CN111398588A - Use method of immunochromatography kit for rapidly detecting novel coronavirus N protein - Google Patents

Use method of immunochromatography kit for rapidly detecting novel coronavirus N protein Download PDF

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CN111398588A
CN111398588A CN202010136067.3A CN202010136067A CN111398588A CN 111398588 A CN111398588 A CN 111398588A CN 202010136067 A CN202010136067 A CN 202010136067A CN 111398588 A CN111398588 A CN 111398588A
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林斯
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Beijing Huaketai Biotechnology Co ltd
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Abstract

The invention relates to a use method of an immunochromatographic kit for rapidly detecting novel coronavirus N protein, wherein the immunochromatographic kit comprises a test strip, the test strip comprises a detection line and a quality control line, the detection line is coated with a strain of anti-novel coronavirus N protein antibody, and the quality control line is coated with a goat anti-rabbit polyclonal antibody. The immunochromatography kit for rapidly detecting the novel coronavirus N protein provided by the invention can be used for detecting the novel coronavirus N protein in serum, plasma and whole blood samples so as to diagnose the novel coronavirus pneumonia.

Description

Use method of immunochromatography kit for rapidly detecting novel coronavirus N protein
Technical Field
The invention belongs to the field of in-vitro diagnosis, and relates to a use method of an immunochromatography kit for rapidly detecting novel coronavirus N protein.
Background
2019 the novel coronavirus (SARS-CoV-2) is a new strain of coronavirus which has never been found in human body before, and after people are infected with coronavirus, the common signs of people include respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. 1 month and 26 days, the national drug administration (HSA) carries out emergency approval to pass 4 new coronavirus detection products of 4 enterprises such as Huada gene and the like, but the currently approved novel coronavirus nucleic acid detection reagent is based on a nucleic acid detection mode, a single sample needs 3 hours to give a detection result by adopting a nucleic acid fluorescence Polymerase Chain Reaction (PCR) method, and a nucleic acid sequencing method needs 6 hours to give a detection result, more importantly, the detection flux is called as a maximum restriction factor due to extremely high requirements of gene amplification on laboratory environment, the supply capacity of the novel coronavirus rapid detection service is greatly restricted, and the requirement of epidemic situation prevention and control cannot be fully met.
Disclosure of Invention
In view of the above, the main objective of the present invention is to provide a method for using an immunochromatographic kit for rapidly detecting a novel coronavirus N protein.
In order to achieve the purpose, the invention provides the following technical scheme:
an immunochromatography kit for rapidly detecting novel coronavirus N protein and a using method thereof.
In a particular embodiment of the invention, wherein the blood sample is selected from serum, plasma or whole blood sample.
In a specific embodiment of the present invention, the method for using the same comprises the following steps: and directly dripping the collected blood sample to a sample adding hole of the kit, standing for 5-20 min, and then inserting into a fluorescence immunoassay analyzer for detection or observing under ultraviolet light to obtain a detection result in time.
In a specific embodiment of the present invention, the method for using the same comprises the following steps:
1) diluting the collected blood sample and a release agent according to a volume ratio of 1-10: 10;
2) dropping the sample diluted in the step 1) of 30-100 mu L into a sample adding hole of the immunochromatographic kit for rapidly detecting the novel coronavirus N protein, standing at room temperature for 5-20 min, inserting the immunochromatographic kit for rapidly detecting the novel coronavirus N protein into a fluorescence immunoassay analyzer for detection, and immediately obtaining a detection result.
In a specific embodiment of the invention, the immunochromatographic kit for rapidly detecting the novel coronavirus N protein comprises a test strip, wherein the test strip comprises a detection line and a quality control line, the detection line is coated with a monoclonal antibody against the novel coronavirus N protein, and the quality control line is coated with a goat anti-rabbit polyclonal antibody.
