CN111024954A - Colloidal gold immunochromatography device for combined detection of COVID-19 antigen and antibody and use method thereof - Google Patents
Colloidal gold immunochromatography device for combined detection of COVID-19 antigen and antibody and use method thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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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- 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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- 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
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- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The invention discloses a colloidal gold immunochromatographic device for joint detection of a novel coronavirus COVID-19 antigen and a novel coronavirus COVID-19 antibody and a use method thereof, which can accurately judge the immunological reaction state of an organism by simultaneously detecting the novel coronavirus COVID-19 antigen, the specific IgM antibody and the IgG antibody thereof, thereby giving an accurate detection result according to the detection result, contributing to improvement of the detection rate of the novel coronavirus, avoiding a false negative result, preventing omission, further improving the detection accuracy and providing help for clinical diagnosis and treatment of novel coronavirus pneumonia. The colloidal gold immunochromatographic device has strong specificity, high detection speed and simple and convenient operation, does not need special equipment or operation of professional personnel, and can be applied to preliminary screening in various places such as communities, primary hospitals, airports, customs and even families.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a colloidal gold immunochromatographic device for combined detection of a novel coronavirus COVID-19 antigen and an antibody and a use method thereof.
Background
In 12-month middle ten of 2019, atypical pneumonia (atypicalponia) with unknown etiology is found for the first time in Wuhan, China, and scientific research shows that the atypical pneumonia is triggered by a novel coronavirus COVID-19. Pneumonia infected by the novel coronavirus COVID-19 is mainly manifested by fever, hypodynamia, dry cough and the like, and a few patients are accompanied by upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like. Severe cases often develop dyspnea after 1 week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, and hemorrhagic coagulation dysfunction. 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 technical requirement, false negative easily occurs, a specimen needs special treatment, professional instruments and equipment such as a PCR amplification instrument and gel electrophoresis are required, the detection time of the novel coronavirus is long, a detection result needs to be operated and judged by professional technicians, and the method cannot be applied to early primary screening of communities, primary hospitals, airports, customs, even families and other primary bases.
Therefore, a diagnostic reagent for detecting the novel coronavirus COVID-19, which is earlier, more accurate, 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 combined detection of the novel coronavirus COVID-19 antigen and antibody and the use method thereof, which have the advantages of high accuracy, strong specificity, high detection speed, simple and convenient operation, no need of special equipment and operation of professionals, can be applied to preliminary screening of various places such as communities, primary hospitals, airports, customs and even families, can judge results within minutes, and provide a simpler and faster field detection means for investigation of suspected patients and screening of asymptomatic infectors, thereby preventing the spread of epidemic situations 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 invention provides a colloidal gold immunochromatographic device for combined detection of a novel coronavirus COVID-19 antigen and antibody, which comprises a COVID-19 antigen detection test strip and a COVID-19 antibody detection test strip; the COVID-19 antigen detection test strip comprises a first sample pad, a first combination pad, a first reaction pad and a first water absorption pad which are connected in sequence; the first 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, the first reaction pad is sequentially provided with a first detection line and a first quality control line along the flow direction of a sample, and the first detection line is coated with the novel coronavirus NP protein monoclonal antibody; the COVID-19 antibody detection test strip comprises a second sample pad, a second combination pad, a second reaction pad and a second water absorption pad which are connected in sequence; the second bonding pad is coated with novel coronavirus NP protein and S protein which are marked by colloidal gold; and a second detection line, a third detection line and a second quality control line are arranged on the second reaction pad, an anti-human IgM antibody is coated on the second detection line, and an anti-human IgG antibody is coated on the third detection line.
The grain 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 parts per million, and the concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is (1-3): 1.
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 second detection line, the third detection line and the second quality control line are sequentially arranged on the second reaction pad along the sample flowing direction.
Further, the first reaction pad and the second reaction pad are Millipore 180 nitrocellulose membranes.
Further, the second bonding pad is also coated with chicken IgY marked by colloidal gold; the first quality control line is coated with a goat anti-rabbit IgG antibody; the second quality control line is coated with goat anti-chicken IgY.
Furthermore, the novel coronavirus NP protein monoclonal antibody is a SARS coronavirus NP protein monoclonal antibody.
