CN111458505A - Detection paper and method for simultaneously and quantitatively detecting IgG and IgM contents of new coronavirus - Google Patents
Detection paper and method for simultaneously and quantitatively detecting IgG and IgM contents of new coronavirus Download PDFInfo
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Abstract
The invention discloses detection paper for simultaneously detecting IgG and IgM of a new crown virus and a method thereof.A nitrocellulose membrane in the detection paper is provided with quality control bands which are coated with an anti-human IgM monoclonal antibody, an anti-human IgG monoclonal antibody and a monoclonal antibody coated with a specific protein for resisting the new crown virus 2019-nCoV at intervals, and a specific protein for the new crown virus 2019-nCoV marked by rare earth ion nano microspheres is coated at a combination pad.
Description
Technical Field
The invention relates to detection paper for detecting IgG and IgM contents and a detection method thereof, in particular to detection paper for simultaneously and quantitatively detecting the IgG and IgM contents of new coronavirus and a detection method thereof, belonging to the field of in-vitro detection.
Background
Since the epidemic situation of the coronavirus 2019-nCoV pneumonia occurs, the epidemic situation spreads in the world and seriously threatens the life safety and the body health of human beings, the coronavirus 2019-nCoV has strong infectivity, clinical symptoms appear after infection of most patients, but some patients are asymptomatic infectors, which brings huge challenges to epidemic situation prevention and control and clinical diagnosis. At present, nucleic acid detection and virus gene sequencing are mainly taken as etiology confirmation evidence, due to the influence of various factors such as sampling, operation and reagents, false negative results can occur in nucleic acid detection, the positive detection rate of virus nucleic acids of 2019 novel coronavirus (2019-nCoV) infected patients is only 30-50%, in addition, the nucleic acid detection has high requirements on instrument equipment, detection sites and environmental conditions, the defects of long detection time, low flux and the like exist, large-scale detection of the crowd under the current epidemic situation is inconvenient, and therefore a rapid and convenient detection kit is urgently needed to be developed for clinical detection, so that infected crowds are isolated as soon as possible to block virus propagation, and the serology detection is increased by a novel coronavirus pneumonia diagnosis and treatment scheme (trial seventh edition) issued by the national health committee: the novel coronavirus specific IgM antibody is positive 3-5 days after disease attack, the IgG antibody titer recovery period is 4 times or more higher than that of the acute period, the novel coronavirus specific IgG antibody/IgM antibody detection method comprises immunoassay methods such as an enzyme-linked immunosorbent assay, a chemiluminescence method, a colloidal gold method and the like, venous blood of a patient is detected, safety and convenience are higher than those of throat swab sampling, infection and virus propagation risks of medical workers are greatly reduced, wherein the enzyme-linked immunosorbent assay and the chemiluminescence method have high sensitivity and can carry out quantitative detection, but the requirement on equipment is high, reaction time is generally more than 1 hour, the colloidal gold method is convenient to operate and does not need equipment, a detection result can be obtained by visual observation within 15min, however, the colloidal gold method has the defects of low sensitivity, incapability of quantitative analysis and the like, and in order to quantitatively detect the novel coronavirus specific IgG antibody/IgM antibody, the invention combines nanotechnology and immunochromatography technology, establishes a time-resolved fluorescence immunochromatography detection method for simultaneously and quantitatively detecting the content of IgG and IgM of the new coronavirus 2019-nCoV, and prepares a corresponding detection card, and the technology can make up the defects of high requirement on nucleic acid detection conditions, long time consumption and low positive rate, can overcome the defects of low detection sensitivity and incapability of quantitative analysis of colloidal gold, can be used as an important supplementary detection means for disease diagnosis, is suitable for rapid screening of people, and is beneficial to diagnosis, prevention and control of 2019-nCoV infection.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the detection paper which has the technical characteristics of low cost, low requirements on detection conditions, short time consumption and the like and is used for simultaneously and quantitatively detecting the content of IgG and IgM of the new coronavirus.
