CN111443196A - New coronavirus antibody detection card and manufacturing method thereof - Google Patents
New coronavirus antibody detection card and manufacturing method thereof Download PDFInfo
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Abstract
The invention discloses a new coronavirus antibody detection card and a manufacturing method thereof, wherein the detection card comprises a test strip packaged in a shell, and the shell comprises an observation hole and a sampling hole; the test strip comprises a bottom plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption membrane; the gold label pad is sprayed with a colloidal gold-SPA solution and a colloidal gold-mouse anti-human IgG solution; the nitrocellulose membrane is provided with detection lines and quality control lines at intervals in parallel, the detection lines are coated with novel coronavirus S protein and N protein, and the quality control lines are coated with rabbit anti-sheep secondary antibody. The method has the advantages of short detection time, good stability, simple operation, no need of other instruments and professional operators, intuitive and reliable result judgment, and suitability for detection of large-batch samples of basic medical institutions and disease control departments.
Description
Technical Field
The invention relates to the technical field of new coronavirus antibodies, in particular to a new coronavirus antibody detection card and a manufacturing method thereof.
Background
The novel coronavirus pneumonia is pneumonia caused by novel coronavirus (COVID-19), and some severe patients can have respiratory distress syndrome or sepsis shock and even die. Patients infected with the new coronavirus (COVID-19) have symptoms of fever, hypodynamia, dry cough, gradual dyspnea and the like in the early stage. Has strong infectivity. Can cause major public health safety incidents. At present, no specific medicine aiming at virus modification exists for treatment.
The real-time quantitative fluorescent PCR nucleic acid detection reagent applied in the prior clinic plays an important role in clinical diagnosis and suspected patient investigation. However, this technique is cumbersome to operate, requires specialized operators, takes a long time for testing, and requires centralized sample presentation. The detection requirements of a large number of suspected patients and asymptomatic infectors which grow rapidly cannot be met.
The colloidal gold immunochromatographic assay is a mature immunological detection technology, and is widely applied to clinical detection means at present, and is also widely applied to detection of agricultural and veterinary drugs and disease diagnosis of pets and animals in the field of food safety. The technology is simple and convenient to operate, the time consumed in the whole detection process is short, the interpretation result is visual and intuitive by naked eyes, and detection equipment and more professional detection personnel are not needed. The novel coronavirus IgG/IgM duplex colloidal gold antibody detection card can be used for simultaneously detecting whether a blood sample contains the novel coronavirus IgG/IgM antibody after one-time sampling and sample adding, and can be used as an important reference of a clinician. Can play an important role in the investigation of a large number of suspected patients and asymptomatic infected persons which are rapidly increased at present.
Disclosure of Invention
The invention provides a new coronavirus antibody detection card and a manufacturing method thereof, aiming at solving the problems.
According to a first aspect of embodiments of the present application, there is provided a novel coronavirus antibody detection card comprising: the test strip comprises a test strip encapsulated in a shell, wherein the shell comprises an observation hole and a sample adding hole; the test strip comprises a bottom plate, a sample pad, a gold label pad, a nitrocellulose membrane, a water absorption membrane and a blood filtration membrane; the gold label pad is sprayed with a colloidal gold-SPA solution and a colloidal gold-mouse anti-human IgG solution; the nitrocellulose membrane is provided with detection lines and quality control lines at intervals in parallel, the detection lines are coated with novel coronavirus S protein and N protein, and the quality control lines are coated with rabbit anti-sheep secondary antibody.
According to a second aspect of the embodiments of the present application, there is provided a method for manufacturing a new coronavirus antibody detection card, comprising: preparing a colloidal gold solution;
preparing a colloidal gold-SPA solution;
preparing a colloidal gold-mouse anti-human IgG solution;
preparing a colloidal gold-mouse anti-human IgM solution;
preparing a detection line and a quality control line;
preparing a gold label pad;
preparing a sample pad;
and assembling the detection card.
