CN213633456U - Device and kit for detecting IgM (immunoglobulin M) antibody of anti-novel coronavirus in sample - Google Patents
Device and kit for detecting IgM (immunoglobulin M) antibody of anti-novel coronavirus in sample Download PDFInfo
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- CN213633456U CN213633456U CN202020308108.8U CN202020308108U CN213633456U CN 213633456 U CN213633456 U CN 213633456U CN 202020308108 U CN202020308108 U CN 202020308108U CN 213633456 U CN213633456 U CN 213633456U
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Abstract
The application discloses device of anti novel coronavirus's IgM antibody in detection sample includes: a backing; and a solid support coated with purified S protein antigen, a solid support coated with purified N protein antigen, and a solid support coated with IgM antibody, which are arranged on the backing in this order and spaced from each other. The application also discloses a kit for detecting anti-novel coronavirus IgM antibodies in a sample, comprising: (1) the above-described device; (2) a labeled secondary antibody that specifically binds to the IgM antibody; (3) a buffer solution; (4) substrate solution; (5) and (5) product instructions. The kit provided by the application has the advantages of short operation time, low cost, low machine dependence, capability of simultaneously utilizing the S protein and the N protein for detection, low detection cost, high detection efficiency, high detection accuracy and the like.
Description
Technical Field
The present application relates to devices and kits for detecting IgM antibodies against a novel coronavirus in a sample. More specifically, the present application relates to a device for the detection of IgM antibodies against novel coronavirus SARS-CoV-2 in a sample by immunoblotting, a kit for the detection of IgM antibodies against novel coronavirus SARS-CoV-2 in a sample by immunoblotting, a method for the detection of IgM antibodies against novel coronavirus SARS-CoV-2 in a sample by immunoblotting, a combination of a purified S protein antigen and a purified N protein antigen or the use of the device or the kit for the detection of IgM antibodies against novel coronavirus SARS-CoV-2 in a sample, and the use of a combination of a purified S protein antigen and a purified N protein antigen or the use of the device or the kit for the preparation of a product for the diagnosis of infection with novel coronavirus SARS-CoV-2.
Background
Aiming at the novel coronavirus epidemic situation, how to quickly and accurately detect whether a patient suffers from the novel coronavirus pneumonia is very important.
At present, one method for detecting the novel coronavirus SARS-COV-2 is nucleic acid detection, which is mainly based on fluorescent quantitative PCR (qRT-PCR) to detect viral nucleic acid, and detection genes comprise ORF1ab and N genes, in addition, the nucleic acid detection also comprises a rapid detection method Sherlock based on CRISPR, and recognition genes of the Sherlock are ORF1ab and S genes. At present, the above-mentioned nucleic acid detection method is defined as a gold standard for detecting a novel coronavirus, but the greatest problem of nucleic acid detection at present is false negative. The false negatives of nucleic acid detection are caused by many reasons, including errors in clinical sampling. In fact, in the clinical examination process, the novel coronavirus nucleic acid can be detected in specimens such as nasopharyngeal swabs, sputum, lower respiratory tract secretions, blood, feces and the like, but the most commonly used specimen at present is an upper respiratory tract specimen due to the limitation of the sampling method and the sampling time. However, patients with viral pneumonia have a higher viral load in the lower respiratory tract and therefore the detection rate of lower respiratory secretions is higher. However, obtaining lower respiratory tract samples such as alveolar lavage fluid is operationally demanding, prone to splash, high in risk of spreading, time-consuming, and finally results in the collected samples not reflecting the actual condition of the patient or suspected pneumonia patient. In addition, the limitation of sample transportation is also another cause of false negative in nucleic acid detection. Nucleic acids of various RNA viruses are one of the most difficult biomolecules to stably preserve due to self-degradation and bio-enzyme mediated degradation, which places high demands on storage and transport conditions of samples. However, the current detection conditions cannot realize bedside detection, and a long waiting time is required from sampling to actual experimental detection, so that the real information of a detected person cannot be accurately reflected by a sample to a certain extent. In addition, nucleic acid detection has problems of long time consumption, expensive equipment, and the like.
In addition, another method for detecting the novel coronavirus SARS-COV-2 is protein detection. At present, the kit is mainly used for detecting the new coronavirus IgM/IgG antibody, and the applied methodology mainly comprises immunochromatography (colloidal gold method), chemiluminescence, ELISA, immunofluorescence and the like. However, the detection methods such as colloidal gold cannot detect the S protein and the N protein in the sample at the same time, only can detect two proteins by a single index, and are not superior in terms of experimental operation, product and time consumption.
Meanwhile, the existing research shows that IgM is an antibody which appears in the human immune system firstly in the infection process, and the IgM antibody in the human body for detecting the recessive infection of the novel coronavirus SARS-COV-2 has the advantages of high sensitivity, early diagnosis time, capability of judging whether a suspected person is infected and the like, and has important significance for effectively controlling the large-scale spread of the novel coronavirus SARS-COV-2.
Therefore, it is very desirable to develop a novel kit for detecting the IgM antibody against the novel coronavirus SARS-CoV-2 in a sample, so as to avoid the problems of difficult sample collection operation, inaccurate reaction of the collected sample on the true state of an illness of a suspected pneumonia patient, difficult sample storage and the like in nucleic acid detection, and simultaneously have the advantages of short operation time, low cost, low machine dependence and the like, and simultaneously detect the S protein and the N protein, thereby reducing the detection cost, obviously shortening the detection time and improving the detection efficiency and the detection accuracy.