In a specific embodiment of the invention, the immunochromatographic kit for rapidly detecting the novel coronavirus N protein comprises a test strip, wherein the test strip comprises a detection line and a quality control line, the detection line is coated with a monoclonal antibody against the novel coronavirus N protein, and the quality control line is coated with a goat anti-rabbit polyclonal antibody;
in a specific scheme of the invention, the test strip further comprises a PVC plate, a sample pad, a marker pad, a coating pad and a water absorption pad which are sequentially connected are fixed on the PVC plate, a detection line and a quality control line are sequentially arranged on the coating pad, and the sample pad and the marker pad are connected into a whole.
In one embodiment of the invention, one end of the coating pad close to the detection line is connected with a marker pad, and one end close to the quality control line is connected with a water absorption pad.
In a specific embodiment of the invention, the marker pad is coated with another monoclonal antibody against the novel coronavirus N protein and rabbit IgG marked by the marker, and the molar ratio of the monoclonal antibody against the novel coronavirus N protein to the rabbit IgG marked by the marker is 1: 0.2-4.
In one embodiment of the present invention, the label is a fluorescent microsphere, colloidal gold, colloidal selenium, colored latex, or magnetic microsphere.
In a specific embodiment of the present invention, the immunochromatographic kit for rapid detection of a novel coronavirus N protein further comprises a cartridge for mounting a test strip.
In one embodiment of the present invention, wherein the card case comprises:
a bottom trough connected to the PVC sheet;
the upper cover is connected with the bottom groove, and a sample adding hole for adding samples to the sample pad is arranged on the upper cover;
and the observation window is arranged on the upper cover and is used for data acquisition of the detection line and the quality control line.
In a specific embodiment of the present invention, the immunochromatographic kit for rapid detection of the N protein of the novel coronavirus is obtained by the following steps:
1) preparation of the coating pad: respectively coating a strain of anti-novel coronavirus N protein antibody and a goat anti-rabbit polyclonal antibody on a nitrocellulose membrane, and drying for later use;
2) preparation of the marker pad: mixing a monoclonal antibody of the novel coronavirus N protein marked by the marker and rabbit IgG marked by the marker, spraying the mixture on a glass cellulose membrane, and drying the mixture for later use;
3) assembling the test strip: bonding a coating pad on a PVC plate, overlapping a water absorption pad at one end close to a quality control line on the coating pad, and overlapping a marker pad and a sample pad connected with the marker pad at one end close to a detection line on the coating pad; then cut into test strips with required width, and then put the test strips into the card shell.
The kit may also be called a test card.
The invention has the beneficial effects that:
1. the immunochromatographic kit for rapidly detecting the novel coronavirus N protein provided by the invention can detect the novel coronavirus nucleocapsid (N) protein by adopting a double-antibody sandwich method, can detect samples such as serum, plasma, whole blood and the like, has high detection speed, can obtain a detection result in 15min, is matched with a fluorescence immunochromatographic instrument for use, is simple to operate, and is suitable for basic medical institutions.
2. The immunochromatographic kit for rapidly detecting the novel coronavirus N protein, provided by the invention, can improve the professional requirements of PCR on instrument operation, personnel, environment and the like, and the defect that a detection antibody cannot be screened in an early stage, can accelerate the screening of first-line suspected cases of epidemic situations, rapidly isolate confirmed persons, and effectively reduce social panic.
3. The immunochromatography kit for rapidly detecting the novel coronavirus N protein provided by the invention can be used for detecting the coronavirus nucleocapsid (N) protein in serum, plasma and whole blood samples so as to be used for diagnosing the novel coronavirus pneumonia.
Drawings
FIG. 1 is a schematic view of an immunochromatographic test strip for rapidly detecting a novel coronavirus N protein provided by the present invention;
FIG. 2A is a schematic diagram of the internal structure of an upper cover of the immunochromatographic assay card for rapid detection of N protein of a novel coronavirus according to the present invention;
FIG. 2B is a schematic diagram of the internal structure of a bottom groove of the immunochromatography detection card for rapidly detecting the N protein of the novel coronavirus provided by the present invention;
the test strip detection device comprises a PVC plate 1, a coating pad 2, a marker pad 3, a water absorption pad 4, a detection line 5, a quality control line 6, a marker joint 7, a sample 8, a sample pad 9, a top cover 11, a bottom groove 12, a sample adding hole 13, an observation window 14, a test strip placement area 15, a positioning column 16, a positioning hole 17, a first limiting part 18, a second limiting part 19 and a third limiting part 20.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The following materials or reagents, unless otherwise specified, are commercially available.
