CN111239400A - Colloidal gold immunochromatographic device for detecting COVID-19 and use method thereof - Google Patents
Colloidal gold immunochromatographic device for detecting COVID-19 and use method thereof Download PDFInfo
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/53—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
- 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
Abstract
The invention discloses a colloidal gold immunochromatographic device for detecting novel coronavirus COVID-19 and a using method thereof, and the colloidal gold immunochromatographic device realizes high sensitivity, strong specificity and high accuracy, has the detection sensitivity of 100pg/ml, has high detection speed, simple and convenient operation, does not need special equipment, is convenient to carry, has low requirement on personnel, does not need to be operated by professional personnel, has low detection cost, can be applied to the primary screening of various places such as hospitals, airports, customs, communities, families and the like, can judge results within minutes, and provides a simpler and faster on-site detection means for the investigation of suspected patients and the screening of asymptomatic infectors, thereby preventing the spread of epidemic situations as early as possible.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a colloidal gold immunochromatographic device for detecting a novel coronavirus COVID-19 and a using method thereof.
Background
Until now, COVID-19 has no specific medicine. Some cases report that the therapeutic drugs still need more clinical practice to prove the effect, and the development of related vaccines is also under development, but the time is needed for clinical application. The urgent need is to study effective diagnostic reagents as soon as possible, diagnose early, isolate early, cut off the propagation path, and control the spread of epidemic.
At present, the detection aiming at the novel coronavirus COVID-19 is mainly virus nucleic acid detection based on PCR, the detection principle is that a unique gene sequence of the virus is taken as a detection target, the DNA sequence of the target selected by people is exponentially increased through PCR amplification, each amplified DNA sequence can be combined with a section of fluorescence labeling probe added in advance to generate a fluorescence signal, and the more the amplified target genes are, the stronger the accumulated fluorescence signal is. In the sample without the virus, the fluorescence signal was not increased because the target gene was not amplified. Thus, nucleic acid detection, in essence, determines whether viral nucleic acid is present in a sample by detecting the accumulation of fluorescent signal.
The detection method has high specificity and sensitivity, the samples need special treatment, professional instruments and equipment such as a PCR amplification instrument and gel electrophoresis are required, the detection time of the novel coronavirus needs to be long, and professional technicians are required to operate and judge the detection result, so that the method cannot be applied to early primary screening of primary bases such as community hospitals, schools and even families.
Therefore, a diagnostic reagent for detecting the novel coronavirus COVID-19, which is earlier, more sensitive, faster and more effective, is needed for early differential diagnosis.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides the colloidal gold immunochromatographic device for detecting the novel coronavirus COVID-19 and the using method thereof, which realize high sensitivity, strong specificity, high speed, simple and convenient operation, no special equipment, application to the primary screening of various places such as hospitals, airports, customs, communities, families and the like, can judge the result within minutes, and provide a simpler and faster field detection means for the investigation of suspected patients and the screening of asymptomatic infectors, thereby preventing the spread of epidemic situation as soon as possible.
The technical problem to be solved by the invention is realized by the following technical scheme:
according to one aspect of the invention, the colloidal gold immunochromatographic device for detecting the novel coronavirus COVID-19 comprises a test strip, wherein the test strip comprises a sample pad, a binding pad, a reaction pad and a water absorption pad which are sequentially connected; the combination pad is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by the colloidal gold, and the particle size of the colloidal gold adopted in the colloidal gold marking is 55-65 nm; the concentration of the colloidal gold is five to seven ten-thousandths, and the mass concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is (1-3): 1; and the reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of the sample, and the detection line is coated with a novel coronavirus NP protein monoclonal antibody.
Further, the particle size of the colloidal gold used in the colloidal gold labeling is 60 nm; the concentration of the colloidal gold is six parts per million.
Further, the reaction pad is a Millipore180 nitrocellulose membrane.
Further, the test strip also comprises a bottom plate for bearing the sample pad, the combination pad, the reaction pad and the water absorption pad; the sample pad, the combination pad, the reaction pad and the water absorption pad are mutually connected in an overlapping way; the quality control line is coated with goat anti-rabbit IgG antibody.
Furthermore, the novel coronavirus NP protein monoclonal antibody is a SARS coronavirus NP protein monoclonal antibody.
Further, the sample pad is a glass fiber membrane or a non-woven fabric or a filter paper which is subjected to impregnation treatment by a sample pad treatment solution, and the sample pad treatment solution contains the following components: tween20, Triton x-405, Casein, BSA, PEG-20000 and NaCl.