SUMMERY OF THE UTILITY MODEL
In order to solve the above problems, a first aspect of the present application provides a device for detecting IgM antibodies against a novel coronavirus in a sample, comprising:
a backing; and
and the solid phase support coated with the purified S protein antigen, the solid phase support coated with the purified N protein antigen and the solid phase support coated with the IgM antibody are arranged on the back lining in sequence and are separated from each other.
A second aspect of the present application provides a device for detecting IgM antibodies against a novel coronavirus in a sample, comprising:
a backing; and
the solid phase support coated with the purified N protein antigen, the solid phase support coated with the purified S protein antigen and the solid phase support coated with the IgM antibody are arranged on the back lining in sequence and are separated from each other.
In one embodiment, a handle label spaced apart from the IgM antibody-coated solid support is further provided downstream of the IgM antibody-coated solid support on the backing of the device of the first or second aspect.
In one embodiment, the backing is transparent and/or the backing is made of a material comprising a plastic, preferably PVC.
In one embodiment, the solid support is a membrane.
In one embodiment, the membrane is made of a material comprising nitrocellulose, nylon, PVDF, Nytron, Biodyne or Porex, preferably the membrane is made of nitrocellulose.
In one embodiment, the solid support coated with purified S protein antigen is white and/or the solid support coated with purified N protein antigen is white and/or the solid support coated with IgM antibody is white.
In one embodiment, the handle label is made of plain printing paper and/or the handle label is white.
In one embodiment, the area and/or length of the solid support coated with purified S protein antigen represents between 2% and 15%, preferably between 4% and 6%, more preferably 5% of the area and/or length of the device; and/or the area and/or length of the solid support coated with purified N protein antigen represents 2% to 15%, preferably 4% to 6%, more preferably 5% of the area and/or length of the device; and/or the area and/or length of the IgM antibody coated solid support is 2% to 15%, preferably 4% to 6%, more preferably 5% of the area and/or length of the device.
In one embodiment, the area and/or length of the handle tag is between 5% and 20%, preferably between 10% and 15%, more preferably 12% of the area and/or length of the device.
In one embodiment, the area and/or length of the space between the solid support coated with purified S protein antigen or the solid support coated with purified N protein antigen and the solid support coated with IgM antibody is 40% to 80%, preferably 50% to 70%, more preferably 63% of the area and/or length of the device.
In one embodiment, the area and/or length of the space between the solid support coated with purified S protein antigen and the solid support coated with purified N protein antigen, and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, respectively, comprise from 1% to 5% and from 40% to 80% of the area and/or length of the device, preferably from 1.5% to 3.5% and from 50% to 70%, more preferably from 2% and 63%, respectively.
In one embodiment, the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with purified S protein antigen, and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, respectively, comprise between 1% and 5% and between 40% and 80% of the area and/or length of the device, preferably between 1.5% and 3.5% and between 50% and 70%, respectively, more preferably between 2% and 63%, respectively.
In one embodiment, the area and/or length of the space between the IgM antibody coated solid support and the handle tag is 1% to 5%, preferably 2% to 4%, more preferably 3% of the area and/or length of the device.
A third aspect of the present application provides a kit for detecting IgM antibodies against a novel coronavirus in a sample, comprising:
(1) the apparatus of the first or second aspect above;
(2) a labeled secondary antibody that specifically binds to the IgM antibody;
(3) a buffer solution;
(4) substrate solution;
(5) and (5) product instructions.
In one embodiment, the kit further comprises: an incubation device.
In one embodiment, the incubation device is an incubation tray.
In one embodiment, the labeled secondary antibody is an enzyme conjugate, preferably an alkaline phosphatase labeled anti-human IgM.
In one embodiment, the buffer is a Tris buffer, preferably a 10-fold concentrated Tris buffer.
In one embodiment, the substrate solution is tetrazolyl nitroaniline blue/5-bromo-4-chloropyridine-3-indole-phosphate.
A fourth aspect of the present application provides a method for detecting IgM antibodies against the novel coronavirus SARS-CoV-2 in a sample by immunoblotting, comprising the steps of:
(1) providing the apparatus of the first or second aspect above;
(2) providing a sample;
(3) providing a labeled second antibody;
(4) immunoreacting the sample of step (2), the purified S protein antigen and the purified N protein antigen on the solid support in the device of step (1) with the second antibody of step (3);
(5) detecting the immune reaction result in the step (4) and judging whether the first antibody exists in the sample.
In one embodiment, the labeled secondary antibody is an enzyme conjugate, preferably an alkaline phosphatase labeled anti-human IgM.
In one embodiment, the device according to the first or second aspect above or the kit according to the third aspect above or the method according to the fourth aspect above, the sample is a human body sample, preferably the human body sample is selected from the group consisting of: blood, serum, plasma, urine, body fluids, saliva and other secretions or excretions and tissue or cell extracts, more preferably the human sample is serum and/or plasma.
A fifth aspect of the present application provides a combination of a purified S protein antigen and a purified N protein antigen; or the apparatus of the first or second aspect above; or the use of the kit according to the third aspect above for the detection of IgM antibodies against the novel coronavirus SARS-CoV-2 in a sample.