The invention also aims to provide the method for simultaneously and quantitatively detecting the IgG and IgM contents of the new coronavirus, which has the technical characteristics of convenient detection, suitability for rapid screening of people and the like.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the utility model provides a while quantitative determination new crown virus's IgG and IgM content's detection paper, detection paper includes the bottom plate and follows bottom plate length direction and glues in order and cover absorbent paper, nitrocellulose membrane, combination pad, sample pad on the bottom plate, order in proper order adjacent position contacts and partially overlaps between absorbent paper, nitrocellulose membrane, combination pad and the sample pad, wherein, nitrocellulose membrane glues to cover in the middle part of bottom plate, the nitrocellulose membrane upper berth is equipped with the first detection area that mutual interval and spraying had anti-human IgM monoclonal antibody, the second detection area that spraying had anti-human IgG monoclonal antibody and the quality control area that spraying had the monoclonal antibody of anti-new crown virus 2019-nCoV specific protein, the combination is filled up the specific protein of new crown virus 2019-nCoV that sprays the nanometer microballon mark of rare earth ion, quality control area, the detection of the test paper of the IgG and IgM content, the test paper is followed, The first detection belt and the second detection belt are arranged in parallel;
the combination pad is positioned above the nitrocellulose membrane, and the length of the overlapped part of the combination pad and the nitrocellulose membrane in the length direction of the bottom plate is 2 mm; the sample pad is arranged above the combination pad, and the length of the overlapped part of the sample pad and the combination pad in the length direction of the bottom plate is 2 mm; the absorbent paper is arranged above the nitrocellulose membrane, and the length of the overlapped part of the absorbent paper and the nitrocellulose membrane in the length direction of the bottom plate (4) is 2 mm.
The detection paper is wrapped by a shell, the shell comprises a base and a clamping cover, and a local area observation port and a sample adding port for exposing the detection paper are formed in the clamping cover; the sample adding port is arranged above the sample pad to expose part or all of the sample pad, and the observation port is arranged above the nitrocellulose membrane to expose the first detection band, the second detection band and the quality control band.
As a refinement, the first detection zone is adjacent to the conjugate pad and is separated by a distance of 0.4cm or 0.5 cm; the second detection zone is positioned between the first detection zone (and the quality control zone, the distance between the second detection zone and the first detection zone is 0.4cm or 0.5cm, and the distance between the second detection zone and the quality control zone is 0.4cm or 0.5 cm.
As an improvement, the base is a plastic base, and the clamping cover is a plastic clamping cover.
A preparation method of detection paper for simultaneously and quantitatively detecting IgG and IgM contents of new coronavirus comprises the following steps:
1) pretreating an antigen, namely selecting a specific protein of a commercial new coronavirus 2019-nCoV, and dialyzing the specific protein at 4 ℃ overnight by using 0.05 mol/L phosphate buffer solution with the pH value of 7.2-7.6, wherein the specific protein of the new coronavirus 2019-nCoV is an N protein or an S protein;
2) adding carbodiimide with the final concentration of 20mmol and pretreated 2019-nCoV specific protein into a nano microsphere solution, wherein the mass of the microsphere is 50:1, reacting at room temperature for 2 hours, centrifuging, removing supernatant, adding a sample diluent until the concentration of the microsphere is 1.0mg/ml, spraying the prepared microsphere on a bonding pad formed by a polyester film by using a quantitative film spraying instrument in an amount of 3ul/cm-5ul/cm, drying at 35-38 ℃ for 1 hour under the condition of keeping out of the sun, adding a drying agent, and sealing for later use, wherein the sample diluent is a phosphate buffer solution with the pH of 7.2 and 0.05 mol/L containing 1% BSA;
3) preparing a nitrocellulose membrane, namely using a phosphate buffer solution containing 1% of sucrose and 0.02 mol/L, wherein the pH value is 7.4, respectively diluting an anti-human IgM monoclonal antibody, an anti-human IgG monoclonal antibody and a monoclonal antibody of a specific protein of anti-new coronavirus 2019-nCoV to the concentration of 1mg/ml, using a quantitative membrane spraying instrument to spray the three on the nitrocellulose membrane at intervals of 1ul/cm, drying for 1h at the temperature of 35-38 ℃, adding a drying agent, and sealing for later use;
4) assembling detection paper: laying a nitrocellulose membrane in the middle of the bottom plate, and then laying absorbent paper at one end close to the quality control band to enable the absorbent paper and the nitrocellulose membrane to be partially overlapped; laying a bonding pad on one end adjacent to the second detection zone, bonding the nitrocellulose membrane and the bonding pad, finally attaching the sample pad, cutting with a cutting machine according to the width of 0.4cm, and placing into a shell.