Further, the sample pad treatment solution contains the following components in percentage by mass: 0.05% -0.3% Tween20, 0.4% -0.6% Triton x-405, 0.4% -0.6% Casein, 0.4% -0.6% BSA, 0.05% -0.2% PEG-20000 and 1% -3% NaCl.
Further, the pH of the sample pad treatment solution is 8.4 to 8.6.
Further, the colloidal gold immunochromatographic device further comprises a sample diluent which is a 0.1M PBS solution containing 0.05-0.2% of Tween20 in volume fraction and 0.4-0.6% of Casein in volume fraction.
According to another aspect of the present invention, there is provided a method for using the above gold immunochromatographic device, comprising the steps of:
adding the sample into the sample diluent for processing to obtain a sample solution;
dripping a proper amount of sample solution onto a sample pad of the test strip, wherein the sample solution moves to the end of the water absorption pad; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 according to the color development conditions of the detection line and the control line, wherein the judging method comprises the following steps: 1) positive: the quality control line and the detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19; 2) negative: the quality control line presents a red strip, and the detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19; 3) and (3) failure: and the quality control line and the detection line do not present red strips or only the detection line presents red strips, which indicates that the test strip is invalid.
Further, the sample comprises at least one of a nasopharyngeal swab, sputum, alveolar lavage, blood, urine.
Further, the sample comprises at least two of a nasopharyngeal swab, sputum, alveolar lavage, blood, urine.
The invention has the following beneficial effects:
in the invention, colloidal gold with the particle size of 55-65nm is adopted in the colloidal gold mark, so that the concentration of the colloidal gold is five to seven ten thousandths, and the mass concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is (1-3): 1, the binding efficiency of antigen and antibody can be increased, the sensitivity of detecting the novel coronavirus COVID-19 by colloidal gold immunochromatography is effectively improved, and the on-site rapid identification of the novel coronavirus COVID-19 can be realized.
In the invention, a sample pad treatment solution scheme and a sample diluent scheme are designed, so that detection of various sample types such as nasopharyngeal swabs, sputum, alveolar lavage fluid, blood, urine and the like is realized; treat various samples with this application specific sample diluent respectively, specific sample pad treatment fluid is handled in the sample pad adoption this application simultaneously, can make each sample solution composition of dropwise add on the test paper strip unanimous relatively, match with the reaction system of test paper strip easily, the interference of sample to the detection has effectively been reduced, realize the same test paper strip and detect many sample types, can let the check-out time extension, can effectively detect the patient of 3-10 days morbidity, can help improving novel coronavirus's relevance ratio, avoid "false negative" result, further promote the inspection accuracy.
The colloidal gold immunochromatographic device provided by the invention has the advantages of high sensitivity, strong specificity, high accuracy, detection sensitivity up to 100pg/ml, high detection speed, simplicity and convenience in operation, no need of special equipment, convenience in carrying, low requirement on personnel, no need of operation by professional personnel, low detection cost, capability of being applied to preliminary screening in various places such as hospitals, airports, customs, communities, families and the like, capability of judging results within minutes, and capability of providing a simpler, more convenient and faster on-site detection means for investigation of suspected patients and screening of asymptomatic infectors, thereby preventing epidemic spread as early as possible.
Detailed Description
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
Unless otherwise defined, terms used in the present specification have the same meaning as those generally understood by those skilled in the art, but in case of conflict, the definitions in the present specification shall control.
The use of "including," "comprising," "containing," "having," or other variations thereof herein, is meant to encompass the non-exclusive inclusion, as such terms are not to be construed. The term "comprising" means that other steps and ingredients can be added that do not affect the end result. The term "comprising" also includes the terms "consisting of …" and "consisting essentially of …". The compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any of the additional or optional ingredients, components, steps, or limitations described herein.
All numbers or expressions referring to quantities of ingredients, process conditions, etc. used in the specification and claims are to be understood as modified in all instances by the term "about". All ranges directed to the same component or property are inclusive of the endpoints, and independently combinable. Because these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range.
As introduced in the background art, for the detection of the novel coronavirus COVID-19, the nucleic acid detection has the advantages of good detection specificity, high detection sensitivity and the like, but is long in time consumption, needs professional technicians to operate and judge detection results, and cannot be applied to early primary screening of primary bases such as community hospitals, schools and even families. There is a need for a diagnostic reagent for early, more sensitive, faster and more effective detection of the novel coronavirus COVID-19 for early differential diagnosis.