The technical scheme provided by the embodiment of the application can have the following beneficial effects: the application designs a new coronavirus antibody detection card and a manufacturing method thereof, the detection card can be used as an important reference of a clinician, can be used for checking infected persons, has the advantages of short detection time, good stability, simple operation, no need of other instruments and personnel for professional operation, and intuitive and reliable result judgment, and is suitable for detection of large-batch samples of primary medical institutions and disease control departments.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a schematic side view of a test strip according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a two-card connector of the detection card according to the embodiment of the present invention;
FIG. 3 is an IgG/IgM antibody negative pattern of the detection card of the embodiment of the present invention;
FIG. 4 is a graph showing IgG antibody positivity and IgM antibody negativity of the detection card according to the example of the present invention;
FIG. 5 is a graph showing that the test card of the example of the present invention is IgG antibody negative and IgM antibody positive;
FIG. 6 is a graph showing IgG antibody positivity and IgM antibody positivity of the test card according to the example of the present invention;
FIG. 7 is a diagram of an invalid card for a test card according to an embodiment of the present invention;
FIG. 8 is a schematic flow chart of the manufacture of a new coronavirus antibody detection card according to an embodiment of the present invention;
FIG. 9 is a schematic flow chart of the production of a colloidal gold solution according to an embodiment of the present invention;
FIG. 10 is a schematic flow chart of the fabrication of a colloidal gold-SPA solution in accordance with an embodiment of the present invention;
FIG. 11 is a schematic flow chart of the preparation of a colloidal gold-mouse anti-human IgG solution according to an embodiment of the present invention;
FIG. 12 is a schematic flow chart of the preparation of colloidal gold-mouse anti-human IgM solution according to an embodiment of the present invention;
FIG. 13 is a schematic flow chart of the manufacturing of the inspection line and the quality control line according to the embodiment of the present invention;
fig. 14 is a schematic flow chart of manufacturing a gold pad according to an embodiment of the invention.
Description of reference numerals:
100. a test strip; 10. a base plate; 20. a sample pad; 30. a gold label pad; 40. a nitrocellulose membrane; 41. detecting lines; 42. a quality control line; 50. a water-absorbing film; 60. a blood filtration membrane.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without any inventive step, are within the scope of the present invention.
It is also to be understood that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
It should be further understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.
Referring to fig. 1-7, the present invention discloses a new coronavirus antibody detection card, wherein the new coronavirus antibody detection card comprises a test strip 100 enclosed in a casing (not shown in the figure), the casing is provided with an observation hole and a sample adding hole, and a user can add a blood sample to be detected from the sample adding hole. The test strip 100 comprises a bottom plate 10, a sample pad 20, a gold-labeled pad 30, a nitrocellulose membrane 40, a water absorption membrane 50 and a blood filtration membrane 60, wherein the blood filtration membrane, the sample pad, the gold-labeled pad, the nitrocellulose membrane and the water absorption membrane are sequentially arranged on the bottom plate. The gold label pad is sprayed with colloidal gold-SPA (staphylococcus Protein A) solution and colloidal gold-mouse anti-human IgG solution; a detection line 41 and a quality control line 42 are arranged on the nitrocellulose membrane at intervals in parallel, the detection line is coated with novel coronavirus S protein and N protein, and the quality control line is coated with rabbit anti-sheep secondary antibody. Whether the new coronavirus antibody detection card of this application contains novel coronavirus IgG/IgM antibody in can detecting the blood sample simultaneously, it can regard as clinician's important reference to can be used for investigating the infector, it is short, stable good to have a check-out time, easy operation, need not the personnel of other instruments and professional operation, and the result is judged directly perceived reliably, is suitable for the detection of basic level medical institution and the big sample of the big batch of disease control department. A novel coronavirus (COVID-19) S protein and an N protein expressed by genetic engineering are coated on a detection line at the same time, so that the binding capacity of a target antibody is improved; in addition, the gold-labeled pad is sprayed with colloidal gold-mouse anti-human IgG solution and colloidal gold-SPA (staphylococcus Protein A) solution, so that the capture capacity of the antibody in the sample is improved.
During operation, only 10ul of whole blood or serum and plasma samples are needed and added into the diluent to be fully and uniformly mixed. Then 80ul of the well mixed sample is added into the sample loading air. The test card is laid flat for 10 minutes and the result is interpreted. Referring to fig. 2-7, in reading the result, the antibody is determined to be positive when red bands appear on both the detection line T and the quality control line C, and the antibody is determined to be negative when only the red band appears on the quality control line C. And when no red strip exists at the position of the quality control line C and the detection line T or no red strip exists at the position of the quality control line C, the detection card fails.
Referring to fig. 8, the present invention further provides a method for manufacturing a new coronavirus antibody detection card, which comprises steps S101-S108.
S101, preparing a colloidal gold solution.
S102, preparing a colloidal gold-SPA solution.
S103, preparing a colloidal gold-mouse anti-human IgG solution.
S104, preparing a colloidal gold-mouse anti-human IgM solution.
And S105, preparing a detection line and a quality control line.
S106, preparing a gold-labeled pad.
S107, preparing a sample pad.