A sixth aspect of the present application provides a combination of a purified S protein antigen and a purified N protein antigen; or the apparatus of the first or second aspect above; or the use of a kit according to the third aspect above for the preparation of a product for the diagnosis of a novel coronavirus SARS-CoV-2 infection.
The application obtains the following beneficial technical effects:
(1) the kit adopts the immunoblotting method to detect the protein antibody in human serum or plasma, thereby avoiding the problems of difficult sample collection operation, inaccurate response of the collected sample to the real illness state of a suspected pneumonia patient, difficult sample storage and the like in nucleic acid detection;
(2) the kit has the advantages of short operation time, low cost, low machine dependence and the like, and solves the problems of long time consumption, expensive detection instrument and the like of nucleic acid detection;
(3) the kit can detect the purified S protein antigen and the purified N protein antigen simultaneously, reduces the detection cost, obviously shortens the detection time, and improves the detection efficiency and the detection accuracy.
Drawings
Fig. 1 is a schematic view of a detection apparatus according to embodiment 1 of the present application.
Detailed Description
The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the application and the examples included therein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification, including definitions, will control.
The term "prepared from …" as used herein is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion.
The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4," "1 to 3," "1-2 and 4-5," "1-3 and 5," and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
In addition, the indefinite articles "a" and "an" preceding an element or component of the application are provided without limitation to the number requirement (i.e. the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the stated number clearly indicates that the singular form is intended.
Device for measuring the position of a moving object
A first aspect of the present application provides a device for detecting IgM antibodies against a novel coronavirus in a sample, comprising:
a backing; and
and the solid phase support coated with the purified S protein antigen, the solid phase support coated with the purified N protein antigen and the solid phase support coated with the IgM antibody (also called a coated quality control band) which are arranged on the back lining in sequence are separated from each other.
The terms "S protein" and "N protein" as used herein are two structural proteins located on the novel coronavirus SARS-CoV-2. The novel coronavirus SARS-CoV-2 is composed of a variety of structural proteins, including spike protein (S protein), small envelope protein (E protein), membrane protein (M protein), and nucleocapsid protein (N protein), wherein N protein is the most abundant and conserved protein, which is mainly distributed inside the virus. The S protein is a class I viral fusion protein on the virion coat, playing a key role in viral infection by recognizing host cell receptors and mediating viral and cell membrane fusion. The S protein is synthesized as a precursor protein, consisting of about 1,300 amino acids, and then cleaved into an amino (N) -terminal S1 subunit and a carboxy (C) -terminal S2 subunit). Three S1/S2 heterodimers assemble to form a trimeric spike, protruding from the viral envelope. The trimeric S glycoprotein, consisting of three S1-S2 heterodimers, binds to the cellular receptor angiotensin converting enzyme 2(ACE2) and mediates fusion of viral and cell membranes through pre-to post-fusion conformational transitions. Further, in the present applicationThe S protein and N protein of (1) are specifically as follows (seehttps://www.ncbi.nlm.nih.gov/)：
| Type (B) | Gene ID | Protein ID |
| S protein | 43740568 | YP_009724390.1 |
| N protein | 43740575 | YP_009724397.2 |
The term "purified" as used herein means that the purified S protein or the purified N protein is obtained by the following method: the crude protein extract flows through a nickel-containing affinity chromatography column by utilizing the specific binding effect of a histone label on the target protein and the nickel column, the target protein is specifically bound to the nickel-containing column, the target protein is separated from non-target protein, and then the high-purity target protein is eluted.
The term "antibody" as used herein refers to an immunoglobulin molecule that recognizes and specifically binds through at least one antigen recognition site located within the variable region of the immunoglobulin molecule.
The term "IgM antibody" as used herein is a protective antibody that is immunogenic. It has been shown that IgM is the first antibody to appear in the human immune system during infection with the novel coronavirus SARS-CoV-2. The IgM antibody in the body of a person with recessive infection of the novel coronavirus SARS-CoV-2 has the advantages of high sensitivity, early diagnosis time, capability of judging whether a suspected person is infected, and the like.
The term "solid support" as used herein refers to a material which can immobilize proteins, in particular purified S proteins and purified N proteins, on a solid phase. In a preferred embodiment, the solid support is a membrane, preferably the membrane is formed from a material comprising nitrocellulose, nylon, PVDF, Nytron, Biodyne or Porex, more preferably the membrane is formed from nitrocellulose.
The term "immunoblotting" as used herein is a method in which a protein is transferred to a solid support and then detected using an antibody.
The term "sample" as used herein is used in its broadest sense. In certain instances, samples include cells, organisms, specimens or cultures obtained from any source, as well as biological and environmental samples. Wherein a biological sample may be obtained from an animal (including a human) and refers to a biological material or composition found therein, including but not limited to bone marrow, interstitial fluid, urine, cerebrospinal fluid, nucleic acids, DNA, blood, serum, platelets, plasma, tissue, and purified or filtered forms thereof; environmental samples include, but are not limited to, surface materials, soil, water, crystals, and industrial samples. In a preferred embodiment, the sample is a human sample; preferably, the human sample is selected from the group consisting of: blood, serum, plasma, urine, body fluids, saliva and other secretions or excretions and tissue or cell extracts; more preferably, the human sample is selected from serum and/or plasma.