Based on this, the first objective of the invention is to provide a colloidal gold immunochromatographic device for detecting a novel coronavirus COVID-19, which has the advantages of high sensitivity, strong specificity, high speed, simple operation, no need of special equipment, applicability to primary screening in various places such as hospitals, airports, customs, communities, families and the like, capability of judging results within minutes, and capability of providing a simpler and faster field detection means for investigation of suspected patients and screening of asymptomatic infectors, thereby preventing epidemic spread as soon as possible.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
a colloidal gold immunochromatographic device for detecting a novel coronavirus COVID-19 comprises a test strip and a sample diluent; the test strip is including consecutive sample pad, combination pad, reaction pad and the pad that absorbs water, sample pad, combination pad, reaction pad and the pad that absorbs water overlap each other and connect, sample pad, combination pad, reaction pad and the pad that absorbs water are located on the bottom plate.
Specifically, the sample pad, the combination pad, the reaction pad and the water absorption pad are all arranged above the bottom plate, the sample pad is arranged at the edge of one end of the bottom plate, and the combination pad is arranged on the inner side of the sample pad and partially overlapped with the sample pad; the combination pad is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by the colloidal gold; the water absorption pad is arranged at the other end of the bottom plate, and the reaction pad is arranged between the combination pad and the water absorption pad and is partially overlapped with the combination pad and the water absorption pad respectively; the reaction pad is provided with a detection line and a quality control line, the detection line is arranged at one end of the reaction pad close to the combination pad, and the control line is sprayed at one end close to the water absorption pad.
Combined pad
The combination pad is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by the colloidal gold, wherein the particle size of the colloidal gold adopted in the colloidal gold marking is 55-65nm, and more preferably, the particle size is 60 nm; the concentration of the colloidal gold is five to seven ten-thousandths, and the mass concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is (1-3): 1, more preferably, the ratio of the concentrations is 2: 1.
The colloidal gold immunochromatographic technique is a simple, convenient and quick immunological detection method which is developed rapidly in recent years, but at present, the domestic research reports about the detection of the novel coronavirus COVID-19 by the colloidal gold immunochromatographic technique are less, no novel coronavirus COVID-19 colloidal gold immunochromatographic product is used for detecting antigens, and how to improve the detection sensitivity is still a technical problem aiming at the detection of the novel antigen, namely the novel coronavirus COVID-19. The inventor finds in research that even if the novel coronavirus COVID-19 colloidal gold immunochromatographic test strip is prepared by referring to the existing SARS coronavirus colloidal gold immunochromatographic test strip, the detection sensitivity is not high, and the accuracy is poor. The inventors have unexpectedly found that by using colloidal gold having a particle size of 55 to 65nm in the colloidal gold labeling, the concentration of the colloidal gold is five to seven parts per million, and the ratio of the mass concentration between the novel coronavirus NP protein monoclonal antibody and the rabbit IgG antibody is (1 to 3): 1, the binding efficiency of antigen and antibody can be increased, the sensitivity of detecting the novel coronavirus COVID-19 by colloidal gold immunochromatography is effectively improved, and the on-site rapid identification of the novel coronavirus COVID-19 can be realized.
In the embodiments of the present application, the material of the bonding pad is not particularly limited, and a material known to those skilled in the art may be used, and preferably, the bonding pad is a glass fiber film.
In the test paper strip, the binding pad is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by colloidal gold, and the novel coronavirus NP protein monoclonal antibody can be specifically bound with novel coronavirus COVID-19. The sensitivity requirement of test strip detection is met, and simultaneously, the requirement of combining goat anti-rabbit polyclonal antibody can also be met.
The colloidal gold particles with good quality are the premise of obtaining the colloidal gold test strip with excellent performance, the selection of the colloidal gold particles with proper size is the key for preparing the colloidal gold test strip, and the colloidal gold particles with proper particle size must be selected by comprehensively considering a detection sample. In the prior art, the particle size of colloidal gold adopted in colloidal gold labeling is usually not more than 40nm in colloidal gold immunochromatography, but the invention verifies and finds that when the particle size of the colloidal gold is 55-65nm, the binding performance of the novel coronavirus NP protein monoclonal antibody and the colloidal gold is more ideal, the novel coronavirus NP protein monoclonal antibody can absorb the novel coronavirus COVID-19 in a sample more effectively, and the sensitivity of detecting the novel coronavirus COVID-19 by the colloidal gold immunochromatography can be greatly improved.
