sensitivity, good detection performance, low cost and simple use method, and can quickly, simply and conveniently detect the effect of the new corona vaccine after inoculation without professional technical personnel and professional equipment under the multi-scene conditions of hospitals, clinics and even at home; the sample demand is small; can improve the effective inoculation rate of the vaccine, prevent infection caused by poor inoculation effect of the vaccine and has good clinical application prospect.
Description
Technical Field
The invention relates to the technical field of rapid immunochromatographic assay, in particular to a test strip for double-line detection of an antibody after vaccination with a new coronavirus.
Background
The novel coronavirus, namely severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), SARS coronavirus in 2003 and MERS coronavirus in 2012 belong to the family beta of Coronaviridae, and the virus is also classified as the seventh coronavirus infecting human by the world health organization. The virus has a latent period of 7-14 days after infection, and has pneumonia after disease occurrence, and common symptoms of fever, cough, myalgia or fatigue; less common symptoms include phlegm, headache, hemoptysis, diarrhea, dyspnea, etc. The symptoms are similar to the common cold, with dyspnea and eventual death if not treated in time. SARS-CoV-2 is transmitted by droplets, air, with the possibility of fecal oral and maternal-infant transmission. The virus infection has no age preference, and the elderly (98 years old) or infants (30 hours) have confirmed diagnosis cases; meanwhile, the conditions of family aggregation outbreak, asymptomatic infectors and the like exist, and the health and the life of human beings are seriously harmed.
At this stage, successful control of the new coronavirus needs to be dependent on vaccine immunization. The vaccination can gradually establish the group immunity in the population, block epidemic situation epidemic, and is particularly important in the current epidemic situation prevention and control. The key index for evaluating the immunity of the population is the amount of the neutralizing antibody, and whether the immunity is formed is judged by detecting whether the titer of the neutralizing antibody reaches the standard. The novel corona vaccines that can be prepared are of the following type: attenuated vaccines, inactivated vaccines, protein subunit vaccines, viral vector vaccines (replicating/non-replicating/antigen presenting cells involved), nucleic acid vaccines (mRNA, DNA), virus-like particle vaccines, and the like. At present, the domestic vaccine uses a new coronavirus inactivated vaccine, and the vaccine is obtained by inactivating and purifying the new coronavirus cultured in vitro. The main principle is as follows: the RBD region of the S protein of the new coronavirus is combined with ACE2 on the surface of a human cell to be amplified in vivo to cause human infection, and the inactivated virus can promote a human body to generate an anti-RBD antibody which can be combined with the RBD to block the combination of the RBD and ACE2, so that the aim of inhibiting the amplification of the virus in vivo is finally fulfilled.
According to the data in the clinical trial phase III of the new corona vaccine, the protective efficacy of the new corona vaccine is 79.34%, and the positive conversion rate of the neutralizing antibody is 99.52%. That is, 99.52% of vaccinated people can produce neutralizing antibodies, while 79.34% of vaccinated people can produce protective efficacy against the body because the neutralizing antibodies produced by vaccination have a certain titer. However, not all people are vaccinated with the vaccine to produce protective efficacy, and some people cannot avoid new coronavirus infection because the protective efficacy produced by vaccination is insufficient, i.e., the purpose of prevention is not achieved. Therefore, after vaccination, it is a problem to be solved to determine whether the organism produces neutralizing antibodies, and whether the efficacy of producing neutralizing antibodies can achieve protective efficacy.
The chemiluminescence method, the colloidal gold/colloidal selenium method and the enzyme-linked immunosorbent assay can be used for detecting a neutralizing antibody, but the inventor firstly finds that when the neutralizing antibody is detected by using the colloidal gold, the detection result of the test strip is invalid due to the adoption of the traditional control band coating strategy; meanwhile, a single detection band cannot simultaneously solve the problems of two aspects of the body reaction to the vaccine and the formation of protection, namely the problem of detecting whether the body generates neutralizing antibodies and whether the neutralizing antibodies can provide protective efficacy.