Furthermore, the novel coronavirus NP protein and S protein are prepared by a gene recombination method.
Further, the preparation method of the novel coronavirus NP protein comprises the following steps: cloning a novel coronavirus COVID-19NP protein gene sequence, connecting the protein sequence with an expression vector pET-30a, constructing a recombinant expression vector pET-30a-NP, transforming the recombinant expression vector pET-30a-NP into an expression bacterium E.coli BL21(DE3) after sequencing identification is correct, inducing protein expression by IPTG, collecting a sample, and performing ultrasonic disruption and Ni column purification; wherein the N end of the expression vector pET-30a is provided with a 6 XHis tag.
Further, the preparation method of the novel coronavirus S protein comprises the following steps: cloning a gene sequence of an RBD region in a novel coronavirus COVID-19S protein; the synthesized gene sequence is inserted into pcDNA3.1 expression vector with IL-33 signal peptide and mouse Fc fragment, HEK293S cell is transfected by the expression vector, and then Protein purification is carried out by Protein G affinity purification column.
According to another aspect of the present invention, there is provided a method for using the above gold immunochromatographic device, comprising the steps of:
respectively dripping a proper amount of blood sample solution on sample pads of a COVID-19 antigen detection test strip and a COVID-19 antibody detection test strip; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 antigen and the novel coronavirus COVID-19 antibody according to the color development conditions of the detection line and the quality control line, wherein the judgment method comprises the following steps: for the COVID-19 antigen detection test strip, 1) positive: the first quality control line and the first detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19 antigen; 2) negative: the first quality control line presents a red strip, and the first detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19 antigen; 3) and (3) failure: the first quality control line and the first detection line do not present red strips or only present red strips, which indicates that the COVID-19 antigen detection test strip is invalid; for the test strip for detection of antibody with COVID-19, 1) positive: the second quality control line and the second detection line both present red bands, and the third detection line does not present red bands, which indicates that the sample contains the novel coronavirus COVID-19 IgM antibody; the second quality control line and the third detection line both present red bands, and the second detection line does not present red bands, which indicates that the sample contains the novel coronavirus COVID-19 IgG antibody; 2) negative: the second quality control line presents a red strip, and the second detection line and the third detection line do not present red strips, which indicates that the sample does not contain the novel coronavirus COVID-19 antibody; 3) and (3) failure: and the second quality control line, the second detection line and the third detection line do not present red strips or the second quality control line does not present red strips, which indicates that the COVID-19 antibody detection test strip is invalid.
The invention has the following beneficial effects:
according to the invention, the immunological reaction state of the organism can be accurately judged by simultaneously detecting the COVID-19 antigen of the novel coronavirus, the specific IgM antibody and the IgG antibody thereof, so that an accurate detection result is given according to the detection result, the detection rate of the novel coronavirus is favorably improved, a false negative result is avoided, the detection omission is prevented, the detection accuracy is further improved, and the clinical diagnosis and treatment of the novel coronavirus pneumonia are facilitated. The colloidal gold immunochromatographic device has strong specificity, high detection speed and simple and convenient operation, does not need special equipment or professional operation, can be applied to preliminary screening of various places such as communities, primary hospitals, airports, customs and even families, can judge results within minutes, and provides a simpler and faster on-site detection means for investigation of suspected patients and screening of asymptomatic infectors, thereby preventing epidemic spread as soon as possible.
Adopting colloidal gold with the particle size of 55-65nm in the colloidal gold labeling to ensure that the concentration of the colloidal gold is five to seven ten thousandths, and the concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is (1-3): simultaneously, a binding pad of the COVID-19 antibody detection test strip is coated with novel coronavirus NP protein, S protein and chicken IgY which are marked by colloidal gold, so that the binding efficiency of an antigen 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.
Drawings
FIG. 1 is a schematic structural diagram of a gold immunochromatographic device of the present invention;
FIG. 2 is a schematic structural diagram of the COVID-19 antigen detection test strip of the present invention;
FIG. 3 is a schematic structural diagram of the COVID-19 antibody detection test strip of the present invention.