In the prior art, the concentration of colloidal gold is usually one ten thousandth in the colloidal gold labeling, and in the embodiment of the application, the concentration of the colloidal gold is five to seven ten thousandth in the colloidal gold labeling, and more preferably, the concentration of the colloidal gold is six ten thousandth, so that the sensitivity of detecting the novel coronavirus COVID-19 by the colloidal gold immunochromatography can be greatly improved.
In the present invention, a specific preparation method of the colloidal gold is not particularly limited, and may be a preparation method known to those skilled in the art, for example: the preparation method adopts a trisodium citrate reduction method and the like.
In the present application, the colloidal gold-labeled novel coronavirus NP protein monoclonal antibody and the colloidal gold-labeled rabbit IgG antibody are prepared by mixing the novel coronavirus NP protein monoclonal antibody and the rabbit IgG antibody, and then adding a colloidal gold solution to label the mixture.
In the present invention, the specific labeling process is not particularly limited, and a method known to those skilled in the art may be used.
According to the latest research article "Evolution of the novel coronavirus from the one of Wuhan outreak and modeling of bits spike protein for the risk of humantransmission", it was confirmed that COVID-19 is similar to SARS coronavirus in 2003, which belongs to the family Coronaviridae, the subfamily Coronaviridae, β.
In the present application, the novel coronavirus NP protein monoclonal antibody is a SARS coronavirus NP protein monoclonal antibody, and the SARS coronavirus NP protein monoclonal antibody can be used for antigen diagnosis of the novel coronavirus due to high amino acid homology between the novel coronavirus NP protein and the SARS coronavirus NP protein.
The inventor finds that the constructed colloidal gold immune test strip has good specificity and high sensitivity by adopting the monoclonal antibody of the NP protein of the SARS coronavirus as the novel monoclonal antibody of the NP protein of the coronavirus.
In the present application, the source of the monoclonal antibody against SARS coronavirus NP protein is not particularly limited, and commercially available products satisfying the above requirements, which are well known to those skilled in the art, may be used, or those satisfying the above requirements may be prepared by a method commonly used by those skilled in the art. For example, the monoclonal antibody against the SARS coronavirus NP protein can be obtained by animal immunoscreening using a recombinant SARS NP protein, the recombinant SARS NP protein can be prepared by a conventional method, the animal can be immunized by a conventional method, and the manual can be referred to for the detailed preparation method.
Reaction pad
And the reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of the sample, and the detection line is coated with a novel coronavirus NP protein monoclonal antibody.
The reaction pad is a Millipore180 nitrocellulose membrane. Incidentally, the chromatography speed of the 180. sup. th nitrocellulose membrane was 180s/4 cm. In the colloidal gold test strip, the reaction pad is used as a bearing body of a detection line and a quality control line and is also a place for generating immunoreaction, so that the type of the reaction pad has obvious influence on the aspects of specificity, sensitivity, color development, background color and the like of a detection result. In order to improve the specificity and sensitivity of detection and facilitate observation, the invention carries out optimization investigation on various different types of reaction pads, and the result shows that the Millipore180 nitrocellulose membrane is adopted as the reaction pad, so that the background color development is avoided, the color development depth of a detection line is avoided, the non-specific color development is avoided, the climbing speed is reduced, the reaction time is prolonged, and the detection sensitivity can be greatly improved.
The quality control line is coated with goat anti-rabbit IgG antibody. The goat anti-rabbit secondary antibody is selected, the titer of the antibody is superior to that of a goat anti-mouse antibody, and the preparation and purification of the antibody in large scale are facilitated.
Sample pad
The sample pad is a glass fiber membrane or non-woven fabric or filter paper which is subjected to dipping treatment by sample pad treatment liquid, and the sample pad treatment liquid contains the following components: tween20, Triton x-405, Casein, BSA, PEG-20000 and NaCl.
Specifically, the mass fractions of the substances in the sample pad treatment solution are 0.05% -0.3% of Tween20, 0.4% -0.6% of Triton x-405, 0.4% -0.6% of Casein, 0.4% -0.6% of BSA, 0.05% -0.2% of PEG-20000 and 1% -3% of NaCl, and the pH value of the sample pad treatment solution is 8.4-8.6; more preferably, the mass fractions of the respective substances included in the sample pad treatment solution are 0.1% Tween20, 0.5% Triton x-405, 0.5% Casein, 0.5% BSA, 0.1% PEG-20000, and 1.5% NaCl, respectively.