Aiming at the technical problems, the inventor develops a test strip which can detect whether an vaccinee body contains an anti-RBD antibody after the new crown vaccination, judge whether the vaccine generates protective efficacy and further detect the antibody after the new crown vaccination, and the test strip can quickly, simply and conveniently detect the effect after the new crown vaccination under the multi-scene conditions of hospitals, clinics, even at home and the like without professional technicians and professional equipment.
Disclosure of Invention
Aiming at the technical problem, the invention provides a test strip for double-line detection of antibodies after vaccination of new corona, which comprises a bottom plate 1, a sample pad 2, a combination pad 3, a reaction pad 4 and a water absorption pad 5, wherein the sample pad 2, the combination pad 3, the reaction pad 4 and the water absorption pad 5 are sequentially lapped on the bottom plate 1, the reaction pad 4 is provided with a detection belt and a control belt, the detection belt is arranged at one end close to the combination pad 3, the control belt is arranged at one end close to the water absorption pad 5, the detection belt comprises a vaccination reaction detection belt 6 and/or an immunity effect detection belt 7, the vaccination reaction detection belt 6 is coated with a novel coronavirus surface spike glycoprotein Receptor Binding Domain (RBD) protein, the immunity detection belt 7 is coated with a colloidal gold labeled novel coronavirus receptor ACE2 protein and mouse IgG, the control belt comprises a first control belt 8 and/or a second control belt 9, are coated with goat anti-mouse IgG antibody.
Preferably, the labeled amount of the ACE2 protein is 6-8 mug of the ACE2 protein per 1ml of 4/ten thousand colloidal gold solution, the labeled amount of a goat anti-mouse antibody is 0.5mg/ml, and the labeled amount of an RBD protein is 0.1 mg/ml.
Preferably, the test strip comprises a vaccination response test strip 6 and an immunity efficacy test strip 7, the control strip comprises a first control strip 8, the vaccination response test strip 6 and the immunity efficacy test strip 7, the first control strip 8 is arranged on the reaction pad 4 in sequence, and the vaccination response test strip 6 and the immunity efficacy test strip 7 can be respectively combined with the anti-RBD antibody, so as to display different test results.
Preferably, the test strip comprises two test strips with different detection zones and control zones, and the reaction pad 4 of the first test strip is provided with a vaccination reaction detection zone 6 and a first control zone 8; the reaction pad of the second test strip is provided with an immunity effect detection zone 7 and a second control zone 9, the two test strips can also be arranged to share the same sample pad 2 and the water absorption pad 5, and the combination pad 3 and the reaction pad 4 are separated by polyethylene materials so as to be convenient for detecting different indexes.
Preferably, the test paper strip be two-way detection test paper strip, sample pad 2 set up in bottom plate 1's intermediate position, combination pad 3, reaction pad 4 and water absorption pad 5 all have the setting in sample pad 2's both sides, combination pad 3, reaction pad 4 and water absorption pad 5 are taken on sample pad 2 in proper order towards two different directions about respectively, reaction pad 4 of test paper strip left end on be provided with immunity efficiency and detect area 7 and second control band 9, reaction pad 4 of test paper strip right-hand member on be provided with vaccination reaction and detect area 6 and first control band 8, the sample dropwise add on sample pad 2 back, can remove to both ends to in order to detect different indexes.
Preferably, the reaction pad 4 is provided with a nitrocellulose membrane, and the vaccination reaction detection strip 6, the immunity efficacy detection strip 7, the first control strip 8 and the second control strip 9 are coated on the nitrocellulose membrane.
Preferably, the interval between the vaccination response test strip 6 and the immunopotency test strip 7, the interval between the immunopotency test strip 7 and the first control strip 8, the interval between the vaccination response test strip 6 and the first control strip 8, and the interval between the immunopotency test strip 7 and the second control strip 9 are all 2-4 mm.
Preferably, the joining length of the sample pad 2 and the binding pad 3 is 1-2mm, the joining length of the binding pad 3 and the reaction pad 4 is 1-2mm, and the joining length of the reaction pad 4 and the absorbent pad 5 is 1-2 mm.