And S108, assembling the detection card.
Referring to fig. 9, in an alternative embodiment, the preparation of the gold colloid solution includes the following steps.
S1011, adding 100ml of ultrapure water into a 300ml triangular flask, and placing the triangular flask on a constant-temperature heating magnetic stirrer for heating.
S1011, boiling ultrapure water, and then adding 1ml of 2% chloroauric acid solution.
S1012, after being heated by ultrapure water and boiled again, 1.5ml of 2% trisodium citrate dihydrate solution is rapidly added.
And S1013, continuously heating the ultrapure water to boiling, and stopping heating after 15 min.
And S1014, returning to room temperature, adding ultrapure water, and returning to the original volume.
S1014, storing at 4 ℃ for later use.
Referring to FIG. 10, the preparation of the gold colloid-SPA solution includes steps S1021-S1025.
And S1021, under the stirring of a magnetic stirrer, using 0.2 mol/L of potassium carbonate to adjust the pH value of the colloidal gold solution to 6.0.
S1022, adding 16 micrograms of SPA into each milliliter of colloidal gold solution, stirring at room temperature for 60min, and adding 10% BSA solution until the final concentration is 1%.
And S1023, continuously stirring at room temperature for 60min, centrifuging at the rotation speed of 5000r/min for 10min at the temperature of 4 ℃ after stirring is stopped, and collecting the sediment at the bottom of the centrifuge tube.
S1024, centrifuging the upper layer colloidal gold solution at the rotating speed of 8500r/min for 20min in the environment of 4 ℃, collecting the sediment at the bottom of the centrifugal tube, and discarding the supernatant.
And S1025, collecting the colloidal gold precipitate twice, redissolving 1/25 of the volume of the colloidal gold solution when the colloidal gold precipitate is marked by the redissolution, and storing the colloidal gold solution for later use at the temperature of 4 ℃.
Referring to FIG. 11, a colloidal gold-mouse anti-human IgG solution is prepared, including steps S1031-S1035.
And S1031, adjusting the pH of the colloidal gold solution to 8.0 by using 0.2 mol/L of potassium carbonate under the stirring of a magnetic stirrer.
S1032, adding 16 micrograms of mouse anti-human IgG monoclonal antibody into each milliliter of colloidal gold solution.
S1033, stirring at room temperature for 60min, adding 10% BSA solution to a final concentration of 1%, and continuing stirring at room temperature for 60 min.
S1034, after stirring is stopped, centrifuging at the rotating speed of 5000r/min for 10min in the environment of 4 ℃, collecting the sediment at the bottom of the centrifugal tube, centrifuging the upper-layer colloidal gold solution at the rotating speed of 8500r/min for 20min in the environment of 4 ℃, collecting the sediment at the bottom of the centrifugal tube, and discarding the supernatant.
And S1035, redissolving the colloidal gold precipitate collected twice by using redissolving solution according to 1/25 of the volume of the colloidal gold solution during marking, and storing at 4 ℃ for later use.
Referring to FIG. 12, the preparation of colloidal gold-mouse anti-human IgM solution includes steps S1041-S1045.
S1041, adjusting the pH of the colloidal gold solution to 8.0 by 0.2 mol/L of potassium carbonate under the stirring of a magnetic stirrer.
S1042, adding 16 microgram of mouse anti-human IgM monoclonal antibody into each milliliter of colloidal gold solution.
S1043, stirring for 60min at room temperature, adding 10% BSA solution to a final concentration of 1%, and continuing stirring for 60min at room temperature.
S1044, after stirring is stopped, centrifuging at the rotating speed of 5000r/min for 10min in the environment of 4 ℃, collecting the sediment at the bottom of the centrifugal tube, centrifuging at the rotating speed of 8500r/min for 20min at the temperature of 4 ℃ for the upper layer of the colloidal gold solution, collecting the sediment at the bottom of the centrifugal tube, and discarding the supernatant.
S1045, re-dissolving the twice collected colloidal gold precipitate with a re-dissolving solution according to 1/25 of the volume of the colloidal gold solution when the colloidal gold precipitate is marked, and storing the colloidal gold precipitate for later use in an environment at 4 ℃.
Wherein the above complex solution is 20mM PB buffer solution with pH 7.4, wherein BSA content is 1%, Tween-20 is 0.5%, PVP-40 is 1.5%, trehalose is 2%, and sucrose is 5%.
Referring to FIG. 13, the preparation of the detection line and the quality control line includes steps S1051-S1052.