The sample pad treatment solutions with different compositions all affect the reaction speed of the colloidal gold test strip, the background color of the reaction pad, the hydration uniformity, the specificity, the sensitivity and the like, and especially when the colloidal gold test strip is used for the joint detection of various samples, the requirements on the reaction system of the test strip are different due to different sample matrixes. The applicant finds that the sample pad treatment solution containing Tween20, Triton x 405, Casein, BSA, PEG-20000 and NaCl has a good matrix interference resistance effect and an optimal detection effect, and is helpful for a colloidal gold immunochromatography device to realize combined detection of novel coronavirus COVID-19 in various samples and increase of the detection rate of the novel coronavirus.
Sample diluent
The sample diluent is a 0.1M PBS solution containing a volume fraction of 0.05% to 0.2% Tween20 and a volume fraction of 0.4% to 0.6% Casein, more preferably the sample diluent is a 0.1MPBS solution containing 0.1% Tween20 and 0.5% Casein.
It should be noted that the colloidal gold immunochromatographic device includes a sample diluent reagent bottle for storing a sample diluent.
In the prior art, samples for detecting the novel coronavirus COVID-19 generally comprise nasopharyngeal swabs, sputum, alveolar lavage fluid, blood, urine and the like, and the appearance time of the novel coronavirus COVID-19 antigen in different types of samples is not consistent. The inventor finds that the detection time can be prolonged by adopting the same test strip to separately detect a plurality of samples respectively and detecting through the combination of a plurality of sample types, the patient with the disease can be effectively detected for 3-10 days, the detection rate of the novel coronavirus can be improved, the false negative result is avoided, and the detection accuracy is further improved.
However, because the types of the samples are different, the related components in the samples are not the same, including pH, protein, ionic strength and the like, and the requirements on the reaction system of the test strip are also different, and when the samples of different types are detected by the combination of the same test strip, the sample matrix has high interference. In order to solve the problems, the inventor finds out after intensive research that various samples are respectively treated by using the specific sample diluent in the application, meanwhile, the sample pad is treated by using the specific sample pad treatment liquid in the application, so that the components of each sample solution dripped onto the test strip are relatively consistent, the sample solution is easily matched with a reaction system of the test strip, the interference of the samples on detection is effectively reduced, the detection of multiple sample types by using the same test strip is realized, the detection time can be prolonged, patients with diseases in 3-10 days can be effectively detected, the detection rate of novel coronavirus can be improved, the false negative result is avoided, and the detection accuracy is further improved.
Water absorption pad
The absorbent pad can be made of any material that is capable of absorbing liquid, but the absorbent capacity should be sufficiently great. Materials that may be used include, but are not limited to, absorbent cotton pads, absorbent silica pads, or absorbent sponge pads.
Base plate
The bottom plate is used for bearing the sample pad, the combination pad, the reaction pad and the water absorption pad; the bottom plate can be various non-absorbent sheets with a supporting function, and by way of example, the bottom plate can be a PVC plate, a PP plate, a PE plate or a PU plate, and is preferably a PVC plate.
The detection principle of the test strip is as follows: the test paper strip detects whether a sample contains a novel coronavirus COVID-19 antigen according to the principle of a double-antibody sandwich method. After a sample is dripped on the sample pad of the test strip, the sample solution moves to the combination pad under the action of chromatography, and the gold-labeled antibody is dissolved when flowing through the combination pad. When the sample contains the novel coronavirus COVID-19 antigen, the novel coronavirus COVID-19 antigen is combined with the gold-labeled antibody to form a gold-labeled antibody-antigen complex, the gold-labeled antibody is immobilized due to the fact that the chromatographic reaction complex moves to a detection line of a reaction pad to form the gold-labeled antibody-antigen-antibody complex, a red line is displayed on the detection line and is in positive reaction, and the redundant gold-labeled antibody moves to a quality control line and is captured by goat anti-rabbit IgG to be in the red quality control line.
In a second aspect, the present invention provides a method for using the above gold immunochromatographic device, comprising the steps of:
adding the sample into the sample diluent for processing to obtain a sample solution;
it should be noted that, the step of adding the sample to the sample diluent for processing is to add the collected sample to the sample diluent for mixing.