The vaccination reaction detection zone 6 and the immunity effect detection zone 7 are used for detecting whether the organism generates neutralizing antibodies after the new corona vaccine is injected and whether the organism generates protective effect after the new corona vaccine is injected, and the positions of the two zones can be exchanged. According to the technical scheme, RBD protein is coated at the position of a detection strip, and an anti-RBD antibody detection test strip is prepared by combining the RBD protein coated at the position of the detection strip and the ACE2 labeled with colloidal gold; meanwhile, ACE2 can be coated at the position of the detection belt, and the RBD is marked by colloidal gold; in order to realize different detection purposes, ACE2 or RBD with different concentrations can be coated at the position of the detection zone, and S protein or nucleoprotein of new coronavirus can be coated at the same time, so as to achieve the purpose of detecting whether the organism responds to the vaccine.
The invention has the beneficial effects that: the test strip has good sensitivity, good detection performance, low cost and simple use method, and can quickly, simply and conveniently detect the effect of the new corona vaccine after inoculation without professional technical personnel and professional equipment under the multi-scene conditions of hospitals, clinics and even at home; the sample demand is small, and the vaccine reaction of the organism and the protective force of the organism can be simultaneously detected only by a small amount of blood samples, so that the feedback can be obtained in time; in addition, the existing corona novacells are various in types, such as inactivated vaccines, mRNA vaccines, adenovirus vector corona novacells, genetic engineering recombinant vaccines, virus-like particle vaccines and the like, and different groups of people have different responses and intensities to different types of vaccines; the test strip can judge whether the detected person has response or protection to the injected new corona vaccine; if protection indicates that the vaccine has immunity against the new coronavirus; if there is a response without protection, the injected species of vaccine can be boosted to achieve protection; if no protection is available, no response is given, which indicates that other kinds of vaccines need to be replaced for immunization. Taking the inoculation of inactivated new corona vaccine as an example; if the control band and the detection band 6 are detected to be developed at a certain time point after inoculation, the detection band 7 is not developed, and the inactivated vaccine needs to be enhanced; if the color of the detection band and the control band indicates that protection is formed; if only the control zone is colored, the organism does not respond to the inactivated vaccine, and other kinds of vaccines such as mRNA vaccine need to be replaced for inoculation if the protection force needs to be obtained; in a word, the kit provided by the invention can improve the effective vaccination rate of the vaccine, prevent infection caused by poor vaccination effect, and has good clinical application prospect. The test strip can preliminarily evaluate the effect of the vaccinated people after the vaccination without going out, and provide a basis for adjusting the new crown protection strategy in the next step.
Drawings
FIG. 1 is a perspective view of a test strip for double-line detection of antibodies after vaccination with neocorona vaccine
1. Bottom plate 2, sample pad 3, combined pad 4, reaction pad 5, water absorption pad 6, vaccination reaction detection band 7, immunity effect detection band 8, first control band
FIG. 2 is a perspective view of a test strip for detecting antibodies after vaccination with two test strips and a single detection line
1. Bottom plate 2, sample pad 3, combined pad 4, reaction pad 5, water absorption pad 6, vaccination reaction detection band 7, immunity effect detection band 8, first control band 9, second control band
FIG. 3 is a top view of the test strip for bidirectional detection of the antibody after vaccination
2. Sample pad 3, combination pad 4, reaction pad 5, water absorption pad 6, vaccination reaction detection band 7, immunity effect detection band 8, first control band 9, second control band
FIG. 4 is a graph showing the detection effect of the control band coated anti-His antibody test strip
FIG. 5 is a diagram showing the test effect of the goat anti-mouse IgG test strip coated with the control band of the double antibody system
FIG. 6 test strip sensitivity analysis for different amounts of label
1. Test strip sensitivity 2 when the labeled amount is 6 mug and test strip sensitivity when the labeled amount is 8 mug
FIG. 7 clinical negative sample test 1
FIG. 8 clinical negative sample test 2
FIG. 9 is a graph showing the results of measurement of negative specimens 6, 13 and 38 treated with a sample diluent
Detailed Description
The invention is further described with reference to the following figures and examples, which are only intended to illustrate the invention and not to limit the scope of the invention.