Example 1:
1. preparation of novel coronavirus N protein immunochromatography detection kit
1) Fluorescent microsphere labeling another strain of mouse anti-novel coronavirus N protein monoclonal antibody
Adding 0.5M L fluorescent microspheres into 1mg carbodiimide (EDC) and 1mg N-hydroxysuccinimide (NHS), stirring at room temperature and 120r/min for 3h, adding 100 mu L of another mouse anti-novel coronavirus N protein monoclonal antibody, stirring at room temperature and 120r/min for 1h, adding 10mg BSA confining liquid, stirring at 120r/min for 1h, centrifuging at 2-8 ℃ and 12000 r/min for 20min, removing supernatant, re-dissolving the solid precipitate obtained after centrifugation into 1M L by using 0.2M phosphate buffer (pH 7.4), adding 1 mu L Proclin300, and storing at 4 ℃ for later use.
2) Labeling rabbit IgG polyclonal antibody by fluorescent microsphere
Adding 1mg of carbodiimide (EDC) and 1mg of N-hydroxysuccinimide (NHS) into 0.5M L fluorescent microspheres, stirring at room temperature and 120r/min for 3h, then adding 100 mu L mg of goat rabbit IgG polyclonal antibody, stirring at room temperature and 120r/min for 1h, then adding 10mg of BSA confining liquid, continuing stirring at 120r/min for 1h, centrifuging at 2-8 ℃ and 11000r/min for 30 min, removing supernatant, finally re-dissolving the solid precipitate to 1M L by using 0.2M phosphate buffer (pH 7.4), then adding 1 mu L Proclin300, and storing at 4 ℃ for later use.
3) Preparation of coating pad
A mouse anti-novel coronavirus N protein monoclonal antibody and a goat anti-rabbit polyclonal antibody are respectively diluted to 1mg/M L by 0.2M phosphate buffer solution (pH 7.4), membrane scribing is carried out on a nitrocellulose membrane (NC membrane) by a membrane scribing and gold spraying instrument, the mouse anti-novel coronavirus N protein monoclonal antibody is used as a detection line (T line) 5, the goat anti-rabbit polyclonal antibody is used as a quality control line (C line) 6, and then the coating pad 2 is prepared by drying for 4 hours at 37 ℃ and under the environment of the humidity of less than 30%.
4) Preparation of marker pad
① the glass cellulose membrane was soaked in 0.2M phosphate buffer (pH 7.4) for 6h and then dried at 35 ℃ for 8 h.
② A mouse anti-novel coronavirus N protein monoclonal antibody marked by fluorescent microspheres with a molar ratio of 1:1 and fluorescent microspheres marked by fluorescent microsphere rabbit IgG are uniformly mixed, sprayed on a glass cellulose membrane at a speed of 10 mu L/cm, and then placed at 45 ℃ for drying for 2 hours to prepare a marker pad 3, and a place, which is coated with the mouse anti-novel coronavirus N protein monoclonal antibody marked by the fluorescent microspheres and the fluorescent microsphere rabbit IgG, on the marker pad 3 is called a marker junction 7.
5) Assembly of immunochromatography detection kit
Firstly, a nitrocellulose membrane 2 is bonded on a PVC plate 1, then a water absorption pad 4 is lapped at one end close to a quality control line 6 on the nitrocellulose membrane 2, a marker pad 3 and a sample pad 9 connected with the marker pad are lapped at one end close to a detection line 5 of the nitrocellulose membrane 2, a strip cutter is used for cutting the test strip (shown in figure 1) with the thickness of 4mm +/-0.1 mm, and the test strip is put into a card shell to prepare the novel coronavirus N protein immunochromatography detection kit.
The kit may also be called a test card.