Dripping a proper amount of sample solution onto a sample pad of the test strip, wherein the sample solution moves to the end of the water absorption pad; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 according to the color development conditions of the detection line and the control line, wherein the judging method comprises the following steps: 1) positive: the quality control line and the detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19; 2) negative: the quality control line presents a red strip, and the detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19; 3) and (3) failure: and the quality control line and the detection line do not present red strips or only the detection line presents red strips, which indicates that the test strip is invalid.
The sample comprises at least one of nasopharyngeal swab, sputum, alveolar lavage fluid, blood and urine, more preferably, the sample comprises at least two of nasopharyngeal swab, sputum, alveolar lavage fluid, blood and urine, and the sample is separately detected.
In order to better understand the technical solutions, the technical solutions will be described in detail with reference to specific examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.
Example 1
A colloidal gold immunochromatographic device for detecting a novel coronavirus COVID-19 comprises a test strip.
The test strip comprises a bottom plate, and a sample pad, a combination pad, a reaction pad and a water absorption pad which are arranged above the bottom plate and are sequentially connected; the combination pad is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by the colloidal gold, and the particle size of the colloidal gold adopted in the colloidal gold marking is 61 nm; the concentration of the colloidal gold is six parts per million, and the mass concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is 1.5: 1; the reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of a sample, the detection line is coated with a novel coronavirus NP protein monoclonal antibody, and the quality control line is coated with a goat anti-rabbit IgG antibody.
The novel coronavirus NP protein monoclonal antibody is a SARS coronavirus NP protein monoclonal antibody. The reaction pad is a Millipore180 nitrocellulose membrane; the sample pad is a glass fiber film or non-woven fabric or filter paper.
The use method of the colloidal gold immunochromatographic device comprises the following steps:
adding the sample into the sample diluent for processing to obtain a sample solution; wherein the sample is a nasopharyngeal swab.
Dripping a proper amount of sample solution onto a sample pad of the test strip, wherein the sample solution moves to the end of the water absorption pad; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 according to the color development conditions of the detection line and the control line, wherein the judging method comprises the following steps: 1) positive: the quality control line and the detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19; 2) negative: the quality control line presents a red strip, and the detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19; 3) and (3) failure: and the quality control line and the detection line do not present red strips or only the detection line presents red strips, which indicates that the test strip is invalid.
Example 2
A colloidal gold immunochromatographic device for detecting a novel coronavirus COVID-19 comprises a test strip and a sample diluent.
The test strip comprises a bottom plate, and a sample pad, a combination pad, a reaction pad and a water absorption pad which are arranged above the bottom plate and are sequentially connected; the combination pad is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by the colloidal gold, and the particle size of the colloidal gold adopted in the colloidal gold marking is 60 nm; the concentration of the colloidal gold is six parts per million, and the mass concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is 2: 1; the reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of a sample, the detection line is coated with a novel coronavirus NP protein monoclonal antibody, and the quality control line is coated with a goat anti-rabbit IgG antibody.
The novel coronavirus NP protein monoclonal antibody is a SARS coronavirus NP protein monoclonal antibody. The reaction pad is a Millipore180 nitrocellulose membrane.
The sample pad is a glass fiber membrane or non-woven fabric or filter paper which is subjected to dipping treatment by sample pad treatment liquid, and the sample pad treatment liquid contains the following components in percentage by mass: 0.1% Tween20, 0.5% Triton x-405, 0.5% Casein, 0.5% BSA, 0.1% PEG-20000 and 1.5% NaCl, the pH of the sample pad treatment solution being 8.4-8.6.
The sample dilution was a 0.1MPBS solution containing a volume fraction of 0.1% Tween20 and a volume fraction of 0.5% Casein.
The use method of the colloidal gold immunochromatographic device comprises the following steps:
respectively adding the samples into the sample diluent for processing to obtain sample solutions; wherein the sample is nasopharyngeal swab, sputum and alveolar lavage fluid;
dripping a proper amount of sample solution onto a sample pad of the test strip, wherein the sample solution moves to the end of the water absorption pad; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 according to the color development conditions of the detection line and the control line, wherein the judging method comprises the following steps: 1) positive: the quality control line and the detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19; 2) negative: the quality control line presents a red strip, and the detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19; 3) and (3) failure: and the quality control line and the detection line do not present red strips or only the detection line presents red strips, which indicates that the test strip is invalid.