Embodiment I, test strip for double-line detection of antibody after vaccination of new coronavirus and interpretation method
A test strip for double-line detection of antibodies of new coronavirus after vaccination comprises a base plate 1, a sample pad 2, a combination pad 3, a reaction pad 4 and a water absorption pad 5, wherein the sample pad 2, the combination pad 3, the reaction pad 4 and the water absorption pad 5 are sequentially lapped on the base plate 1, the reaction pad 4 is provided with a vaccination reaction detection belt 6, an immunity effect detection belt 7 and a first control belt 8, the vaccination reaction detection belt 6 is arranged at one end close to the combination pad 3, the first control belt 8 is arranged at one end close to the water absorption pad 5, and the immunity effect detection belt 7 is arranged between the vaccination reaction detection belt 6 and the first control belt 8; the joint length of the sample pad 2 and the combination pad 3 is 2mm, the joint length of the combination pad 3 and the reaction pad 4 is 2mm, and the joint length of the reaction pad 4 and the water absorption pad 5 is 2 mm; the reaction pad 4 is provided with a nitrocellulose membrane, the vaccination reaction detection belt 6, the immunity effect detection belt 7 and the first control belt 8 are coated on the nitrocellulose membrane, and the intervals between the vaccination reaction detection belt 6, the immunity effect detection belt 7 and the control belt 8 are all 4 mm.
The vaccination reaction detection zone 6 and the immunity effect detection zone 7 are used for detecting whether the organism generates neutralizing antibodies after the new corona vaccine is injected and whether the organism generates protective effect after the new corona vaccine is injected, and the positions of the two detection lines can be exchanged. The vaccination response detection band 6 is used for detecting whether the organism reacts to the vaccination, and the immunity efficacy detection band 7 is used for detecting whether the organism generates protective efficacy after being vaccinated.
Firstly, when the vaccination reaction detection zone 6, the immunity effect detection zone 7 and the first control zone 8 are all colored, the examinee is the organism which is inoculated with the new corona vaccine and not only reacts, but also the titer of the neutralizing antibody in the body reaches a certain concentration, and the protective effect is generated; secondly, when the vaccination reaction detection zone 6 and the first control zone 8 are colored and the immunity effect detection zone 7 is not colored, the fact that the organism generates a certain reaction after the subject is vaccinated with the new corona vaccine, but the generated neutralizing antibody does not reach a certain titer and does not generate protective effect on the organism is meant; thirdly, when the first control band 8 is colored and neither the vaccination response detection band 6 nor the immunity effect detection band 7 is colored, the examinee is proved to have no response and protective effect after being vaccinated with the new corona vaccine; and fourthly, when the control band 8 does not develop the color, the detection result is invalid no matter whether the vaccination reaction detection band 6 and the immunity efficacy detection band 7 develop the color or not. When the immunity effect detection band 7 does not show the line, namely the detected person does not generate the protection effect after being inoculated with the new corona vaccine, whether the vaccination reaction detection band 6 shows the line or not and whether the organism generates the reaction or not, the type of the vaccine is required to be changed, and the vaccination is carried out again.