S1051, detecting the concentrations of the S protein and the N protein of the novel coronavirus in the line coating liquid to be 0.5 mg/ml; coated on a nitrocellulose membrane with a striper according to the parameters of 1 ul/cm.
S1052, coating the goat anti-mouse secondary antibody contained in the quality control line coating solution with the concentration of 1mg/ml on a nitrocellulose membrane by using a membrane scribing instrument according to the parameter of 1 ul/cm; the coating solution was 0.01M PBS phosphate buffer containing 2% sucrose.
Referring to fig. 14, in an alternative embodiment, the preparation of the gold pad includes the following steps.
S1061, drying the glass fiber soaked by the gold-labeled pad treatment solution in a blast drying box at 37 ℃, sealing, drying and storing the dried gold pad for later use, wherein the gold-labeled pad treatment solution comprises 0.02M Ph8.5 Tris-HC L buffer solution containing 1% BSA, 0.5% PVP-40, 1.5% sucrose and 0.3% Tween-20.
S1062, mixing the prepared colloidal gold-SPA solution and the colloidal gold-mouse anti-human IgG according to a ratio of 1:1, and spraying the mixture on the treated glass fiber by using a gold spraying instrument according to gold spraying parameters of 2.0 ul/cm.
S1063, spraying the prepared colloidal gold-mouse anti-human IgM solution on the treated glass fiber by using a gold spraying instrument according to the gold spraying parameter of 2.0ul/cm, drying the sprayed gold pad in a blast drying oven at 37 ℃, and drying, sealing and storing the dried gold pad.
And the preparation of the sample pad comprises the steps of drying the glass fiber soaked by the sample pad treatment solution in a 37 ℃ air blowing drying box, sealing, drying and storing the dried sample pad for later use.
The assembly of the detection card comprises the step of sequentially sticking the prepared gold pad, the sample pad and the blood filtering membrane to the PVC bottom plate in the environment with the humidity lower than 30 percent. Cutting into strips according to the parameter of 4 mm/strip by a strip cutting machine, and arranging in corresponding detection cards.
The above description is only for the specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive various equivalent modifications or substitutions within the technical scope of the present invention, and these modifications or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. The new coronavirus antibody detection card is characterized by comprising a test strip packaged in a shell, wherein the shell comprises a viewing hole and a sampling hole; the test strip comprises a bottom plate, a sample pad, a gold label pad, a nitrocellulose membrane, a water absorption membrane and a blood filtration membrane; the gold label pad is sprayed with a colloidal gold-SPA solution and a colloidal gold-mouse anti-human IgG solution; the nitrocellulose membrane is provided with detection lines and quality control lines at intervals in parallel, the detection lines are coated with novel coronavirus S protein and N protein, and the quality control lines are coated with rabbit anti-sheep secondary antibody.
2. A method for manufacturing a new coronavirus antibody detection card is characterized by comprising the following steps:
preparing a colloidal gold solution;
preparing a colloidal gold-SPA solution;
preparing a colloidal gold-mouse anti-human IgG solution;
preparing a colloidal gold-mouse anti-human IgM solution;
preparing a detection line and a quality control line;
preparing a gold label pad;
preparing a sample pad;
and assembling the detection card.
3. The method for preparing the new coronavirus antibody detection card of claim 2, wherein the preparation of the colloidal gold solution comprises the following steps:
adding 100ml of ultrapure water into a 300ml triangular flask, and placing the triangular flask on a constant-temperature heating magnetic stirrer for heating;
after the ultrapure water is boiled, 1ml of 2% chloroauric acid solution is added;
after the ultrapure water is heated and boiled again, 1.5ml of 2% trisodium citrate dihydrate solution is rapidly added;
continuously heating the ultrapure water to boil, and stopping heating after 15 min;
returning to room temperature, adding ultrapure water to return to the original volume, and storing at 4 ℃ for later use.
4. The method for preparing the novel coronavirus antibody detection card of claim 3, wherein the preparation of the colloidal gold-SPA solution comprises the following steps:
the pH of the colloidal gold solution is adjusted to 6.0 by 0.2 mol/L of potassium carbonate under the stirring of a magnetic stirrer;
adding 16 micrograms of SPA into each milliliter of colloidal gold solution, stirring at room temperature for 60min, and adding 10 percent BSA solution to a final concentration of 1 percent;
continuously stirring at room temperature for 60min, centrifuging at the rotation speed of 5000r/min for 10min at 4 deg.C after stirring is stopped, and collecting the precipitate at the bottom of the centrifuge tube;
centrifuging the upper layer colloidal gold solution at the rotating speed of 8500r/min for 20min at the temperature of 4 ℃, collecting the sediment at the bottom of a centrifuge tube, and discarding the supernatant;
and (3) carrying out redissolving on the collected colloidal gold precipitates twice by 1/25 of the volume of the colloidal gold solution when the redissolving solution is used for marking, and storing the colloidal gold solution for later use at the temperature of 4 ℃.