In the figure: 1. the sample feeding device comprises a clamping shell, 2, a sample feeding hole, 3, an observation window, 4, a first sample pad, 5, a first combination pad, 6, a first reaction pad, 7, a first water absorption pad, 8, a first detection line, 9, a first quality control line, 10, a first bottom plate, 11, a second sample pad, 12, a second combination pad, 13, a second reaction pad, 14, a second water absorption pad, 15, a second detection line, 16, a third detection line, 17, a second quality control line, 18 and a second bottom plate.
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.
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.
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 time-consuming, is easy to generate false negative, needs a professional to operate and judge the detection result, and cannot be applied to early primary screening of the basic levels of a community health service center, a basic-level hospital, customs and even families. There is a need for a diagnostic reagent for detecting the novel coronavirus COVID-19 more early, accurately, quickly and effectively for early differential diagnosis.
Based on the above, the first objective of the present invention is to provide a colloidal gold immunochromatographic device for detecting a novel coronavirus covi-19, which has the advantages of high accuracy, strong specificity, high detection speed, simple operation, no need of special equipment and no need of operation of professionals, can be applied to preliminary screening in various places such as communities, primary hospitals, airports, customs and even families, can judge results within minutes, and provides a simpler, faster and on-site 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 combined detection of novel coronavirus COVID-19 antigen and antibody comprises a COVID-19 antigen detection test strip and a COVID-19 antibody detection test strip.
The COVID-19 antigen detection test strip comprises a first sample pad 4, a first combination pad 5, a first reaction pad 6 and a first water absorption pad 7 which are connected in sequence; the first binding pad 5 is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by colloidal gold, the first reaction pad 6 is sequentially provided with a first detection line 8 and a first quality control line 9 along the flow direction of a sample, and the first detection line 8 is coated with the novel coronavirus NP protein monoclonal antibody.
Specifically, the first sample pad 4, the first combination pad 5, the first reaction pad 6 and the first water absorption pad 7 are all arranged above the first base plate 10, the first sample pad 4 is arranged at the edge of one end of the first base plate 10, and the first combination pad 5 is arranged at the inner side of the first sample pad 4 and partially overlapped with the first sample pad 4; the first binding pad 5 is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold and a rabbit IgG antibody marked by colloidal gold; the first water absorption pad 7 is arranged at the other end of the first base plate 10, and the first reaction pad 6 is arranged between the first combination pad 5 and the first water absorption pad 7 and partially overlaps with the two pads respectively; the first reaction pad 6 is provided with a first detection line 8 and a first quality control line 9, the first detection line 8 is arranged at one end, close to the first combination pad 5, of the first reaction pad 6, and the first quality control line 9 is sprayed at one end, close to the first water absorption pad 7.
The COVID-19 antibody detection test strip comprises a second sample pad 11, a second combination pad 12, a second reaction pad 13 and a second water absorption pad 14 which are connected in sequence; the second binding pad 12 is coated with a novel coronavirus NP protein and a novel coronavirus S protein which are labeled by colloidal gold; the second reaction pad 13 is provided with a second detection line 15, a third detection line 16 and a second quality control line 17, the second detection line 15 is coated with an anti-human IgM antibody, and the third detection line 16 is coated with an anti-human IgG antibody.
Specifically, the second sample pad 11, the second conjugate pad 12, the second reaction pad 13 and the second absorbent pad 14 are all disposed above the second base plate 18, the second sample pad 11 is disposed at the edge of one end of the second base plate 18, and the second conjugate pad 12 is disposed inside the second sample pad 11 and partially overlaps the second sample pad 11; the second binding pad 12 is coated with a novel coronavirus NP protein, a novel coronavirus S protein and a novel coronavirus IgY which are labeled with colloidal gold; the second water absorption pad 14 is arranged at the other end of the second bottom plate 18, and the second reaction pad 13 is arranged between the second combination pad 12 and the second water absorption pad 14 and partially overlaps with the two pads respectively; the second reaction pad 13 is provided with a second detection line 15, a third detection line 16 and a second quality control line 17, the second detection line 15 is arranged at one end of the second reaction pad 13 close to the second combination pad 12, the second quality control line 17 is sprayed at one end close to the second water absorption pad 14, and the third detection line 16 is arranged between the second detection line 15 and the second quality control line 17.