Embodiment two, test strip for detecting antibody after new coronavirus vaccination by double test strip and single detection line and interpretation method
The utility model provides a test paper strip of two test paper strip single detection lines detection new coronavirus vaccination after antibody, as shown in figure 2, divide into two test paper strips, includes bottom plate 1, sample pad 2, combination pad 3, reaction pad 4 and absorb water pad 5 respectively, sample pad 2, combination pad 3, reaction pad 4 and absorb water pad 5 overlap joint in proper order on bottom plate 1, be equipped with vaccination reaction on the reaction pad 4 of one of them test paper strip and detect area 6 and first control band 8, be equipped with immunity efficiency on the reaction pad 4 of another test paper strip and detect area 7 and second control band 9. Sample pad 2 and combination pad 3 link up length and be 2mm, combination pad 3 and reaction pad 4 link up length and be 2mm, reaction pad 4 and 5 link up length of absorbing water are 2 mm. The reaction pad 4 is a nitrocellulose membrane, the vaccination reaction detection belt 6, the first control belt 8, the immunity effect detection belt 7 and the second control belt 9 are respectively coated on the nitrocellulose membranes of the two test paper strips, and the intervals between the vaccination reaction detection belt 6 and the first control belt 8, and between the immunity effect detection belt 7 and the second control belt 9 are all 4 mm. The reaction pad 4 is a nitrocellulose membrane, the vaccination reaction detection belt 6, the first control belt 8, the immunity effect detection belt 7 and the second control belt 9 are respectively coated on the nitrocellulose membranes of the two test paper strips, and the intervals between the vaccination reaction detection belt 6 and the first control belt 8, and between the immunity effect detection belt 7 and the second control belt 9 are all 4 mm. The vaccination reaction detection zone 6 and the immunity effect detection zone 7 are used for detecting whether the organism generates neutralizing antibodies after the new corona vaccine is injected and whether the organism generates protective effect after the new corona vaccine is injected, and the positions of the two detection lines can be exchanged.
The vaccination response detection band 6 is used for detecting whether the organism reacts to the vaccination, and the immunity efficacy detection band 7 is used for detecting whether the organism generates protective efficacy after being vaccinated. Firstly, when the vaccination reaction detection band 6, the first control band 8, the immunity effect detection band 7 and the second control band 9 are all colored, the examinee is the organism which is inoculated by the new corona vaccine and not only generates reaction, but also the titer of the neutralizing antibody in vivo reaches a certain concentration, and the protective effect is generated; secondly, when the vaccination reaction detection zone 6, the first control zone 8 and the second control zone 9 are colored and the immunity effect detection zone 7 is not colored, the fact that the body of the subject generates a certain reaction after the vaccination with the new corona vaccine, but the generated neutralizing antibody does not reach a certain titer and does not generate protective effect on the body is meant; thirdly, when the first control band 8 and the second control band 9 are colored, and the vaccination reaction detection band 6 and the immunity effect detection band 7 are not colored, the fact that the organism of the examinee does not react and does not generate protection effect after the examinee is vaccinated with the new corona vaccine is shown; when the first control band 8 and the second control band 9 do not develop color, the detection result is invalid no matter whether the vaccination reaction detection band 6 and the immunity efficacy detection band 7 develop color or not; when the immunity effect detection zone 7 is not developed, namely the tested person is not protected after inoculating new corona vaccine, no matter whether the vaccination reaction detection zone 6 is developed or not and whether the organism is reacted or not, the type of vaccine should be changed, and the vaccination is carried out again.
Example III test strip for bidirectional detection of antibody after vaccination with new coronavirus and interpretation method
The utility model provides a test paper strip of two-way detection new coronavirus vaccination after antibody, as shown in fig. 3, the test paper strip include sample pad 2, combination pad 3, reaction pad 4 and absorb water and fill up 5, sample pad 2 set up in the intermediate position of bottom plate, combination pad 3, reaction pad 4 and absorb water pad 5 overlap joint in proper order on the bottom plate, both sides reaction pad 4 on one side be equipped with vaccination reaction and detect area 6 and first control band 8, be equipped with immunity efficiency and detect area 7 and second control band 9 on the other side. Sample pad 2 be sharing sample pad, its and combination pad 3 link up length is 2mm, combination pad 3 and reaction pad 4 link up length is 2mm, reaction pad 4 and the pad 5 link up length that absorbs water are 2 mm. The reaction pad 4 is a nitrocellulose membrane, the vaccination reaction detection belt 6, the first control belt 8, the immunity effect detection belt 7 and the second control belt 9 are respectively coated on the nitrocellulose membrane at two sides, and the intervals between the vaccination reaction detection belt 6 and the first control belt 8, and between the immunity effect detection belt 7 and the second control belt 9 are all 4 mm. The vaccination reaction detection zone 6 and the immunity effect detection zone 7 are used for detecting whether the organism generates neutralizing antibodies after the new corona vaccine is injected and whether the organism generates protective effect after the new corona vaccine is injected, and the positions of the two detection lines can be exchanged.