The term "solid phase support coated with IgM antibody" is also called coating quality control band, and is used as positive quality control, and whether the normal color development can judge whether the operation is correct or not, and whether the result is reliable or not. In general, the quality control band is strongly colored: the operation is normal, and the result is credible; otherwise, the quality control band is not developed: the operation is abnormal, and the result is not credible. Preferably, the quality control band used in the present application is coated with human IgM, the corresponding enzyme conjugate is alkaline phosphatase-labeled anti-human IgM, and they interact with each other, and then a substrate solution is added to produce a strong color band.
In one embodiment, a handle label spaced apart from the IgM antibody coated solid support is further provided on the backing downstream of the IgM antibody coated solid support.
In one embodiment, the backing is transparent and/or the backing is made of a material comprising a plastic, preferably PVC.
In one embodiment, the solid support is a membrane.
In one embodiment, the membrane is made of a material comprising nitrocellulose, nylon, PVDF, Nytron, Biodyne or Porex, preferably the membrane is made of nitrocellulose. In one embodiment, the membrane is a nitrocellulose membrane, also known as an NC membrane.
In one embodiment, the solid support coated with purified S protein antigen is white and/or the solid support coated with purified N protein antigen is white and/or the solid support coated with IgM antibody is white.
In one embodiment, the handle label is made of plain printing paper and/or the handle label is white.
In one embodiment, the area and/or length of the solid support coated with purified S protein antigen represents between 2% and 15%, preferably between 4% and 6%, more preferably 5% of the area and/or length of the device; and/or the area and/or length of the solid support coated with purified N protein antigen represents 2% to 15%, preferably 4% to 6%, more preferably 5% of the area and/or length of the device; and/or the area and/or length of the IgM antibody coated solid support is 2% to 15%, preferably 4% to 6%, more preferably 5% of the area and/or length of the device.
In one embodiment, the area and/or length of the handle tag is between 5% and 20%, preferably between 10% and 15%, more preferably 12% of the area and/or length of the device.
In one embodiment, the area and/or length of the space between the solid support coated with purified S protein antigen or the solid support coated with purified N protein antigen and the solid support coated with IgM antibody is 40% to 80%, preferably 50% to 70%, more preferably 63% of the area and/or length of the device.
In one embodiment, the area and/or length of the space between the solid support coated with purified S protein antigen and the solid support coated with purified N protein antigen, and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, respectively, comprise from 1% to 5% and from 40% to 80% of the area and/or length of the device, preferably from 1.5% to 3.5% and from 50% to 70%, more preferably from 2% and 63%, respectively.
In one embodiment, the area and/or length of the space between the IgM antibody coated solid support and the handle tag is 1% to 5%, preferably 2% to 4%, more preferably 3% of the area and/or length of the device.
A second aspect of the present application provides a device for detecting IgM antibodies against a novel coronavirus in a sample, comprising:
a backing; and
the solid phase support coated with the purified N protein antigen, the solid phase support coated with the purified S protein antigen and the solid phase support coated with the IgM antibody are arranged on the back lining in sequence and are separated from each other.
The device provided by the second aspect of the present application is substantially the same as the device provided by the first aspect, except that the positions of the solid support coated with purified protein S antigen and the solid support coated with purified protein N antigen are interchanged. Thus, the embodiments described in the first aspect of the present application are equally applicable to the second aspect of the present application.
In the device of the first aspect or the second aspect of the present application, the S protein and the N protein can be simultaneously used for detection, thereby improving the detection accuracy.
Reagent kit
A third aspect of the present application provides a kit for detecting IgM antibodies against a novel coronavirus in a sample, comprising:
(1) the apparatus of the first or second aspect above;
(2) a labeled secondary antibody that specifically binds to the IgM antibody;
(3) a buffer solution;
(4) substrate solution;
(5) and (5) product instructions.
In one embodiment, the kit further comprises: an incubation device.
In one embodiment, the incubation device is an incubation tray.
In one embodiment, the labeled secondary antibody is an enzyme conjugate, preferably an alkaline phosphatase labeled anti-human IgM.
In one embodiment, the buffer is a Tris buffer, preferably a 10-fold concentrated Tris buffer.
In one embodiment, the substrate solution is tetrazolyl nitroaniline blue/5-bromo-4-chloropyridine-3-indole-phosphate.
Detection method
A fourth aspect of the present application provides a method for detecting IgM antibodies against the novel coronavirus SARS-CoV-2 in a sample by immunoblotting, comprising the steps of:
(1) providing the apparatus of the first or second aspect above;
(2) providing a sample;
(3) providing a labeled second antibody;
(4) immunoreacting the sample of step (2), the purified S protein antigen and the purified N protein antigen on the solid support in the device of step (1) with the second antibody of step (3);
(5) detecting the immune reaction result in the step (4) and judging whether the first antibody exists in the sample.
In one embodiment, the labeled secondary antibody is an enzyme conjugate, preferably an alkaline phosphatase labeled anti-human IgM.
In one embodiment, the device according to the first or second aspect above or the kit according to the third aspect above or the method according to the fourth aspect above, the sample is a human body sample, preferably the human body sample is selected from the group consisting of: blood, serum, plasma, urine, body fluids, saliva and other secretions or excretions and tissue or cell extracts, more preferably the human sample is serum and/or plasma.