The card housing is selected from the prior art, for example, the card housing (as shown in fig. 2) may include: a bottom tank 12 connected to the PVC plate 1; an upper cover 11 connected to the bottom tank 12, the upper cover 11 being provided with a sample application hole 13 for applying a sample to the sample pad 9; and the observation window 14 is arranged on the upper cover 11 and is used for data acquisition of the detection line 5 and the quality control line 6.
As shown in fig. 2B, the bottom tank 12 includes: a plurality of positioning holes 17 which are symmetrically distributed and positioned on the inner surface of the test strip, wherein a plurality of first limiting parts 18 used for limiting the test strip to move transversely and second limiting parts 19 used for limiting the test strip to move longitudinally are arranged among the plurality of positioning holes 17; the first limiting part 18 and the second limiting part 19 which are symmetrically arranged enclose a paper strip placing area 15 (a dotted line area) for placing the test paper strip;
as shown in fig. 2A, the upper cover 11 includes: a plurality of positioning posts 16 which cooperate with a plurality of said positioning holes 17, so as to cooperate to fix the upper cover 11 and the bottom slot 12 together; the upper cover 11 further includes a third limiting portion 20 for limiting the up and down movement of the test strip.
An observation window 14 for data acquisition is arranged above the coated pad 2 to expose all the detection lines 5 and the quality control lines 6 for collecting detection results; and the observation window 14 is arranged on the upper cover 11 at a position corresponding to the middle part of the test strip placement area 15. The upper cover 11 is provided with a sample adding hole at a position corresponding to the sample pad 9, so that the sample 8 is dripped on the sample pad 9. The distance between the detection line and the sample adding hole is 15-25 mm.
2. Detection of
Taking a sample to be detected with the thickness of 60 mu L, filling a standard substance or the sample to be detected on a marker pad through a sample adding hole, standing for 15min at room temperature, enabling an antigen to be mixed with the marker for reaction, carrying out chromatography along a nitrocellulose membrane, respectively reacting with a test line and a quality control line, inserting the sample into a fluorescence immunochromatography analyzer for detection, automatically calculating a T/C value of the sample by the analyzer, and judging whether the sample is positive or negative through the range of the sample and the normal value.
The ultraviolet light may also be called ultraviolet light, and an ultraviolet lamp may be specifically used.
3. Evaluation of Performance
1) Precision degree
Two samples with the configuration concentration of (300 +/-75) ng/M L and (100 +/-25) ng/M L are respectively detected by using kits of 3 different batches, the detection is repeated for 10 times for each batch, the average value M and the standard deviation SD of 30 times of concentration value measurement results are calculated, and the detection results of the calculated variation coefficient CV. are shown in the following table 1.
TABLE 1
Figure BDA0002397369810000061
Figure BDA0002397369810000071
As can be seen from the data in Table 1, the batch-to-batch variation coefficients are all less than 10.92%, which indicates that the immunochromatography kit for rapidly detecting the novel coronavirus N protein has high precision.
2) Sensitivity of the probe
The polypeptide fragments synthesized by the N protein of the novel coronavirus are configured, the concentrations are 50ng/m L, 100ng/m L and 200ng/m L, and the detection results are shown in the following table 2.
TABLE 2
Figure BDA0002397369810000072
As can be seen from the detection results in Table 2, the immunochromatographic kit for rapidly detecting the N protein of the novel coronavirus meets the requirement of sensitivity.
Example 2:
serum/plasma/whole blood sample testing
The sample types taken include serum/plasma/whole blood.
After the serum/plasma/whole blood sample is collected, the sample to be detected is directly dripped to a sample inlet of a detection card to be kept stand for 15min without sample pretreatment, then the sample is inserted into a fluorescence immunochromatography analyzer for detection, the T/C value of the sample is automatically calculated by the analyzer, and the negative and positive are judged according to the range of the normal value. In addition, when two fluorescence bands appear under the irradiation of ultraviolet light, the fluorescence band is positive. The results of the clinical tests are shown in Table 5 below.