The vaccination response detection band 6 is used for detecting whether the organism reacts to the vaccination, and the immunity efficacy detection band 8 is used for detecting whether the organism generates protection efficacy after being vaccinated. Firstly, when the vaccination reaction detection band 6, the first control band 8, the immunity effect detection band 7 and the second control band 9 are all colored, the examinee is the organism which is inoculated by the new corona vaccine and not only generates reaction, but also the titer of the neutralizing antibody in vivo reaches a certain concentration, and the protective effect is generated; secondly, when the vaccination reaction detection zone 6, the first control zone 8 and the second control zone 9 are colored and the immunity effect detection zone 7 is not colored, the fact that the body of the subject generates a certain reaction after the vaccination with the new corona vaccine, but the generated neutralizing antibody does not reach a certain titer and does not generate protective effect on the body is meant; thirdly, when the first control band 8 and the second control band 9 are colored, and the vaccination reaction detection band 6 and the immunity effect detection band 7 are not colored, the fact that the organism of the examinee does not react and does not generate protection effect after the examinee is vaccinated with the new corona vaccine is shown; when the first control band 8 and the second control band 9 do not develop color, the detection result is invalid no matter whether the vaccination reaction detection band 6 and the immunity efficacy detection band 7 develop color or not; fifthly, when the immunity effect detection zone 7 does not show the line, namely the subject does not generate the protection effect after being inoculated with the new corona vaccine, no matter whether the vaccination reaction detection zone 6 shows the line or not, whether the organism generates the reaction or not, the type of the vaccine is required to be changed, and the vaccination is carried out again.
Example four, a method of using a test strip for detecting antibodies after vaccination with a novel coronavirus
When the test strip is used, the detection test strip coated with the film is horizontally placed, the sample is dripped on the sample pad 2, the sample gradually chromatographs to the combination pad 3 based on the capillary action and the suction action of the water absorption pad 5, combines with the particles marked on the combination pad 3 to react, and chromatographs to the reaction pad 4 together with the marked particles, and then reacts with substances coated on the vaccination reaction detection belt 6, the immunity effect detection belt 7 and the first control belt 8 to develop color, and redundant samples and marked particles chromatographed to the water absorption pad 5.
Example V Effect of antibody labeling on the detection Effect
The test strip for detecting the effect of the new corona vaccine after vaccination comprises a sample pad, a combination pad, a reaction pad and a water absorption pad, wherein the combination pad is fixed with a novel coronavirus ACE2 protein (Fipeng biology, Cat: ACE2-PS-Ag1, production date: 20201130), and the reaction pad is coated with two detection bands and a control band, such as the test strip prepared in the first embodiment.
In the previous study, when the anti-His antibody is coated on the control band and the RBD protein (Fipeng organism, cat # ncov-PS-Ag28, production date: 20201210) is coated on the detection band, the negative can be developed normally, but as the concentration of the neutralizing antibody in the sample increases, the control band becomes shallower along with the shallower detection line (as shown in FIG. 4), so that the result of the test strip cannot be judged normally.
The inventor coats goat anti-mouse antibody on the control band, coats RBD antibody on the detection band, and adds mouse IgG while marking ACE2 protein with colloidal gold, so that the mouse IgG is marked with the colloidal gold. During detection, the gold colloidal particles labeled with ACE2 protein and mouse IgG (Luoyangbo-Otto center of Experimental materials, cat # 20201227) were captured on the control band, so that the control band developed color without affecting the normal color development of the detection band (see FIG. 5).
EXAMPLE VI Effect of protein labeling amount on assay results
The test agent for detecting the effect after the new crown vaccination comprises a dispersion medium and a novel coronavirus ACE2 protein marked by colloidal gold in the dispersion medium. The novel coronavirus ACE2 protein marked by colloidal gold is obtained by combining colloidal gold and recombinant protein. The recombinant protein is composed of a novel coronavirus ACE2 protein and a tag protein, and the tag protein is a His tag.