Use of
A fifth aspect of the present application provides a combination of a purified S protein antigen and a purified N protein antigen; or the apparatus of the first or second aspect above; or the use of the kit according to the third aspect above for the detection of IgM antibodies against the novel coronavirus SARS-CoV-2 in a sample.
A sixth aspect of the present application provides a combination of a purified S protein antigen and a purified N protein antigen; or the apparatus of the first or second aspect above; or the use of a kit according to the third aspect above for the preparation of a product for the diagnosis of a novel coronavirus SARS-CoV-2 infection.
The present application includes the following embodiments:
embodiment 1. a device for the detection of IgM antibodies against a novel coronavirus SARS-CoV-2 in a sample by immunoblotting, comprising:
a backing; and
and the solid phase support coated with the purified S protein antigen, the solid phase support coated with the purified N protein antigen and the solid phase support coated with the IgM antibody are arranged on the back lining in sequence and are separated from each other.
a backing; and
the solid phase support coated with the purified N protein antigen, the solid phase support coated with the purified S protein antigen and the solid phase support coated with the IgM antibody are arranged on the back lining in sequence and are separated from each other.
Embodiment 4. the device of embodiment 1 or 2, wherein the backing is transparent and/or the backing is made of a material comprising plastic, preferably PVC.
Embodiment 6. the device of embodiment 5, wherein the membrane is made of a material comprising nitrocellulose, nylon, PVDF, Nytron, Biodyne or Porex, preferably the membrane is made of nitrocellulose.
Embodiment 7. the device according to embodiment 1 or 2, wherein the solid support coated with purified S protein antigen is white and/or the solid support coated with purified N protein antigen is white and/or the solid support coated with IgM antibody is white.
Embodiment 8 the device of embodiment 3, wherein the handle label is made of plain printing paper and/or the handle label is white.
Embodiment 9. the device according to embodiment 1 or 2, wherein the area and/or length of the solid support coated with purified S protein antigen represents 2% to 15%, preferably 4% to 6%, more preferably 5% of the area and/or length of the device; and/or the area and/or length of the solid support coated with purified N protein antigen represents 2% to 15%, preferably 4% to 6%, more preferably 5% of the area and/or length of the device; and/or the area and/or length of the IgM antibody coated solid support is 2% to 15%, preferably 4% to 6%, more preferably 5% of the area and/or length of the device.
Embodiment 10. the device of embodiment 3, wherein the area and/or length of the handle label is 5% -20%, preferably 10% -15%, more preferably 12% of the area and/or length of the device.
Embodiment 11. the device according to embodiment 1, wherein the area and/or length of the space between the solid support coated with purified S protein antigen and the solid support coated with purified N protein antigen and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, respectively, account for 1% -5% and 40% -80% of the area and/or length of the device, preferably 1.5% -3.5% and 50% -70%, respectively, more preferably 2% and 63%, respectively.
Embodiment 13. the device of embodiment 3, wherein the area and/or length of the space between the IgM antibody coated solid support and the handle tag is 1% to 5%, preferably 2% to 4%, more preferably 3% of the area and/or length of the device.
Embodiment 14. a kit for detecting IgM antibodies against a novel coronavirus SARS-CoV-2 in a sample by immunoblotting, comprising:
(1) the device according to any one of embodiments 1-13;
(2) a labeled secondary antibody that specifically binds to the IgM antibody;
(3) a buffer solution;
(4) substrate solution;
(5) and (5) product instructions.
Embodiment 15. the kit of embodiment 14, further comprising: an incubation device.
Embodiment 16 the kit of embodiment 15, wherein the incubation device is an incubation tray.
Embodiment 17. the kit of embodiment 14, wherein the labeled secondary antibody is an enzyme conjugate, preferably an alkaline phosphatase-labeled anti-human IgM.
Embodiment 18. the kit of embodiment 14, wherein the buffer is a Tris buffer, preferably a 10-fold concentrated Tris buffer.
Embodiment 19. the kit of embodiment 14, wherein the substrate solution is tetrazolyl nitroanilide blue/5-bromo-4-chloropyridine-3-indole-phosphate.
Embodiment 20. method for the detection of IgM antibodies against the novel coronavirus SARS-CoV-2 in a sample by immunoblotting, comprising the steps of:
(1) providing a device according to any one of embodiments 1-13;
(2) providing a sample;
(3) providing a labeled second antibody;
(4) immunoreacting the sample of step (2), the purified S protein antigen and the purified N protein antigen on the solid support in the device of step (1) with the second antibody of step (3);
(5) detecting the immune reaction result in the step (4) and judging whether the first antibody exists in the sample.
Embodiment 21. the method of embodiment 20, wherein the labeled secondary antibody is an enzyme conjugate, preferably an alkaline phosphatase labeled anti-human IgM.
Embodiment 22. the device according to any one of embodiments 1 to 13 or the kit according to any one of embodiments 14 to 19 or the method according to embodiment 20 or 21, wherein the sample is a human sample, preferably the human sample is selected from the group consisting of: blood, serum, plasma, urine, body fluids, saliva and other secretions or excretions and tissue or cell extracts, more preferably the human sample is serum and/or plasma.
Embodiment 23. a combination of purified S protein antigen and purified N protein antigen; or the device according to any one of embodiments 1-13; or the kit according to any of embodiments 14 to 19, for use in the detection of IgM antibodies against the novel coronavirus SARS-CoV-2 in a sample.