5. The method of claim 4, wherein the complex solution is 20mM PB buffer solution with pH 7.4, wherein BSA content is 1%, Tween-20 is 0.5%, PVP-40 is 1.5%, trehalose is 2%, and sucrose is 5%.
6. The method for preparing the new coronavirus antibody detection card of claim 2, wherein the preparation of the colloidal gold-mouse anti-human IgG solution comprises:
the pH of the colloidal gold solution is adjusted to 8.0 by 0.2 mol/L of potassium carbonate under the stirring of a magnetic stirrer;
adding 16 micrograms of mouse anti-human IgG monoclonal antibody into each milliliter of colloidal gold solution;
stirring at room temperature for 60min, adding 10% BSA solution to a final concentration of 1%, and stirring at room temperature for 60 min;
after stirring is stopped, centrifuging for 10min at the rotating speed of 5000r/min in the environment of 4 ℃, collecting the bottom sediment of the centrifuge tube, centrifuging the upper-layer colloidal gold solution for 20min at the rotating speed of 8500r/min in the environment of 4 ℃, collecting the bottom sediment of the centrifuge tube, and discarding the supernatant;
and (3) redissolving the twice collected colloidal gold precipitates by using redissolving solution according to 1/25 of the volume of the colloidal gold solution during marking, and storing the colloidal gold precipitates for later use at the temperature of 4 ℃.
7. The method for preparing the new coronavirus antibody detection card of claim 2, wherein the preparation of the colloidal gold-mouse anti-human IgM solution comprises:
the pH of the colloidal gold solution is adjusted to 8.0 by 0.2 mol/L of potassium carbonate under the stirring of a magnetic stirrer;
adding 16 micrograms of mouse anti-human IgM monoclonal antibody into each milliliter of colloidal gold solution;
stirring at room temperature for 60min, adding 10% BSA solution to a final concentration of 1%, and stirring at room temperature for 60 min;
after stirring is stopped, centrifuging for 10min at the rotating speed of 5000r/min in the environment of 4 ℃, collecting the bottom sediment of the centrifuge tube, centrifuging for 20min at the rotating speed of 8500r/min at the temperature of 4 ℃ for the upper layer of the colloidal gold solution, collecting the bottom sediment of the centrifuge tube, and discarding the supernatant;
the twice collected colloidal gold precipitates were redissolved with a redissolution according to 1/25 of the volume of the colloidal gold solution at the time of labeling, and stored at 4 ℃ for further use.
8. The method for manufacturing the new coronavirus antibody detection card of claim 2, wherein the preparation of the detection line and the quality control line comprises the following steps:
the concentrations of the S protein and the N protein of the novel coronavirus in the detection line coating liquid are both 0.5 mg/ml; coating on a nitrocellulose membrane by a membrane scratching instrument according to the parameter of 1 ul/cm;
the quality control line coating solution contains goat anti-mouse secondary antibody with the concentration of 1mg/ml, and is coated on the nitrocellulose membrane by a membrane scribing instrument according to the parameter of 1 ul/cm; the coating solution was 0.01M PBS phosphate buffer containing 2% sucrose.
9. The method for preparing the new coronavirus antibody detection card of claim 8, wherein the preparation of the gold pad comprises the following steps:
drying the glass fiber soaked by the gold label pad treatment solution in a 37 ℃ blast drying oven, sealing and drying the dried gold pad for later use;
mixing the prepared colloidal gold-SPA solution and the colloidal gold-mouse anti-human IgG according to the proportion of 1:1, and spraying the mixture on the treated glass fiber by using a gold spraying instrument according to the gold spraying parameter of 2.0 ul/cm;
and spraying the prepared colloidal gold-mouse anti-human IgM solution on the treated glass fiber by using a gold spraying instrument according to the gold spraying parameter of 2.0ul/cm, drying the sprayed gold pad in a blast drying oven at 37 ℃, and drying, sealing and storing the dried gold pad.
10. The method for preparing a new coronavirus antibody detection card according to claim 2, wherein the preparation of the sample pad comprises:
and (3) drying the glass fiber soaked by the sample pad treatment solution in a 37 ℃ blast drying oven, and sealing, drying and storing the dried sample pad for later use.
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