When preparing a test strip for detecting whether an organism reacts to a vaccine, the amount of recombinant ACE2 protein corresponding to 4/ten thousand per 1ml of colloidal gold solution is 6 mug, a dispersion medium consists of phosphate buffer solution, bovine serum albumin, sucrose, trehalose and tween, and the balance of water, wherein the concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.2; the final mass concentration of bovine serum albumin is 1.5%, the final mass concentration of sucrose is 3%, the final mass concentration of trehalose is 3%, and the final mass concentration of tween is 2%; in phosphate buffer, Na2HPO4·12H20.26% of O, NaH2PO4·2H2The O mass content is 0.044%, the quality control line on the reaction pad is coated with 0.5mg/ml of goat anti-mouse IgG antibody, and the RBD protein on the detection line is coated with 0.1 mg/ml. The detection effect is shown in figure 6, and the sensitivity of the detection neutralizing antibody can reach 5 mug/ml.
When the test strip for detecting whether the body generates protective force after being inoculated with vaccine is prepared, the amount of recombinant ACE2 protein corresponding to each 1ml of 4/ten thousand colloidal gold solution is 8 mug, the dispersion media are the same, the quality control line on the reaction pad is coated with 0.5mg/ml goat anti-mouse antibody, and the detection line is coated with 0.1mg/ml RBD protein. The detection effect is shown in FIG. 6, and the sensitivity of the detection neutralizing antibody can reach 20 mug/ml.
Similarly, if two tests are carried out on one test strip, the colloidal gold solution is combined with a fixed amount of recombinant ACE2 protein, and RBD proteins with different concentrations are coated on two detection lines of the reaction pad. The marked ACE2 protein and the RBD protein coated by the detection line can be interchanged to achieve the same detection effect.
EXAMPLE VII verification of test strip clinical samples for detection of neutralizing antibodies
131 negative serum samples are collected clinically, the whole serum sample is dripped into a sample pad, and the sample flows through a combination pad, a detection line on a nitrocellulose membrane, a control line and redundant serum in sequence and is absorbed by absorbent paper. The results are shown in FIGS. 7 and 8, in which the color development of Nos. 6, 13 and 38 was poor, and the color development effect was shown in FIG. 9 after dilution at a ratio of 1:1 using a sample diluent (10mMPB plus 20% FBS). In the full serum detection of 131 negative serum samples, 128 samples are good in color development and are directly judged to be negative, 3 samples with lighter color development are adjusted by a diluent, the final color development is good, and the samples are judged to be negative, namely the negative coincidence rate is 100%.
Because the test strip is a competition method, the quality control line and the detection line show the line at the same time, and the test strip is judged to be negative. All negative sample detection results are negative, wherein the color development of individual samples is poor, and the color development effect is improved after the sample diluent is used. The test result of the negative sample proves that the test strip has good detection performance on clinical negative samples.
In conclusion, the test strip disclosed by the invention has the advantages of good sensitivity, good detection performance, low cost and simple use method, and can be used for quickly, simply and conveniently detecting the effect of the new corona vaccination under the multi-scene conditions of hospitals, clinics and even at home without professional technicians and professional equipment; the sample demand is small, and the vaccine reaction of the organism and the protective force of the organism can be simultaneously detected only by a small amount of blood samples, so that the feedback can be obtained in time; in addition, the existing corona novacells are various in types, such as inactivated vaccines, mRNA vaccines, adenovirus vector corona novacells, genetic engineering recombinant vaccines, virus-like particle vaccines and the like, and different groups of people have different responses and intensities to different types of vaccines; the test strip can judge whether the detected person has response or protection to the injected new corona vaccine; if protection indicates that the vaccine has immunity against the new coronavirus; if there is a response without protection, the injected species of vaccine can be boosted to achieve protection; if no protection is available, no response is given, which indicates that other kinds of vaccines need to be replaced for immunization. Can improve the effective inoculation rate of the vaccine, prevent infection caused by poor inoculation effect of the vaccine and has good clinical application prospect.