Immunoglobulin M (IgM) and immunoglobulin G (IgG) are the two most important antibodies in the human body, and antibody detection of the novel coronavirus mainly comprises IgM and IgG antibodies. After the organism is infected with the novel coronavirus COVID-19, the organism is not ready to cause immune response, so that only the novel coronavirus COVID-19 antigen can be detected at the moment. Thereafter, an immune response is elicited by the body, with the first immunoglobulin appearing being IgM antibodies, followed by IgG antibodies. Therefore, by detecting the existence or nonexistence of the novel coronavirus COVID-19 antigen and the specific IgM antibody and IgG antibody in the organism, the immune reaction state of the organism to the novel coronavirus COVID-19 antigen can be diagnosed. If only virus antigen can be detected, the organism is just infected for several days; if IgM antibody with virus specificity is detected in blood, the organism is proved to be infected with the novel coronavirus COVID-19 recently, but due to the fact that half-life period of the IgM is short, detection of the IgM is negative, the organism cannot be proved to be not infected with the novel coronavirus COVID-19, IgG antibody with long half-life period and highest content needs to be detected to clearly diagnose, and IgG antibody positive indicates that infection time is long. The antigen of the novel coronavirus COVID-19 has a highest value in a body, the concentration of the antigen can change along with the change of time, and the antigen can not be detected. Therefore, according to the invention, the immunological reaction state of the organism can be accurately judged by simultaneously detecting the novel coronavirus COVID-19 antigen and the specific IgM antibody and IgG antibody thereof, so that an accurate detection result is given according to the detection result, the detection rate of the novel coronavirus is favorably improved, a false negative result is avoided, the detection omission is prevented, the detection accuracy is further improved, and the clinical diagnosis and treatment of the novel coronavirus pneumonia are facilitated. The colloidal gold immunochromatographic device has strong specificity, high detection speed and simple and convenient operation, does not need special equipment or professional operation, can be applied to preliminary screening of various places such as communities, primary hospitals, airports, customs and even families, can judge results within minutes, and provides a simpler and faster on-site detection means for investigation of suspected patients and screening of asymptomatic infectors, thereby preventing epidemic spread as soon as possible.
First and second bond pads 5 and 12
The first binding pad 5 is coated with a novel coronavirus NP protein monoclonal antibody labeled with colloidal gold and a rabbit IgG antibody labeled with colloidal gold, wherein the particle size of the colloidal gold used in the colloidal gold labeling is 55-65nm, and more preferably, the particle size is 60 nm; the concentration of the colloidal gold is five to seven parts per million, and more preferably, the concentration of the colloidal gold is six parts per million; the 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 second binding pad 12 is coated with a novel coronavirus NP protein and a novel coronavirus S protein which are labeled by colloidal gold; the particle size of the colloidal gold adopted in the colloidal gold labeling is 55-65nm, and more preferably, the particle size is 60 nm; the concentration of the colloidal gold is five to seven parts per million, and more preferably, the concentration of the colloidal gold is six parts per million.
The colloidal gold immunochromatographic technique is a simple, convenient and quick immunological detection method which is developed rapidly in recent years, but at present, domestic research reports about the detection of the novel coronavirus COVID-19 by the colloidal gold immunochromatographic technique are few, and no colloidal gold immunochromatographic device for simultaneously detecting the novel coronavirus COVID-19 antigen and specific IgM antibody and IgG antibody thereof exists on the market. Aiming at the detection of the novel coronavirus COVID-19, how to improve the detection sensitivity is still a technical problem. The inventor finds that the detection sensitivity is not high even if the novel coronavirus COVID-19 colloidal gold immunochromatographic test strip is prepared by referring to the existing coronavirus colloidal gold immunochromatographic test strip. In order to solve the technical problem of low sensitivity of a colloidal gold immunochromatographic test strip for detecting the novel coronavirus COVID-19 in the prior art, the inventor further researches and discovers that the concentration of colloidal gold is five to seven ten thousandths by adopting the colloidal gold with the particle size of 55-65nm in a colloidal gold label, and the concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is (1-3): the novel coronavirus NP protein and the novel coronavirus S protein marked by the colloidal gold are coated on the binding pad of the COVID-19 antibody detection test strip, so that the binding efficiency of an antigen antibody can be increased, the sensitivity of detecting the novel coronavirus COVID-19 by colloidal gold immunochromatography is effectively improved, and the field rapid identification of the novel coronavirus COVID-19 can be realized.