A detection method of detection paper for simultaneously detecting IgG and IgM of a new coronavirus comprises the following steps:
a) dropwise adding 20ul of sample into the sample addition port, adding 50ul of sample diluent, and immediately detecting after waiting for 15 min;
b) scanning the fluorescence intensity of the first detection zone, the second detection zone and the quality control zone by using a fluorescence detector, if the quality control line zone has a fluorescence emission peak, indicating that the detection paper is effective, otherwise, indicating that the detection paper is ineffective;
c) and (4) calculating the concentrations of IgG and IgM of the new coronavirus in the sample on the standard curve according to fluorescence counting, and judging whether the result is positive or negative.
As an improvement, the specific protein of the new coronavirus comprises a 2019-nCoV specific N protein or a 2019-nCoV specific S protein, or the specific protein of the new coronavirus is a mixture of the 2019-nCoV specific N protein and the specific S protein.
As a modification, the dilution is 0.05 mol/L phosphate buffer containing 1% BSA at pH 7.2.
Has the advantages that: 1. the nitrocellulose membrane is provided with a first detection band sprayed with an anti-human IgM monoclonal antibody, a second detection band sprayed with an anti-human IgG monoclonal antibody and a quality control band sprayed with a monoclonal antibody of a specific protein of anti-new coronavirus 2019-nCoV, and the specific protein of the new coronavirus 2019-nCoV marked by rare earth ion nano microspheres is sprayed at a combination pad; the detection paper can creatively realize the combination of a time-resolved fluorescence immunity technology, an immunochromatography technology and a nanotechnology, so that a time-resolved fluorescence immunity chromatography method capable of simultaneously and quantitatively detecting the contents of IgG and IgM of the new coronavirus 2019-nCoV is formed, the problem that the existing colloidal gold technology cannot be used for quantification is solved, the sensitivity is higher, and convenience is brought to clinical use;
2. the detection paper is used for quantitatively detecting the contents of IgG and IgM of the new coronavirus 2019-nCoV, a first detection band sprayed with an anti-human IgM monoclonal antibody and a second detection band sprayed with an anti-human IgG monoclonal antibody are arranged on the same detection test paper, the two detection bands are arranged in parallel, and the contents of IgG and IgM of the new coronavirus 2019-nCoV are relatively and respectively detected, so that the sample consumption is reduced, and the working efficiency is improved;
3. the nanometer microsphere contains europium ions, so that the problem of quenching of rare earth ions in a sample solution is solved, the fluorescence intensity emitted by the rare earth ions is improved, and the detection sensitivity is improved;
4. the detection card with the shell is convenient to store and carry and is convenient for detection operation in an operation process.
Drawings
FIG. 1 is a schematic cross-sectional view of a test paper of the present invention taken along its length;
FIG. 2 is a schematic plan view of a test paper of the present invention;
fig. 3 is a plan view schematically illustrating the overall structure of the present invention.
Detailed Description
The present invention will be further described with reference to the drawings attached to the specification, but the present invention is not limited to the following examples.
Example 1
As shown in fig. 1 and 2, the detection paper for simultaneously and quantitatively detecting IgG and IgM contents of new coronavirus 2019-nCoV comprises a bottom plate 4, and a water absorption paper 1, a nitrocellulose membrane 2, a binding pad 8 and a sample pad 3 which are sequentially adhered on the bottom plate 4 along the length direction of the bottom plate 4; the nitrocellulose membrane 2 is adhered to the middle part of the bottom plate 4, and the absorbent paper 1, the nitrocellulose membrane 2, the combination pad 8 and the sample pad 3 are sequentially contacted with adjacent parts and partially overlapped; the nitrocellulose membrane 2 is provided with a first detection band 7 coated with an anti-human IgM monoclonal antibody, a second detection band 6 coated with an anti-human IgG monoclonal antibody and a quality control band 5 coated with a monoclonal antibody of a specific protein of anti-new coronavirus 2019-nCoV at intervals, and the specific protein of the new coronavirus 2019-nCoV marked by rare earth ion nano microspheres is coated at the position of the combination pad 8; wherein, the first detection belt 7 is arranged close to the combination pad 8, and the quality control belt 5 is arranged close to the absorbent paper 1.
