CN112798797A - Novel coronavirus neutralizing antibody detection test paper - Google Patents
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Abstract
The invention relates to the field of biomedical detection, in particular to novel coronavirus neutralizing antibody detection test paper, a kit, a detection method and application, wherein the detection method comprises the following steps: the sample loading pad, the colloidal gold adsorption pad, the antibody bearing film and the water absorption pad are sequentially overlapped and lapped on the substrate; the antibody bearing film is provided with a detection line T1, a detection line T2 and a quality control line C which are spaced, the detection line T2 is close to the colloidal gold adsorption pad, and the quality control line C is close to the water absorption pad; the colloidal gold adsorption pad is coated with a colloidal gold labeled novel coronavirus S-RBD antigen and a colloidal gold labeled antibody irrelevant to the novel coronavirus, and a detection line T1 is coated with a secondary antibody of the novel coronavirus antibody IgG; the detection line T2 coats angiotensin converting enzyme 2; the quality control line C is coated with a secondary antibody which is specifically combined with the antibody unrelated to the new coronavirus marked by the colloidal gold; the detection test paper can detect the novel coronavirus neutralizing antibody, is simple to operate, short in time, high in specificity and high in sensitivity during detection.
Description
Technical Field
The invention relates to the field of biomedical detection, in particular to novel coronavirus neutralizing antibody detection test paper, a kit, a detection method and application.
Background
With the global development of the global anti-new coronavirus epidemic situation into the persistent stage, except for the new coronavirus vaccine in the clinical test stage, the neutralizing antibody capable of effectively blocking the new coronavirus is discovered in succession, and the vaccine is used as a candidate therapy and brings new hope to the academia.
The antibody is a protein with immune effect produced by stimulating B lymphocyte by antigen in human body, and is mainly present in blood and tissue. Antibodies have a Y-shaped structure, which can help them capture antigen accurately. After the microbe invades human body, it stimulates the body to produce several antibodies, only part of which can recognize the microbe and capture it before it invades cells, protecting the human body from infection. This process is called neutralization and the antibody that exerts its effect is the neutralizing antibody. In the course of combating new coronaviruses, neutralizing antibody therapy is the artificial replenishment of antibodies to support the immune system from infection by new coronaviruses.
Currently, there are three main types of neutralizing antibodies. The first is serotherapy, namely, after a person is infected with new coronavirus, an IgG antibody is generated, and the reason is that the serum of a convalescent patient with new coronary pneumonia is extracted to be used for treating other patients with severe pneumonia. This method is the fastest to apply, but carries some risk. The second is monoclonal antibody, which is an antibody screened and prepared artificially, has single component and strong specificity and is also called biological missile. The monoclonal antibody can accurately identify a certain antigen part of the new coronavirus, and generates a high-efficiency antiviral effect. The last one is a genetic engineering antibody, which modifies the amino acid sequence of the antibody and has stronger curative effect. The neutralizing antibodies are distinguished from vaccines in that they aim to train the human immune system to master the process of destroying new corona viruses, and to effectively eliminate them until the virus invades, putting emphasis on prevention. The neutralizing antibody medicine mainly kills the new coronavirus accurately and efficiently, has quick response and has considerable treatment effect.
The detection of the neutralizing antibody can determine whether the patient can safely return to the work position or participate in more social activities, whether the recovered patient or the asymptomatic infected patient has the risk of re-infection at the present stage is confirmed, and the neutralizing antibody can be widely applied along with the successful development of the vaccine at the later stage and can be used as an evaluation index of the vaccine effect. The existing detection method aiming at the new crown neutralizing antibody mainly comprises detection means such as enzyme-linked immunosorbent assay, chemiluminescence assay and the like, and corresponding kits are available on the market for the two methods. However, the above-mentioned method is complicated and time-consuming in detection operation, and has low specificity and poor sensitivity in detection, and the concentration level of the neutralizing antibody cannot be determined.
Disclosure of Invention
Therefore, the technical problems to be solved by the invention are that the existing detection operation of the novel coronavirus neutralizing antibody is complex and time-consuming, the specificity is low and the sensitivity is poor during detection, and the concentration level of the neutralizing antibody cannot be judged, so that the novel coronavirus neutralizing antibody detection test paper, the kit, the detection method and the application are provided.