In the embodiment of the present application, the material of the first bonding pad 5 and the second bonding pad 12 is not particularly limited, and a material known to those skilled in the art may be used, and preferably, the first bonding pad 5 and the second bonding pad 12 are glass fiber films.
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, in the colloidal gold immunochromatography, the particle size of colloidal gold adopted in the colloidal gold labeling is usually not more than 40nm, but the invention verifies and finds that the sensitivity of detecting the novel coronavirus COVID-19 by the colloidal gold immunochromatography can be greatly improved when the particle size of the colloidal gold is 55-65 nm.
In the prior art, the concentration of colloidal gold is usually one ten thousandth in the colloidal gold labeling, while in the embodiment of the application, the concentration of the colloidal gold is five to seven ten thousandth in the colloidal gold labeling, and by the arrangement, 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 particle size of the colloidal gold can be changed and controlled by adjusting the adding proportion of the chloroauric acid and the trisodium citrate by adopting methods such as a trisodium citrate reduction method preparation method and the like.
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.
In the above test paper for detecting covi-19 antigen, the first binding pad 5 is coated with a colloidal gold labeled monoclonal antibody of novel coronavirus NP protein and a colloidal gold labeled rabbit IgG antibody, and the monoclonal antibody of novel coronavirus NP protein can be specifically bound with novel coronavirus covi-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.
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.
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.
In the invention, the second binding pad 12 is coated with the novel coronavirus NP protein and the novel coronavirus S protein which are labeled by colloidal gold, and the novel coronavirus NP protein and the novel coronavirus S protein have high affinity, good specificity and high sensitivity to a novel coronavirus antibody.
In the present invention, the preparation method of the novel coronavirus NP protein and S protein is not particularly limited, and the novel coronavirus NP protein and S protein can be prepared according to a conventional technique in the art.
Preferably, the novel coronavirus NP protein and S protein are prepared by a gene recombination method.
The preparation method of the novel coronavirus NP protein comprises the following steps: cloning a novel coronavirus COVID-19NP protein gene sequence, connecting the protein sequence with an expression vector pET-30a, constructing a recombinant expression vector pET-30a-NP, transforming the recombinant expression vector pET-30a-NP into an expression bacterium E.coli BL21(DE3) after sequencing identification is correct, inducing protein expression by IPTG, collecting a sample, and performing ultrasonic disruption and Ni column purification; wherein the N end of the expression vector pET-30a is provided with a 6 XHis tag.
The preparation method of the novel coronavirus S protein comprises the following steps: cloning a gene sequence of an RBD region in a novel coronavirus COVID-19S protein; the synthesized gene sequence is inserted into pcDNA3.1 expression vector with IL-33 signal peptide and mouse Fc fragment, HEK293S cell is transfected by the expression vector, and then Protein purification is carried out by Protein G affinity purification column.
A first reaction pad 6 and a second reaction pad 13
The first reaction pad 6 and the second reaction pad 13 are Millipore 180 nitrocellulose membranes. 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 Millipore 180 nitrocellulose membrane is adopted as the reaction pad, so that the background color development is avoided, the color development of the detection line is deep, the non-specific color development is avoided, the climbing speed can be reduced, the reaction time is prolonged, and the sensitivity of detecting the novel coronavirus can be greatly improved.
And a goat anti-rabbit IgG antibody is coated on the first quality control line 9. 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.
The second quality control line 17 is coated with goat anti-chicken IgY.
In the embodiment of the present application, preferably, the second reaction pad 13 is sequentially provided with the second detection line 15, a third detection line 16 and a second quality control line 17 along a sample flowing direction, the second detection line 15 is coated with an anti-human IgM antibody, and the third detection line 16 is coated with an anti-human IgG antibody. The inventors have found in practice that with this arrangement, the sensitivity of detecting the novel coronavirus can be improved.
It should be noted that the anti-human IgG antibody and the anti-human IgM antibody on the second reaction pad 13 may be prepared according to a conventional technique in the art.
In the present invention, the types of the first sample pad 4 and the second sample pad 11 are not particularly limited, and a sample pad generally used in the art for blood sample detection may be used.