In this embodiment, the diameter of the nanoparticle is 200nm, and the diameter of the polymer nanoparticle may generally be set to 10-500nm according to actual needs during use, that is, the diameter of the nanoparticle may be set to 10nm, 255mm, 300mm, or 500nm, or may be set to any other value between these two values.
The quality control band 5, the first detection band 7 and the second detection band 6 are arranged in parallel; wherein the minimum distance from the second detection zone 6 to the quality control zone 5 or the first detection zone 7 is 0.3cm, and the maximum distance from the second detection zone 6 to the quality control zone 5 or the first detection zone 7 is 0.7cm, that is, in this embodiment, the three are all arranged in parallel, and the first detection zone 7 is adjacent to the bonding pad 8; the second detection zone 6 is positioned between the first detection zone 7 and the quality control zone 5, and the distance between the first detection zone and the quality control zone is 0.4 cm.
On the basis of the above embodiment, the absorbent paper 1 is disposed above the nitrocellulose membrane 2, the length of the overlapped portion of the absorbent paper 1 and the nitrocellulose membrane 2 in the length direction of the bottom plate 4 is 2mm, the combination pad 8 is disposed above the nitrocellulose membrane 2, the length of the overlapped portion of the combination pad 8 and the nitrocellulose membrane 2 in the length direction of the bottom plate 4 is 2mm, the sample pad 3 is disposed above the combination pad 8, and the length of the overlapped portion of the sample pad 3 and the combination pad 8 in the length direction of the bottom plate 4 is 2mm, wherein, in order to ensure the smooth use of the detection paper, the length of the combination pad 8 in the length direction of the bottom plate 4 is greater than 4mm, that is, the length of the combination pad 8 is greater than the length of the overlapped portion of the sample pad 3 and the combination pad 8 in the length direction of the bottom plate 4, and the length of the bonding pad 8 is larger than the sum of the lengths of the overlapped part of the bonding pad 8 and the nitrocellulose membrane 2 along the length direction of the bottom plate 41.
Assembling detection paper: laying a nitrocellulose membrane 2 in the middle of a bottom plate, and then laying absorbent paper 1 at one end close to a quality control line to ensure that the absorbent paper 1 is partially overlapped with the nitrocellulose membrane 2; laying a bonding pad 8 at one end adjacent to the second detection line, bonding the nitrocellulose membrane 2 and the bonding pad 8, finally attaching the sample pad 3, cutting by a cutting machine according to the width of 0.4cm, and then filling into a plastic card shell.
Example 2
As shown in fig. 3, on the basis of example 1, this example further provides a time-resolved fluorescence immunochromatography detection card for simultaneously and quantitatively detecting IgG and IgM contents of new coronavirus 2019-nCoV, which comprises the detection paper described in example 1 and a shell covering the detection paper; the shell comprises a base and a card cover 9, wherein the card cover 9 is provided with a local area observation port 10 and a sample adding port 11 for exposing the detection paper; the sample addition port 11 is arranged above the sample pad 3 to expose part or all of the area of the sample pad 3, and the observation port 10 is arranged on the upper part of the nitrocellulose membrane 2 to expose the first detection strip 7, the second detection strip 6 and the quality control strip 5, in this embodiment, the detection card with the shell is not only convenient to store and carry, but also convenient to detect in the operation process; and the sample adding port 11 is arranged to facilitate the addition of sample liquid in the operation process, and the observation port 10 is arranged to facilitate the observation of the detection result.
In this embodiment, the base is preferably a plastic base, and the card cover 9 is preferably a plastic card cover 9; the detection card has light weight and low manufacturing cost.