Example 3
A colloidal gold immunochromatographic device for detecting a novel coronavirus COVID-19 comprises a test strip and a sample diluent.
The test strip comprises a bottom plate, and a sample pad, a combination pad, a reaction pad and a water absorption pad which are arranged above the bottom plate and are sequentially connected; the combination pad is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by the colloidal gold, and the particle size of the colloidal gold adopted in the colloidal gold marking is 55 nm; the concentration of the colloidal gold is five parts per million, and the mass concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is 1: 1; the reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of a sample, the detection line is coated with a novel coronavirus NP protein monoclonal antibody, and the quality control line is coated with a goat anti-rabbit IgG antibody.
The novel coronavirus NP protein monoclonal antibody is a SARS coronavirus NP protein monoclonal antibody. The reaction pad is a Millipore180 nitrocellulose membrane.
The sample pad is a glass fiber membrane or non-woven fabric or filter paper which is subjected to dipping treatment by sample pad treatment liquid, and the sample pad treatment liquid contains the following components in percentage by mass: 0.05% Tween20, 0.4% Triton x-405, 0.4% Casein, 0.4% BSA, 0.05% PEG-20000 and 1% NaCl, the pH of the sample pad treatment solution being 8.4-8.6.
The sample dilution was a 0.1M PBS solution containing a volume fraction of 0.05% Tween20 and a volume fraction of 0.4%% Casein.
The use method of the colloidal gold immunochromatographic device comprises the following steps:
respectively adding the samples into the sample diluent for processing to obtain sample solutions; the sample is nasopharyngeal swab and urine;
dripping a proper amount of sample solution onto a sample pad of the test strip, wherein the sample solution moves to the end of the water absorption pad; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 according to the color development conditions of the detection line and the control line, wherein the judging method comprises the following steps: 1) positive: the quality control line and the detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19; 2) negative: the quality control line presents a red strip, and the detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19; 3) and (3) failure: and the quality control line and the detection line do not present red strips or only the detection line presents red strips, which indicates that the test strip is invalid.
Example 4
A colloidal gold immunochromatographic device for detecting a novel coronavirus COVID-19 comprises a test strip and a sample diluent.
The test strip comprises a bottom plate, and a sample pad, a combination pad, a reaction pad and a water absorption pad which are arranged above the bottom plate and are sequentially connected; the combination pad is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by the colloidal gold, and the particle size of the colloidal gold adopted in the colloidal gold marking is 65 nm; the concentration of the colloidal gold is seven ten-thousandth, and the mass concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is 3: 1; the reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of a sample, the detection line is coated with a novel coronavirus NP protein monoclonal antibody, and the quality control line is coated with a goat anti-rabbit IgG antibody.
The novel coronavirus NP protein monoclonal antibody is a SARS coronavirus NP protein monoclonal antibody. The reaction pad is a Millipore180 nitrocellulose membrane.
The sample pad is a glass fiber membrane or non-woven fabric or filter paper which is subjected to dipping treatment by sample pad treatment liquid, and the sample pad treatment liquid contains the following components in percentage by mass: 0.3% Tween20, 0.6% Triton x-405, 0.6% Casein, 0.6% BSA, 0.2% PEG-20000 and 3% NaCl, the pH of the sample pad treatment solution being 8.4-8.6.
The sample dilution was a 0.1MPBS solution containing a volume fraction of 0.2% Tween20 and a volume fraction of 0.6% Casein.
The use method of the colloidal gold immunochromatographic device comprises the following steps:
respectively adding the samples into the sample diluent for processing to obtain sample solutions; wherein the sample is sputum, alveolar lavage fluid and blood;
dripping a proper amount of sample solution onto a sample pad of the test strip, wherein the sample solution moves to the end of the water absorption pad; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 according to the color development conditions of the detection line and the control line, wherein the judging method comprises the following steps: 1) positive: the quality control line and the detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19; 2) negative: the quality control line presents a red strip, and the detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19; 3) and (3) failure: and the quality control line and the detection line do not present red strips or only the detection line presents red strips, which indicates that the test strip is invalid.
Novel coronavirus COVID-19 colloidal gold immunochromatographic device performance evaluation
(1) Sensitivity detection
The colloidal gold immunochromatographic device for detecting the novel coronavirus COVID-19 is used for detecting the novel coronavirus COVID-19 with different concentrations. The results showed that the sensitivity of the colloidal gold immunochromatographic device to the novel coronavirus COVID-19 was 100 pg/ml.