A first absorbent pad 7 and a second absorbent pad 14
The first absorbent pad 7 and the second absorbent pad 14 can be made of any material that can absorb liquid, but the absorption capacity should be sufficiently large. Materials that may be used include, but are not limited to, absorbent cotton pads, absorbent silica pads, or absorbent sponge pads.
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 colloidal gold immunochromatographic device further comprises a card shell 1, wherein the COVID-19 antigen detection test strip and the COVID-19 antibody detection test strip are arranged in the card shell 1 side by side;
be provided with application of sample hole 2 and observation window 3 on the card shell 1, application of sample hole 2 with first sample pad 4 and second sample pad 11 are corresponding, observation window 3 with first reaction pad 6 and second reaction pad 13 are corresponding.
The detection principle of the COVID-19 antigen detection 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.
The principle of the test strip for detecting the COVID-19 antibody provided by the invention for detecting whether the sample contains the COVID-19 antibody is as follows: 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 antibody, the novel coronavirus COVID-19 antibody is combined with the gold-labeled antigen to form a gold-labeled antigen-antibody complex, and the complex moves to the detection line of the reaction pad due to chromatography; when the novel coronavirus COVID-19 antibody contained in the sample is an IgM antibody, a gold-labeled antigen-antibody-anti-human IgM antibody complex is formed on the second detection line 15, the gold-labeled antibody is immobilized, and a red line is displayed on the second detection line 15; when the novel coronavirus covi-19 antibody contained in the sample is an IgG antibody, a gold-labeled antigen-antibody-anti-human IgG antibody complex is formed on the third detection line 16, the gold-labeled antibody is immobilized, and a red line is displayed on the third detection line 16.
In a second aspect, the present invention provides a method for using the above gold immunochromatographic device, comprising the steps of:
respectively dripping a proper amount of blood sample solution on sample pads of a COVID-19 antigen detection test strip and a COVID-19 antibody detection test strip; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 antigen and the novel coronavirus COVID-19 antibody according to the color development conditions of the detection line and the quality control line, wherein the judgment method comprises the following steps: for the COVID-19 antigen detection test strip, 1) positive: the first quality control line and the first detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19 antigen; 2) negative: the first quality control line presents a red strip, and the first detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19 antigen; 3) and (3) failure: the first quality control line and the first detection line do not present red strips or only present red strips, which indicates that the COVID-19 antigen detection test strip is invalid; for the test strip for detection of antibody with COVID-19, 1) positive: the second quality control line and the second detection line both present red bands, and the third detection line does not present red bands, which indicates that the sample contains the novel coronavirus COVID-19 IgM antibody; the second quality control line and the third detection line both present red bands, and the second detection line does not present red bands, which indicates that the sample contains the novel coronavirus COVID-19 IgG antibody; 2) negative: the second quality control line presents a red strip, and the second detection line and the third detection line do not present red strips, which indicates that the sample does not contain the novel coronavirus COVID-19 antibody; 3) and (3) failure: and the second quality control line, the second detection line and the third detection line do not present red strips or the second quality control line does not present red strips, which indicates that the COVID-19 antibody detection test strip is invalid.
Specifically, the results of the various tests are explained in Table 1.
The application of the detection sample is blood, and the problem that the exposure risk of the medical staff is high when the upper respiratory tract sample is collected by the existing nucleic acid detection is avoided.
The blood includes human serum, plasma and whole blood samples.
Colloidal gold immunochromatography device performance evaluation for combined detection of COVID-19 antigen and antibody
(1) Sensitivity detection
The colloidal gold immunochromatographic device provided by the invention 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%; in 2 cases, the nucleic acid detection in the initial stage is negative, the detection in the later stage is positive, and the detection results in the two cases are positive.
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 (10)
1. A colloidal gold immunochromatographic device for combined detection of a novel coronavirus COVID-19 antigen and antibody is characterized by comprising a COVID-19 antigen detection test strip and a COVID-19 antibody detection test strip; the COVID-19 antigen detection test strip comprises a first sample pad, a first combination pad, a first reaction pad and a first water absorption pad which are connected in sequence; the first 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, the first reaction pad is sequentially provided with a first detection line and a first quality control line along the flow direction of a sample, and the first detection line is coated with the novel coronavirus NP protein monoclonal antibody; the COVID-19 antibody detection test strip comprises a second sample pad, a second combination pad, a second reaction pad and a second water absorption pad which are connected in sequence; the second bonding pad is coated with novel coronavirus NP protein and S protein which are marked by colloidal gold; and a second detection line, a third detection line and a second quality control line are arranged on the second reaction pad, an anti-human IgM antibody is coated on the second detection line, and an anti-human IgG antibody is coated on the third detection line.