On the basis of embodiment 1, this embodiment further provides a method for preparing the detection paper described in embodiment 1, including the following steps:
(1) pretreating antigen by selecting specific protein (N protein or S protein) of commercial new coronavirus 2019-nCoV, and dialyzing overnight at 4 ℃ with 0.05 mol/L phosphate buffer solution with pH of 7.2-7.6;
(2) adding carbodiimide (EDC) (with the final concentration of 20mmol) and pretreated 2019-nCoV specific protein (the mass of the microsphere: the mass of an antibody is 50:1) into a nano microsphere solution, reacting at room temperature for 2 hours, centrifuging, removing supernatant, adding a sample diluent (0.05 mol/L containing 1% BSA and a phosphate buffer solution with the pH value of 7.2) to the microsphere concentration of 1.0mg/ml for later use, spraying the prepared microspheres on a binding pad 8 formed by a polyester film by using a quantitative film spraying instrument in an amount of 3ul/cm-5ul/cm, drying at 35-38 ℃ for 1 hour under the condition of avoiding light, adding a drying agent, and sealing for later use;
(3) preparing a nitrocellulose membrane 2, namely using a phosphate buffer solution containing 1% of sucrose and 0.02 mol/L, wherein the pH value is 7.4, respectively diluting an anti-human IgM monoclonal antibody, an anti-human IgG monoclonal antibody and a monoclonal antibody of a specific protein of anti-new coronavirus 2019-nCoV to the concentration of 1mg/ml, spraying the three on the nitrocellulose membrane 2 at a certain interval by using a quantitative membrane spraying instrument in the amount of 1ul/cm, drying for 1h at 35-38 ℃, adding a drying agent, and sealing for later use;
(4) assembling detection paper: laying a nitrocellulose membrane 2 in the middle of a bottom plate, and then laying absorbent paper 1 at one end adjacent to a quality control band 6 to ensure that the absorbent paper 1 is partially overlapped with the nitrocellulose membrane 2; laying a bonding pad 8 at one end adjacent to the second detection line, bonding the nitrocellulose membrane 2 and the bonding pad 8, finally attaching the sample pad 3, cutting by a cutting machine according to the width of 0.4cm, and then filling into a plastic card shell.
Example 3
This example provides a method for quantitatively detecting the IgG and IgM contents of a new coronavirus 2019-nCoV with the paper strip described in the examples, comprising the following steps:
(1) drawing a standard curve:
and (3) diluting IgG and IgM of the new coronavirus 2019-nCoV with high concentration into a series of standard products, actually detecting according to a certain gradient, wherein the smaller the gradient value is, the finer the division is, and the detection result takes a fluorescence signal value as a vertical coordinate and the concentration of the standard product as a horizontal coordinate, establishes an equation and fits a standard curve.
(2) Sample detection:
dropping 20ul of sample (serum or plasma or whole blood) into the sample hole of the detection paper, adding 50ul of sample diluent (0.05 mol/L containing 1% BSA and pH 7.2 phosphate buffer), waiting for 15min, and immediately detecting;
scanning the fluorescence intensity of the first detection zone 7, the second detection zone 6 and the quality control zone 5 by using a fluorescence detector, if a fluorescence emission peak appears in the quality control line 5, indicating that the detection paper is effective, otherwise, indicating that the detection paper is ineffective,
and (4) calculating (reading) the concentrations of IgG and IgM of the new coronavirus 2019-nCoV in the sample on a standard curve according to fluorescence counting, and judging the negative and positive results.
Finally, it should be noted that the present invention is not limited to the above embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (6)
1. The utility model provides a while quantitative determination new coronavirus IgG and IgM content's detect paper which characterized in that: the detection paper comprises a bottom plate (4), and absorbent paper (1), a nitrocellulose membrane (2), a combination pad (8) and a sample pad (3) which are sequentially adhered to the bottom plate (4) along the length direction of the bottom plate (4), wherein adjacent parts among the absorbent paper (1), the nitrocellulose membrane (2), the combination pad (8) and the sample pad (3) are contacted and partially overlapped, the nitrocellulose membrane (2) is adhered to the middle part of the bottom plate (4), a first detection belt (7) which is mutually spaced and sprayed with an anti-human IgM monoclonal antibody, a second detection belt (6) which is sprayed with an anti-human IgG monoclonal antibody and a quality control belt (8) which is sprayed with a monoclonal antibody which is anti-new crown virus 2019-nCoV specific protein are paved on the nitrocellulose membrane (2), and the specific protein of the new crown virus 2019-nCoV which is marked by a nano microsphere which is sprayed with rare earth ions is paved on the combination pad (8), the quality control belt (8), the first detection belt (7) and the second detection belt (6) are arranged in parallel;
the combination pad (8) is positioned above the nitrocellulose membrane (2), and the length of the overlapped part of the combination pad (8) and the nitrocellulose membrane (2) in the length direction of the bottom plate (4) is 2 mm; the sample pad (3) is arranged above the combination pad (8), and the length of the overlapped part of the sample pad (3) and the combination pad (8) in the length direction of the bottom plate (4) is 2 mm; the absorbent paper (1) is arranged above the nitrocellulose membrane (2), and the length of the overlapped part of the absorbent paper (1) and the nitrocellulose membrane (2) in the length direction of the bottom plate (4) is 2 mm.