Therefore, the invention provides the following technical scheme:
a novel coronavirus neutralizing antibody detection test paper comprises: the sample loading device comprises a substrate and a sample loading pad, a colloidal gold adsorption pad, an antibody bearing film and a water absorption pad which are sequentially overlapped and lapped on the substrate; the antibody bearing film is provided with a detection line T1, a detection line T2 and a quality control line C which are spaced, the detection line T2 is close to the colloidal gold adsorption pad, and the quality control line C is close to the water absorption pad;
the colloidal gold adsorption pad is coated with a receptor binding domain RBD antigen of the colloidal gold labeled novel coronavirus S protein and a colloidal gold labeled antibody unrelated to the novel coronavirus;
the detection line T1 is coated with a secondary antibody of a novel coronavirus antibody IgG;
the detection line T2 is coated with angiotensin converting enzyme 2;
the control line C coats a secondary antibody that specifically binds to an antibody unrelated to the colloidal gold-labeled neocoronavirus.
Optionally, the second antibody of the novel coronavirus antibody IgG is a mouse anti-human IgG monoclonal antibody; or
The colloidal gold labeled antibody irrelevant to the new coronavirus is a rabbit IgG antibody; or
The secondary antibody specifically combined with the new coronavirus irrelevant antibody marked by the colloidal gold is goat anti-rabbit IgG polyclonal antibody.
The invention provides a preparation method of the novel coronavirus neutralizing antibody detection test paper, which comprises the following steps:
sequentially overlapping the sample loading pad, the colloidal gold adsorption pad, the antibody bearing film and the water absorption pad on the substrate;
coating a receptor binding domain RBD antigen of the colloidal gold-labeled novel coronavirus S protein and a colloidal gold-labeled antibody unrelated to the novel coronavirus on the colloidal gold adsorption pad;
a secondary antibody of a novel coronavirus antibody IgG, angiotensin converting enzyme 2 and a secondary antibody specifically combined with the antibody irrelevant to the novel coronavirus labeled by colloidal gold are respectively coated on a detection line T1, a detection line T2 and a quality control line C on an antibody bearing film.
Optionally, the second antibody of the novel coronavirus antibody IgG is a mouse anti-human IgG monoclonal antibody; or
The colloidal gold labeled antibody irrelevant to the new coronavirus is a rabbit IgG antibody; or
The secondary antibody specifically combined with the new coronavirus irrelevant antibody marked by the colloidal gold is goat anti-rabbit IgG polyclonal antibody.
Optionally, the coating concentration of the goat anti-rabbit IgG polyclonal antibody is: 2.0-2.5 mg/ml; or
The coating concentration of the mouse anti-human IgG monoclonal antibody is as follows: 0.5-0.6 mg/ml; or
Colloidal gold labeling concentration of receptor binding domain RBD antigen of novel coronavirus S protein: 4-5 mug/ml; or
Colloidal gold labeling concentration of rabbit IgG: 4-5 μ g/ml.
The invention provides a novel coronavirus neutralizing antibody detection device, which comprises a shell, and novel coronavirus neutralizing antibody detection test paper arranged in the shell or novel coronavirus neutralizing antibody detection test paper prepared by the preparation method;
along the chromatography direction of the novel coronavirus neutralizing antibody detection test paper, a sample hole is formed in the position, corresponding to the sample pad, of the shell, and observation ports are formed in the positions of the detection line T2, the detection line T1 and the quality control line C.
The invention provides a novel coronavirus neutralizing antibody detection kit, which comprises the novel coronavirus neutralizing antibody detection test paper, the novel coronavirus neutralizing antibody detection test paper prepared by the preparation method or the novel coronavirus neutralizing antibody detection device.
The invention relates to a novel test paper for detecting coronavirus neutralizing antibodies, the novel test paper for detecting coronavirus neutralizing antibodies prepared by the preparation method, or application of the novel test paper for detecting coronavirus neutralizing antibodies in preparation of products for detecting the novel coronavirus neutralizing antibodies.
The invention provides a detection method for non-disease diagnosis of a novel coronavirus neutralizing antibody, which is a novel coronavirus neutralizing antibody detection test paper or a novel coronavirus neutralizing antibody detection device prepared by the preparation method.