2. The gold immunochromatographic device according to claim 1, wherein the particle size of the gold used in the gold label is 55 to 65 nm; the concentration of the colloidal gold is five to seven parts per million, and the concentration ratio of the novel coronavirus NP protein monoclonal antibody to the rabbit IgG antibody is (1-3): 1.
3. the colloidal gold immunochromatographic device according to claim 1, wherein the second detection line, the third detection line and the second quality control line are provided on the second reaction pad in this order along the direction of flow of the sample.
4. The colloidal gold immunochromatographic device according to claim 1, wherein the first and second reaction pads are Millipore 180 nitrocellulose membranes.
5. The gold immunochromatographic device of claim 1, wherein the second conjugate pad is further coated with gold-labeled chicken IgY; the first quality control line is coated with a goat anti-rabbit IgG antibody; the second quality control line is coated with goat anti-chicken IgY.
6. 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.
7. The colloidal gold immunochromatographic device according to claim 1, wherein the novel coronavirus NP protein and S protein are prepared by a gene recombination method.
8. The colloidal gold immunochromatographic device according to claim 7, wherein the novel coronavirus NP protein is prepared by the method comprising: cloning a novel coronavirus COVID-19NP protein gene sequence, connecting the protein sequence with an expression vector pET-30a, constructing a recombinant expression vector pET-30a-NP, transforming the recombinant expression vector pET-30a-NP into an expression bacterium E.coli BL21(DE3) after sequencing identification is correct, inducing protein expression by IPTG, collecting a sample, and performing ultrasonic disruption and Ni column purification; wherein the N end of the expression vector pET-30a is provided with a 6 XHis tag.
9. The colloidal gold immunochromatographic device according to claim 7, wherein the novel coronavirus S protein is prepared by the method comprising: cloning a gene sequence of an RBD region in a novel coronavirus COVID-19S protein; the synthesized gene sequence is inserted into pcDNA3.1 expression vector with IL-33 signal peptide and mouse Fc fragment, HEK293S cell is transfected by the expression vector, and then Protein purification is carried out by Protein G affinity purification column.
10. The method of using the colloidal gold immunochromatographic device according to any one of claims 1 to 9, comprising the steps of:
respectively dripping a proper amount of blood sample solution on sample pads of a COVID-19 antigen detection test strip and a COVID-19 antibody detection test strip; after a period of time, judging whether the sample contains the novel coronavirus COVID-19 antigen and the novel coronavirus COVID-19 antibody according to the color development conditions of the detection line and the quality control line, wherein the judgment method comprises the following steps: for the COVID-19 antigen detection test strip, 1) positive: the first quality control line and the first detection line both present red bands, which indicates that the sample contains the novel coronavirus COVID-19 antigen; 2) negative: the first quality control line presents a red strip, and the first detection line does not present a red strip, which indicates that the sample does not contain the novel coronavirus COVID-19 antigen; 3) and (3) failure: the first quality control line and the first detection line do not present red strips or only present red strips, which indicates that the COVID-19 antigen detection test strip is invalid; for the COVID-19 antibody detection test strip, 1) positive: the second quality control line and the second detection line both present red bands, and the third detection line does not present red bands, which indicates that the sample contains the novel coronavirus COVID-19 IgM antibody; the second quality control line and the third detection line both present red bands, and the second detection line does not present red bands, which indicates that the sample contains the novel coronavirus COVID-19 IgG antibody; 2) negative: the second quality control line presents a red strip, and the second detection line and the third detection line do not present red strips, which indicates that the sample does not contain the novel coronavirus COVID-19 antibody; 3) and (3) failure: and the second quality control line, the second detection line and the third detection line do not present red strips or the second quality control line does not present red strips, which indicates that the COVID-19 antibody detection test strip is invalid.
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