The detection paper is wrapped by a shell, the shell comprises a base and a clamping cover (9), and a local area observation port (10) and a sample adding port (11) for exposing the detection paper are formed in the clamping cover (9); the sample adding port (11) is arranged above the sample pad (3) to expose part or all of the sample pad (3), and the observation port (10) is arranged above the nitrocellulose membrane (2) to expose the first detection band (7), the second detection band (6) and the quality control band (8).
2. The detection paper for simultaneously and quantitatively detecting the IgG and IgM contents of a novel coronavirus according to claim 1, wherein: the first detection belt (7) is adjacent to the combination pad (8) and is separated by 0.4cm or 0.5 cm; the second detection belt (6) is positioned between the first detection belt (7) and the quality control belt (8), the distance between the second detection belt (6) and the first detection belt (7) is 0.4cm or 0.5cm, and the distance between the second detection belt (6) and the quality control belt (8) is 0.4cm or 0.5 cm.
3. The detection paper for simultaneously and quantitatively detecting the IgG and IgM contents of a novel coronavirus according to claim 1, wherein: the base is a plastic base, and the clamping cover (9) is a plastic clamping cover.
4. A preparation method of detection paper for simultaneously and quantitatively detecting the IgG and IgM contents of new coronavirus is characterized by comprising the following steps:
1) pretreating an antigen, namely selecting a specific protein of a commercial new coronavirus 2019-nCoV, and dialyzing the specific protein at 4 ℃ overnight by using 0.05 mol/L phosphate buffer solution with the pH value of 7.2-7.6, wherein the specific protein of the new coronavirus 2019-nCoV is an N protein or an S protein;
2) adding carbodiimide with the final concentration of 20mmol and pretreated 2019-nCoV specific protein into a nano microsphere solution, wherein the mass of the microsphere is 50:1, reacting at room temperature for 2 hours, centrifuging, removing supernatant, adding a sample diluent until the concentration of the microsphere is 1.0mg/ml for later use, spraying the prepared microsphere on a bonding pad (8) formed by a polyester film by using a quantitative film spraying instrument in an amount of 3ul/cm-5ul/cm, drying at 35-38 ℃ for 1 hour under the condition of keeping out of the sun, adding a drying agent, and sealing for later use, wherein the sample diluent is 0.05 mol/L containing 1% BSA and a phosphate buffer solution with the pH value of 7.2;
3) preparing a nitrocellulose membrane (2), namely using a phosphate buffer solution containing 1% of sucrose and 0.02 mol/L with the pH value of 7.4 to respectively dilute an anti-human IgM monoclonal antibody, an anti-human IgG monoclonal antibody and a monoclonal antibody of a specific protein of anti-new coronavirus 2019-nCoV to the concentration of 1mg/ml, using a quantitative membrane spraying instrument to spray the three on the nitrocellulose membrane (2) at intervals of 1ul/cm, drying for 1h at the temperature of 35-38 ℃, adding a drying agent, and sealing for later use;
4) assembling the test strip: paving a nitrocellulose membrane (2) in the middle of a bottom plate (4), and then paving absorbent paper (1) at one end close to a quality control band (8) to ensure that the absorbent paper (1) is partially overlapped with the nitrocellulose membrane (2); a conjugate pad (8) was laid on the end adjacent to the second detection tape (6), the nitrocellulose membrane (2) and the conjugate pad (8) were attached, and finally the sample pad (3) was attached, cut by a cutter in a width of 0.4cm, and then packed in a housing.