Embodiment 24. a combination of purified S protein antigen and purified N protein antigen; or the device according to any one of embodiments 1-13; or the use of a kit according to any of embodiments 14 to 19 for the preparation of a product for the diagnosis of a novel coronavirus SARS-CoV-2 infection.
PREFERRED EMBODIMENTS
The following detailed description of the preferred embodiments of the present application, taken in conjunction with the accompanying drawings, is intended to be illustrative, and not restrictive, and it is intended that all such modifications and equivalents fall within the scope of the present application.
Example 1
The detection device (see the attached figure 1) has the following specific structure:
the backing 1 is a transparent PVC plate with the length of 100mm and the width of 2 mm; and
the white nitrocellulose membrane 2 coated with the purified S protein antigen, the white nitrocellulose membrane 3 coated with the purified N protein antigen, the white nitrocellulose membrane 4 coated with the IgM antibody (namely a coated quality control band) and the white handle label 5 made of common printing paper are arranged on the back lining in sequence and are separated from each other;
wherein the white nitrocellulose membrane 2 coated with the purified S protein antigen is 5mm long and 2mm wide, and the distance between the upper end of the nitrocellulose membrane and the upper end of the backing 1 is 5 mm;
the white nitrocellulose membrane 3 coated with the purified N protein antigen is 5mm long and 2mm wide, and the distance between the upper end of the white nitrocellulose membrane 2 coated with the purified S protein antigen and the lower end of the white nitrocellulose membrane 2 is 2mm (namely, the interval is 2 mm);
the IgM antibody-coated white nitrocellulose membrane 4 is 5mm long and 2mm wide, and the distance between the upper end thereof and the lower end of the purified N protein antigen-coated white nitrocellulose membrane 3 is 63mm (i.e., the interval is 63 mm);
the white handle label 5 made of common printing paper is 12mm long and 2mm wide, and the distance between the upper end of the white handle label and the lower end of the white nitrocellulose membrane 4 coated with the IgM antibody is 3mm (namely, the interval is 3 mm).
The above S protein and N protein are specifically as follows (seehttps://www.ncbi.nlm.nih.gov/)：
| Type (B) | Gene ID | Protein ID |
| S protein | 43740568 | YP_009724390.1 |
| N protein | 43740575 | YP_009724397.2 |
The kit comprises the following components:
(1) the above-mentioned detection device;
(2) a labeled secondary antibody that specifically binds to the IgM antibody, the labeled secondary antibody being an alkaline phosphatase-labeled anti-human IgM;
(3) a buffer that is a 10-fold concentrated Tris buffer;
(4) the substrate solution is tetrazole nitroaniline blue/5-bromo-4-chloropyridine-3-indole-phosphate;
(5) a product description;
(6) the plates were incubated.
Operation process
Note that: all reagents must be equilibrated at room temperature (18-25 ℃) for about 30 minutes before use. The kit after unsealing also needs to be stored at 2-8 ℃ and care is taken to avoid pollution.
Detection apparatus of example 1: can be used directly. To prevent condensation on the film on the test device, the package is opened after equilibration to room temperature. Taking out the detection device, immediately sealing the original package and storing the package at 2-8 ℃.
Preparation of sample buffer: the buffer (i.e., 10-fold concentrated Tris buffer) was diluted with distilled water at a volume ratio of 1:9 to obtain a sample buffer.
Use of enzyme conjugate (alkaline phosphatase-labeled anti-human IgM): the desired enzyme conjugate was pipetted from the vial and diluted 1:9 by volume with the above sample buffer for use. If necessary, incubate a test device and dilute 0.15mL of enzyme conjugate with 1.35mL of sample buffer. The diluted enzyme conjugate should be used up on the same working day.
Preparation of a washing buffer: in use, a required amount of buffer (i.e., 10-fold concentrated Tris buffer) is pipetted from a vial and diluted with distilled water at a volume ratio of 1:9 to obtain a washing buffer. If necessary, incubate a test device and dilute 1mL of concentrated buffer with 9mL of distilled water. The wash buffer should be used up on the same working day.
Substrate solution (tetrazolyl nitroaniline blue/5-bromo-4-chloropyridine-3-indole-phosphate, NBT/BCIP): the test paper is directly used and is sensitive to light, a bottle cap is covered immediately after the test paper is used, and the test paper is incubated in a dark place in the experimental process.
Sample preparation: human serum.
The specific operation flow is as follows in sequence:
(1) pretreatment: the detection device was removed and placed in an incubation well. The face of the detecting device with the number faces upwards. Adding 1.5mL of sample buffer solution into the incubation grooves respectively, and after incubating for 5 minutes on a rocking table at room temperature, sucking off the liquid in the incubation grooves;
(2) sample incubation: adding 1.5mL of diluted samples into the incubation grooves respectively, and incubating for 15 minutes at room temperature (18-25 ℃) on a rocking table;
(3) cleaning: the liquid in the tank was aspirated off, and the NC membrane on the detection device was washed 3 times 5 minutes each time on a rocking bed with 1.5mL of washing buffer;
(4) incubation of enzyme conjugate: absorbing the liquid in the tank, adding 1.5mL of enzyme conjugate with the titer of 5000, and incubating for 15 minutes at room temperature (18-25 ℃) on a rocking table;
(5) cleaning: the liquid in the tank was aspirated off, and the NC membrane on the detection device was washed 3 times 5 minutes each time on a rocking bed with 1.5mL of washing buffer;
(6) substrate incubation: adding 1.5mL of substrate solution into the incubation grooves respectively, and incubating for 10 minutes on a rocking table at room temperature (18-25 ℃) in a dark place;
(7) and (4) terminating: sucking off liquid in the tank, and cleaning the detection device with distilled water for 3 times, each time for 1 minute;
(8) and (4) judging the result through visual inspection: placing the detection device in a result judgment template, and observing the strength of color development to judge a result after air drying; or the result is judged by Euromen EUROLineScan software.