Optionally, adding a sample to a sample loading pad of the detection test paper, and then adding a diluent to the sample loading pad, wherein the detection test paper is marked as test paper A; meanwhile, adding a diluent to another test paper as a negative control, wherein the test paper is marked as test paper B; observing the test paper A and the test paper B after 15 minutes, and obtaining an invalid result after 20 minutes;
optionally, when a mauve strip appears at the position of the quality control line C of the test paper A and the detection line T2, the mauve strip does not appear in the detection line T1, the color development result of the test paper A is consistent with that of the test paper B, and the detection result is negative;
when the test paper A has a light purple red strip at the detection line T1 and a purple red strip at the detection line T2, the detection line T2 has a darker color than the detection line T1, the test paper A detection line T2 has a lighter color than the test paper B detection line T2, and the detection result shows that the sample contains the novel coronavirus neutralizing antibody with low titer;
when the test paper A has light purple red strips at the detection lines T1 and T2, the color development depths of the detection lines T1 and T2 are equivalent, the color development of the test paper A detection line T2 is lighter than that of the test paper B detection line T2, and the detection result shows that the sample contains the titer of the novel coronavirus neutralizing antibody;
when the A test paper has a purple red strip at a T1 detection line and does not have a purple red strip at a T2 detection line, the detection result shows that the sample contains the novel coronavirus neutralizing antibody with high titer;
and if the quality control line C of the test paper A and the test paper B does not have a purple red strip, indicating that the operation process is incorrect or fails.
The technical scheme of the invention has the following advantages:
1. the novel coronavirus neutralizing antibody detection test paper provided by the invention comprises a substrate, and a sample loading pad, a colloidal gold adsorption pad, an antibody bearing film and a water absorption pad which are sequentially overlapped and lapped on the substrate; the antibody bearing film is provided with a detection line T1, a detection line T2 and a quality control line C which are spaced, the detection line T2 is close to the colloidal gold adsorption pad, and the quality control line C is close to the water absorption pad; the colloidal gold adsorption pad is coated with a receptor binding domain RBD antigen of the colloidal gold labeled novel coronavirus S protein and a colloidal gold labeled antibody unrelated to the novel coronavirus; the detection line T1 is coated with a secondary antibody of a novel coronavirus antibody IgG; the detection line T2 is coated with angiotensin converting enzyme 2; the quality control line C is coated with a secondary antibody which is specifically combined with the antibody unrelated to the new coronavirus marked by the colloidal gold; in the detection test paper, a colloidal gold pad is pre-coated with a receptor binding domain (S-RBD) antigen of a novel coronavirus S protein, mouse anti-human IgG monoclonal antibody, angiotensin converting enzyme 2(ACE-2) and goat anti-rabbit IgG antibody are respectively coated at nitrocellulose membrane detection lines T1, T2 and a quality control line, a T1 detection line applies a capture method, a T2 detection line applies a principle of a competitive inhibition method, a sample neutralization antibody can be qualitatively detected, and the concentration level of the neutralization antibody in the sample can be detected at the same time, wherein the principle is as follows:
when detecting positive samples, the novel coronavirus neutralizing antibody in the samples is combined with the receptor binding domain (S-RBD) antigen of the novel coronavirus S protein marked by the colloidal gold to form a complex, and the complex moves forwards along the paper strip due to chromatography:
when a mauve strip appears on the test paper quality control line C and the detection line T2, the mauve strip does not appear on the detection line T1, the color development result of the test paper A is consistent with that of the test paper B, and the detection result is negative;
when the test paper A has a light purple red strip at the detection line T1 and a purple red strip at the detection line T2, the detection line T2 has a darker color than the detection line T1, the test paper A detection line T2 has a lighter color than the test paper B detection line T2, and the detection result shows that the sample contains the novel coronavirus neutralizing antibody with low titer;
when the test paper A has light purple red strips at the detection lines T1 and T2, the color development depths of the detection lines T1 and T2 are equivalent, the color development of the test paper A detection line T2 is lighter than that of the test paper B detection line T2, and the detection result shows that the sample contains the titer of the novel coronavirus neutralizing antibody;
when the A test paper has a purple red strip at a T1 detection line and does not have a purple red strip at a T2 detection line, the detection result shows that the sample contains the novel coronavirus neutralizing antibody with high titer;
and if the quality control line C of the test paper A and the test paper B does not have a purple red strip, indicating that the operation process is incorrect or fails.