5. A method for simultaneously quantitatively detecting IgG and IgM contents of a novel coronavirus by using the detection paper as set forth in any one of claims 1 to 3, the method comprising the steps of:
a) dropwise adding 20ul of sample into the sample adding port (10), adding 50ul of sample diluent, and immediately detecting after waiting for 15 min;
b) scanning the fluorescence intensity of the first detection zone (7), the second detection zone (6) and the quality control zone (8) by using a fluorescence detector, if a fluorescence emission peak appears in the quality control zone (8), the test strip is effective, otherwise, the test strip is ineffective;
c) and (4) calculating the concentrations of IgG and IgM of the new coronavirus in the sample on the standard curve according to fluorescence counting, and judging whether the result is positive or negative.
6. The detection method of detection paper for simultaneously and quantitatively detecting the IgG and IgM contents of a new coronavirus according to claim 5, wherein the specific protein of the new coronavirus comprises a 2019-nCoV specific N protein or a 2019-nCoV specific S protein, or the specific protein of the new coronavirus is a mixture of the 2019-nCoV specific N protein and the specific S protein;
the dilution was 0.05 mol/L phosphate buffer containing 1% BSA at pH 7.2.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111999507A (en) * | 2020-08-25 | 2020-11-27 | 中国人民解放军军事科学院军事医学研究院 | Fluorescence immunochromatography test paper for detecting novel coronavirus antibody |
| CN112180083A (en) * | 2020-09-30 | 2021-01-05 | 厦门稀土材料研究所 | IgM antibody detection kit, detection card thereof and preparation method of detection card |
| CN112305218A (en) * | 2020-08-18 | 2021-02-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Novel coronavirus antibody colloidal gold immune lateral chromatography detection method and application thereof |
| CN112730832A (en) * | 2021-02-04 | 2021-04-30 | 厦门稀土材料研究所 | COVID-19 antigen detection card, preparation method and application thereof |
| CN112881681A (en) * | 2020-12-09 | 2021-06-01 | 安徽医科大学 | Indirect ELISA detection method for SARS-CoV-2N protein IgA |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104181298A (en) * | 2014-09-02 | 2014-12-03 | 秦志浩 | Human parainfluenza virus (HPIVs) IgM antibody detection test strip and preparation method thereof |
| CN204514928U (en) * | 2014-11-20 | 2015-07-29 | 江苏省原子医学研究所 | Detect the test strips of pepsinogen I and II and apply its test card |
| CN110007096A (en) * | 2019-05-06 | 2019-07-12 | 江苏硕世生物科技股份有限公司 | A kind of dengue virus IgG/IgM antibody test strip, kit and preparation method thereof |
-
2020
- 2020-04-07 CN CN202010263635.6A patent/CN111458505A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104181298A (en) * | 2014-09-02 | 2014-12-03 | 秦志浩 | Human parainfluenza virus (HPIVs) IgM antibody detection test strip and preparation method thereof |
| CN204514928U (en) * | 2014-11-20 | 2015-07-29 | 江苏省原子医学研究所 | Detect the test strips of pepsinogen I and II and apply its test card |
| CN110007096A (en) * | 2019-05-06 | 2019-07-12 | 江苏硕世生物科技股份有限公司 | A kind of dengue virus IgG/IgM antibody test strip, kit and preparation method thereof |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112305218A (en) * | 2020-08-18 | 2021-02-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Novel coronavirus antibody colloidal gold immune lateral chromatography detection method and application thereof |
| CN111999507A (en) * | 2020-08-25 | 2020-11-27 | 中国人民解放军军事科学院军事医学研究院 | Fluorescence immunochromatography test paper for detecting novel coronavirus antibody |
| CN111999507B (en) * | 2020-08-25 | 2024-04-30 | 中国人民解放军军事科学院军事医学研究院 | Fluorescent immunochromatography test paper for detecting novel coronavirus antibody |
| CN112180083A (en) * | 2020-09-30 | 2021-01-05 | 厦门稀土材料研究所 | IgM antibody detection kit, detection card thereof and preparation method of detection card |
| CN112881681A (en) * | 2020-12-09 | 2021-06-01 | 安徽医科大学 | Indirect ELISA detection method for SARS-CoV-2N protein IgA |
| CN112730832A (en) * | 2021-02-04 | 2021-04-30 | 厦门稀土材料研究所 | COVID-19 antigen detection card, preparation method and application thereof |
| CN112730832B (en) * | 2021-02-04 | 2022-05-24 | 厦门稀土材料研究所 | COVID-19 antigen detection card, preparation method and application thereof |
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