The criteria for visual result judgment and software result interpretation are as follows:
wherein, the negative result is the patient without infection, the positive result is the patient with infection, the positive degree can qualitatively reflect the disease degree of the patient, and the two are positively correlated.
Example 2
Substantially the same as in example 1, except that the positions of the white nitrocellulose membrane 2 coated with a purified S protein antigen and the white nitrocellulose membrane 3 coated with a purified N protein antigen in example 1 were interchanged.
Comparative example 1
The conventional nucleic acid detection kit is specifically operated as follows (using fluorescent quantitative PCR (polymerase chain reaction)):
first, a sample is collected, and conventional sample types include pharyngeal swab, nasal swab, sputum, bronchial lavage, alveolar lavage, and the like, and viral RNA is extracted from the sample and reverse-transcribed into DNA.
Then, a specific sequence containing Taqman probe is used as a primer, if a target sequence exists in the sample, the probe is combined with the template during PCR reaction, and fluorescence is emitted. For each amplified DNA strand, a fluorescent molecule is produced.
Then, the relationship between the cycle number (Ct value) at which the fluorescence reaches a predetermined threshold and the viral nucleic acid concentration was monitored by a fluorescence quantitative PCR instrument, and the Ct value decreased as the viral nucleic acid concentration increased.
And finally, judging whether the sample is positive or negative according to the set Ct value.
Test results
19 samples with positive nucleic acid and positive clinical diagnosis were selected, and the detection rate was determined by using the kit of example 1 and using Euromen EUROLineScan software. The test results are shown in table 1 below.
TABLE 1
As can be seen from Table 1 above, all of the 19 positive samples were detected, i.e., the detection rate was 100%. However, with the conventional nucleic acid kit of comparative example 1, the detection rate was only 30% to 50%.
To sum up, this application for prior art, has obtained following beneficial technological effect:
1. the sample can be serum, plasma or whole blood, is easy to obtain, and can be finished by a common nurse; the samples required by the traditional nucleic acid reagent mainly comprise respiratory tract samples such as nasal swabs, pharyngeal swabs, sputum and bronchoalveolar lavage fluid (BALF); nasal swabs and pharyngeal swabs are relatively easy to obtain, but have low detectable rate, and alveolar lavage fluid is an ideal sample, but has technical difficulty, needs to be operated by professional personnel, has high infection risk and has low patient acceptance.
2. The detection rate is extremely high.
3. Because IgM antibodies exist in the initial stage of infection and gradually disappear along with the disease course, IgM antibodies can be detected, the method helps to judge the infection period of a patient, and traditional nucleic acid detection can only detect whether viruses exist and cannot distinguish the length of the infection period.
The foregoing examples are merely illustrative and serve to explain some of the features of the present disclosure. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the application. As used in the claims, the term "comprising" and its grammatical variants are also logically inclusive of different and varying phrases, such as, but not limited to, "consisting essentially of" or "consisting of. Where desired, numerical ranges are provided and sub-ranges therebetween are included. Variations in these ranges are also self-explanatory to those skilled in the art and should not be considered to be dedicated to the public, but rather should be construed to be covered by the appended claims where possible. And that advances in science and technology will result in possible equivalents or sub-substitutes not currently contemplated for reasons of inaccuracy in language representation, and such changes should also be construed where possible to be covered by the appended claims.
Claims (37)
1. Device for detecting IgM antibodies against a novel coronavirus in a sample, comprising:
a backing; and
and the solid phase support coated with the purified S protein antigen, the solid phase support coated with the purified N protein antigen and the solid phase support coated with the IgM antibody are arranged on the back lining in sequence and are separated from each other.
2. The device of claim 1, wherein the area and/or length of the space between the solid support coated with purified S protein antigen and the solid support coated with purified N protein antigen, and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, account for 1% -5% and 40% -80% of the area and/or length of the device, respectively.
3. The device of claim 1, wherein the area and/or length of the space between the solid support coated with purified S protein antigen and the solid support coated with purified N protein antigen, and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, respectively, accounts for 1.5% -3.5% and 50% -70% of the area and/or length of the device.
4. The device of claim 1, wherein the area and/or length of the space between the solid support coated with purified S protein antigen and the solid support coated with purified N protein antigen, and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, respectively, accounts for 2% and 63% of the area and/or length of the device.
5. Device for detecting IgM antibodies against a novel coronavirus in a sample, comprising:
a backing; and
the solid phase support coated with the purified N protein antigen, the solid phase support coated with the purified S protein antigen and the solid phase support coated with the IgM antibody are arranged on the back lining in sequence and are separated from each other.
6. The device of claim 5, wherein the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with purified S protein antigen, and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, account for 1% -5% and 40% -80% of the area and/or length of the device, respectively.