TABLE 5
Figure BDA0002397369810000081
Figure BDA0002397369810000091
As can be seen from the clinical test data in table 5, all of the 20 negative samples were detected as negative and consistent with the clinical diagnosis; all 20 positive samples are detected to be positive, and the total positive coincidence rate is 100%.
The above-mentioned embodiments are merely examples provided to fully illustrate the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (13)

1. The use method of the immunochromatographic kit for rapidly detecting the novel coronavirus N protein is characterized in that a sample detected by the method is a blood sample.
2. The method of use of claim 1, wherein the blood sample is selected from the group consisting of a serum, plasma, or whole blood sample.
3. Use according to claim 1, characterized in that it comprises the following steps: and directly dripping the collected blood sample to a sample adding hole of the kit, standing for 5-20 min, and then inserting into a fluorescence immunoassay analyzer for detection or observing under ultraviolet light to obtain a detection result immediately.
4. Use according to claim 1, characterized in that it comprises the following steps:
1) diluting the collected blood sample and a release agent according to a volume ratio of 1-10: 10;
2) dropping the sample diluted in the step 1) of 30-100 mu L into a sample adding hole of the immunochromatography kit for rapidly detecting the novel coronavirus N protein, standing at room temperature for 5-20 min, inserting the immunochromatography kit for rapidly detecting the novel coronavirus N protein into a fluorescence immunoassay analyzer for detection, and immediately obtaining a detection result.
5. The use method of claim 1, wherein the immunochromatographic kit for rapid detection of the N protein of the novel coronavirus comprises a test strip, the test strip comprises a detection line and a quality control line, the detection line is coated with a monoclonal antibody against the N protein of the novel coronavirus, and the quality control line is coated with a polyclonal antibody against goat rabbit.
6. The use method of claim 1, wherein the immunochromatographic kit for rapid detection of the N protein of the novel coronavirus comprises a test strip, the test strip comprises a detection line and a quality control line, the detection line is coated with a monoclonal antibody against the N protein of the novel coronavirus, and the quality control line is coated with a polyclonal antibody against goat rabbit.
7. The use method of claim 5 or 6, wherein the test strip further comprises a PVC plate, a sample pad, a marker pad, a coating pad and a water absorption pad are fixed on the PVC plate and sequentially connected, a detection line and a quality control line are sequentially arranged on the coating pad, and the sample pad and the marker pad are integrally connected.
8. The use of claim 7, wherein the coated pad is attached to a label pad at the end near the detection line and a bibulous pad at the end near the quality control line.
9. The use of claim 8, wherein the marker pad is coated with another monoclonal antibody against the novel coronavirus N protein and rabbit IgG labeled with a marker at a molar ratio of 1: 0.2-4.
10. The method of use of claim 9, wherein the label is a fluorescent microsphere, colloidal gold, colloidal selenium, colored latex, or a magnetic microsphere.
11. The use of claim 10, wherein the immunochromatographic kit for rapid detection of the N protein of the novel coronavirus further comprises a cartridge for the cartridge test strip.
12. The method of use of claim 11, wherein the card housing comprises:
a bottom trough connected to the PVC sheet;
the upper cover is connected with the bottom groove, and a sample adding hole for adding samples to the sample pad is formed in the upper cover;
and the observation window is arranged on the upper cover and is used for data acquisition of the detection line and the quality control line.
13. The use of claim 12, wherein the immunochromatographic kit for rapid detection of the N protein of the novel coronavirus is obtained by the following steps:
1) preparation of the coating pad: respectively coating a strain of anti-novel coronavirus N protein antibody and a goat anti-rabbit polyclonal antibody on a nitrocellulose membrane, and drying for later use;
2) preparation of the marker pad: mixing a monoclonal antibody of the novel coronavirus N protein marked by the marker and rabbit IgG marked by the marker, spraying the mixture on a glass cellulose membrane, and drying the mixture for later use;
3) assembling the test strip: bonding a coating pad on a PVC plate, overlapping a water absorption pad at one end close to a quality control line on the coating pad, and overlapping a marker pad and a sample pad connected with the marker pad at one end close to a detection line on the coating pad; then cut into test strips with required width, and then put the test strips into the card shell.
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