Claims (8)
1. A test strip for double-line detection of antibodies after new coronavirus vaccination comprises a base plate (1), a sample pad (2), a combination pad (3), a reaction pad (4) and a water absorption pad (5), wherein the sample pad (2), the combination pad (3), the reaction pad (4) and the water absorption pad (5) are sequentially lapped on the base plate (1), the reaction pad (4) is provided with a detection belt and a control belt, the detection belt is arranged at one end close to the combination pad (3), the control belt is arranged at one end close to the water absorption pad (5), the test strip is characterized by comprising a vaccination reaction detection belt (6) and/or an immunity detection belt (7), the vaccination reaction detection belt (6) is coated with a novel coronavirus surface spike glycoprotein receptor binding Region (RBD) protein, and the immunity detection belt is coated with a colloidal gold labeled novel coronavirus receptor 2 protein and mouse IgG, the control band comprises a first control band (8) and/or a second control band (9) which are coated with goat anti-mouse IgG antibodies.
2. The test strip for the two-line detection of antibodies after the vaccination with the neocoronaviruse of claim 5, wherein the labeled amount of ACE2 protein is 6-8 μ g of ACE2 protein, 0.5mg/ml of goat anti-mouse antibody and 0.1mg/ml of RBD protein per 1ml of 4/ten thousand colloidal gold solution.
3. The strip according to claim 2, wherein the test strip comprises a vaccination response test strip (6) and an immunoefficacy test strip (7), the control strip comprises a first control strip (8), the vaccination response test strip (6) and the immunoefficacy test strip (7), and the first control strip (8) is sequentially disposed on the reaction pad (4).
4. The test strip for the two-line detection of the antibody after the vaccination of the new coronavirus according to claim 2, wherein the test strip comprises two test strips with different detection bands and control bands, and the reaction pad (4) of the first test strip is provided with a vaccination reaction detection band (6) and a first control band (8); an immunity effectiveness detection belt (7) and a second control belt (9) are arranged on the reaction pad of the second test strip.
5. The test strip for the two-line detection of the antibody after the vaccination of the new coronavirus according to claim 2, wherein the test strip is a two-way test strip, the sample pad (2) is arranged at the middle position of the bottom plate (1), the combination pad (3), the reaction pad (4) and the water absorption pad (5) are arranged on two sides of the sample pad (2), the reaction pad (4) at the left end of the test strip is provided with an immune efficacy detection zone (7) and a second control zone (9), and the reaction pad (4) at the right end of the test strip is provided with a vaccination reaction detection zone (6) and a first control zone (8).
6. The test strip for the two-line detection of antibodies after the vaccination with the new coronavirus according to any one of claims 1-5, wherein the sample pad (2) is connected to the binding pad (3) for a length of 1-2mm, the binding pad (3) is connected to the reaction pad (4) for a length of 1-2mm, and the reaction pad (4) is connected to the absorbent pad (5) for a length of 1-2 mm.
7. The test strip for the two-line detection of the antibodies after the vaccination of the new coronavirus according to any one of claims 1 to 5, wherein the reaction pad (4) is provided with a nitrocellulose membrane, and the vaccination reaction detection strip (6), the immune efficacy detection strip (7), the first control strip (8) and the second control strip (9) are coated on the nitrocellulose membrane.
8. The strip for the two-line detection of neutralizing antibodies after vaccination with a novel coronavirus according to claim 7, wherein the intervals between the vaccination response detection zone (6) and the immunopotency detection zone (7), between the immunopotency detection zone (7) and the first control zone (8), between the vaccination response detection zone (6) and the first control zone (8), and between the immunopotency detection zone (7) and the second control zone (9) are 2-4 mm.
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| CN113267634A (en) * | 2021-07-20 | 2021-08-17 | 南京申基医药科技有限公司 | Test strip for combined detection of IgG, IgM and neutralizing antibody, preparation method thereof and kit |
| CN113281523A (en) * | 2021-07-21 | 2021-08-20 | 南京申基医药科技有限公司 | SARS-CoV-2 neutralizing antibody test paper strip and its preparation method and kit |
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