7. The device of claim 5, wherein the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with purified S protein antigen, and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, respectively, accounts for 1.5% -3.5% and 50% -70% of the area and/or length of the device.
8. The device of claim 5, wherein the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with purified S protein antigen, and the area and/or length of the space between the solid support coated with purified N protein antigen and the solid support coated with IgM antibody, respectively, account for 2% and 63% of the area and/or length of the device.
9. The device of claim 1 or 5, wherein a handle label is further provided on the backing downstream of the IgM antibody-coated solid support, spaced from the IgM antibody-coated solid support.
10. The device of claim 9, wherein the handle label is made of plain printing paper and/or the handle label is white.
11. The device of claim 9, wherein the area and/or length of the handle label is between 5% and 20% of the area and/or length of the device.
12. The device of claim 9, wherein the area and/or length of the handle label is 10% -15% of the area and/or length of the device.
13. The device of claim 9, wherein the area and/or length of the handle label is 12% of the area and/or length of the device.
14. The device of claim 9, wherein the area and/or length of the space between the IgM antibody coated solid support and the handle label is 1-5% of the area and/or length of the device.
15. The device of claim 9, wherein the area and/or length of the space between the IgM antibody coated solid support and the handle label is 2-4% of the area and/or length of the device.
16. The device of claim 9, wherein the area and/or length of the space between the IgM antibody coated solid support and the handle label is 3% of the area and/or length of the device.
17. The device of claim 1 or 5, wherein the backing is transparent and/or the backing is made of a material comprising plastic.
18. The device of claim 17, wherein the plastic is PVC.
19. The device of claim 1 or 5, wherein the solid support is a membrane.
20. The device of claim 19, wherein the membrane is made of a material comprising nitrocellulose, nylon, PVDF, Nytron, Biodyne, or Porex.
21. The device of claim 20, wherein the membrane is made of nitrocellulose.
22. The device according to claim 1 or 5, wherein the solid support coated with purified S protein antigen is white and/or the solid support coated with purified N protein antigen is white and/or the solid support coated with IgM antibodies is white.
23. The device of claim 1 or 5, wherein the area and/or length of the solid support coated with purified S protein antigen is from 2% to 15% of the area and/or length of the device; and/or the area and/or length of the solid support coated with purified N protein antigen is 2% -15% of the area and/or length of the device; and/or the area and/or length of the IgM antibody coated solid support is 2-15% of the area and/or length of the device.
24. The device of claim 1 or 5, wherein the area and/or length of the solid support coated with purified S protein antigen is 4% to 6% of the area and/or length of the device; and/or the area and/or length of the solid support coated with purified N protein antigen is 4% -6% of the area and/or length of the device; and/or the area and/or length of the IgM antibody coated solid support is 4% to 6% of the area and/or length of the device.
25. The device of claim 1 or 5, wherein the area and/or length of the solid support coated with purified protein S antigen is 5% of the area and/or length of the device; and/or the area and/or length of the solid support coated with purified N protein antigen comprises 5% of the area and/or length of the device; and/or the area and/or length of the IgM antibody coated solid support is 5% of the area and/or length of the device.
26. Kit for the detection of IgM antibodies against a novel coronavirus in a sample, characterized in that it comprises:
(1) the device of any one of claims 1-25;
(2) a labeled secondary antibody that specifically binds to the IgM antibody;
(3) a buffer solution;
(4) substrate solution;
(5) and (5) product instructions.
27. The kit of claim 26, further comprising: an incubation device.
28. The kit of claim 27, wherein the incubation device is an incubation tray.
29. The kit of claim 26, wherein the labeled second antibody is an enzyme conjugate.
30. The kit of claim 26, wherein the labeled second antibody is an alkaline phosphatase-labeled anti-human IgM.
31. The kit of claim 26, wherein the buffer is Tris buffer.
32. The kit of claim 26, wherein the buffer is a 10-fold concentrated Tris buffer.
33. The kit of claim 26, wherein the substrate solution is tetrazolium nitroaniline blue/5-bromo-4-chloropyridine-3-indole-phosphate.
34. The device of claim 1 or 5 or the kit of claim 26 or 27, wherein the sample is a human sample.
35. The device according to claim 1 or 5 or the kit according to claim 26 or 27, wherein the sample is a human sample selected from one of the group consisting of: blood, serum, plasma, urine, body fluids, and saliva.
36. The device of claim 1 or 5 or the kit of claim 26 or 27, wherein the sample is a human sample, and the human sample is serum or plasma.
37. The device of claim 1 or 5 or the kit of claim 26 or 27, wherein the sample is a human sample diluted with Tris buffer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202020308108.8U CN213633456U (en) | 2020-03-12 | 2020-03-12 | Device and kit for detecting IgM (immunoglobulin M) antibody of anti-novel coronavirus in sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202020308108.8U CN213633456U (en) | 2020-03-12 | 2020-03-12 | Device and kit for detecting IgM (immunoglobulin M) antibody of anti-novel coronavirus in sample |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN213633456U true CN213633456U (en) | 2021-07-06 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202020308108.8U Active CN213633456U (en) | 2020-03-12 | 2020-03-12 | Device and kit for detecting IgM (immunoglobulin M) antibody of anti-novel coronavirus in sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN213633456U (en) |
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- 2020-03-12 CN CN202020308108.8U patent/CN213633456U/en active Active
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