(2) Specificity detection
The colloidal gold immunochromatographic device is used for detecting influenza virus, adenovirus and respiratory syncytial virus, and the result shows that the detection results of the influenza virus, the adenovirus and the respiratory syncytial virus are negative. The test paper strip can be used for specifically detecting the novel coronavirus COVID-19, and has no specific reaction on other components.
(3) Testing the stability;
the test paper prepared by the method is put into a sealed bag, a drying agent is arranged in the sealed bag, the sealed bag is placed in a baking oven at the temperature of 45 ℃ for storage, the test paper is taken out respectively for 7 days, 14 days, 21 days, 28 days and 35 days, and the stability of the test paper is detected. The results show that the detection results are not obviously changed after the storage for 7 days, 14 days, 21 days, 28 days and 35 days at 45 ℃.
(4) Clinical sample testing
At present, the number of clinical samples developed by people is 100, wherein the clinical samples comprise 10 confirmed cases, the nucleic acid detection results of the 10 confirmed cases are positive, the detection results of the detection device are also positive, and the consistency rate reaches 100%; 22 of the cases are suspected cases, and the detection is also carried out, and the specific test results are shown in table 1.
TABLE 1
TABLE 1
TABLE 1
The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.
Claims (9)
1. A colloidal gold immunochromatographic device for detecting a novel coronavirus COVID-19 comprises a test strip, wherein the test strip comprises a sample pad, a binding pad, a reaction pad and a water absorption pad which are sequentially connected; the novel coronavirus NP protein monoclonal antibody marked by colloidal gold and the rabbit IgG antibody marked by the colloidal gold are coated on the combination pad, and the particle size of the colloidal gold adopted in the colloidal gold marking is 55-65 nm; the concentration of the colloidal gold is five to seven ten-thousandths, and the mass concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is (1-3): 1; and the reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of the sample, and the detection line is coated with a novel coronavirus NP protein monoclonal antibody.
2. The colloidal gold immunochromatographic device according to claim 1, wherein the reaction pad is a Millipore180 nitrocellulose membrane.
3. The colloidal gold immunochromatographic device according to claim 1, wherein the test strip further comprises a bottom plate carrying the sample pad, the conjugate pad, the reaction pad and the water-absorbing pad; the sample pad, the combination pad, the reaction pad and the water absorption pad are mutually connected in an overlapping way; the quality control line is coated with goat anti-rabbit IgG antibody.
4. The colloidal gold immunochromatographic device according to claim 1, wherein the novel coronavirus NP protein monoclonal antibody is a SARS coronavirus NP protein monoclonal antibody.
5. The colloidal gold immunochromatographic device according to claim 1, wherein the sample pad is a glass fiber membrane or a nonwoven fabric or a filter paper subjected to an immersion treatment with a sample pad treatment solution containing the following components: tween20, Triton x-405, Casein, BSA, PEG-20000 and NaCl.
6. The colloidal gold immunochromatographic device according to claim 5, wherein the sample pad treatment solution contains the following components in mass fraction: 0.05% -0.3% Tween20, 0.4% -0.6% Triton x-405, 0.4% -0.6% Casein, 0.4% -0.6% BSA, 0.05% -0.2% PEG-20000 and 1% -3% NaCl; the pH of the sample pad treatment solution is 8.4 to 8.6.
7. The gold immunochromatographic device according to claim 1, further comprising a sample diluent which is a 0.1M PBS solution containing 0.05% to 0.2% Tween20 by volume and 0.4% to 0.6% Casein by volume.
8. The method of using the colloidal gold immunochromatographic device according to any one of claims 1 to 7, comprising the steps of:
adding the sample into the sample diluent for processing to obtain a sample solution;
dripping a proper amount of sample solution onto a sample pad of the test strip, wherein the sample solution moves to the end of the water absorption pad; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 according to the color development conditions of the detection line and the control line, wherein the judging method comprises the following steps: 1) positive: the quality control line and the detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19; 2) negative: the quality control line presents a red strip, and the detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19; 3) and (3) failure: and the quality control line and the detection line do not present red strips or only the detection line presents red strips, which indicates that the test strip is invalid.
9. The method of using the gold immunochromatographic device of claim 8, wherein the sample comprises at least one of nasopharyngeal swab, sputum, alveolar lavage, blood, urine.
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