Furthermore, the detection test paper also has the advantages of simple detection operation, short time, high specificity and high sensitivity during detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic view of a novel test strip A for detecting coronavirus neutralizing antibodies in example 1 of the present invention;
FIG. 2 is a schematic view of a detection apparatus for neutralizing antibodies against the novel coronavirus in example 1 of the present invention;
FIG. 3 shows a color chart according to example 1 of the present invention.
Reference numerals:
1-substrate, 2-loading pad, 3-colloidal gold adsorption pad, 4-antibody bearing membrane, 5-water absorption pad, 6-protective membrane, 7-shell, 8-sample hole, 9-observation port, T1-detection line T1, T2-detection line T2 and C-quality control line C.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Goat anti-rabbit IgG polyclonal antibody is purchased from Hangzhou Zhengzhi Biotechnology, Inc., mouse anti-human IgG monoclonal antibody is purchased from Hangzhou Zhengzhi Biotechnology, Inc., angiotensin converting enzyme 2 is purchased from Catalog # hACE2-hFc of Anyuan pharmaceutical technology (Shanghai), Inc., and receptor binding domain RBD antigen (S-RBD) of novel coronavirus S protein is purchased from Catalog # SARS-CoV-2RBD of Anyuan pharmaceutical technology (Shanghai), Inc
EXAMPLE 1 novel coronavirus neutralizing antibody detection test paper
The embodiment provides a novel coronavirus neutralizing antibody detection test paper, which comprises:
as shown in fig. 1, the sample loading device comprises a substrate 1, and a sample loading pad 2, a colloidal gold adsorption pad 3, an antibody bearing film 4 and a water absorption pad 5 which are sequentially overlapped and lapped on the substrate 1; the antibody bearing film 4 is provided with a detection line T1, a detection line T2 and a quality control line C which are arranged at intervals, the detection line T2 is close to the colloidal gold adsorption pad 3, and the quality control line C is close to the water absorption pad 5. The colloidal gold adsorbs 3 a receptor binding domain RBD antigen of the novel coronavirus S protein coated with the colloidal gold label and a colloidal gold-labeled antibody unrelated to the novel coronavirus, wherein the antibody unrelated to the novel coronavirus is selected to be rabbit IgG. The test line T1 is coated with a secondary antibody of the novel coronavirus antibody IgG, in this example a murine anti-human IgG monoclonal antibody. The detection line T2 is coated with angiotensin converting enzyme 2. The control line C coated a secondary antibody specifically binding to the colloidal gold labeled novel coronavirus-unrelated antibody, in this example the secondary antibody was a goat anti-rabbit IgG polyclonal antibody. In the present embodiment, the distance between the detection line T2, the detection line T1 and the quality control line C along the chromatography direction is 4-6mm, and in the present embodiment, the distance is selected to be 5 mm.
Further, the sample loading pad 2 and the absorbent pad 5 are both provided with a protective film 6.
Example 2
This example provides a method for preparing the novel test strip for detecting coronavirus neutralizing antibodies of example 1, which is prepared by the following steps:
1. preparing an antibody bearing membrane:
(1) selecting a nitrocellulose membrane with the aperture of 3-10 mu m, and cutting the membrane into specifications with the width of 2.0cm and the length of 30.5cm according to requirements for later use.
(2) The mouse anti-human IgG monoclonal antibody for the detection line coating is prepared by 0.1M Tris-HCL buffer solution with the pH value of 8.0, the concentration of the antibody is 0.5-0.6 mg/ml, in the embodiment, 0.5mg/ml, the concentration of ACE-2 is 1.5-2.0 mg/ml, in the embodiment, 1.8mg/ml, and the goat anti-rabbit IgG polyclonal antibody for the quality control line coating is prepared by 0.85% sodium chloride buffer solution by mass percent, so that the concentration of the antibody is 2.0-2.5 mg/ml, in the embodiment, 2.2 mg/ml.
(3) Selecting an antibody coating surface of a nitrocellulose membrane and marking, coating a detection line and a quality control line solution to be coated on a membrane in parallel and uniformly, and controlling the distance between the detection line and the quality control line to be C-T1: 4.0mm, T1-T2: 4.0mm, and drying the nitrocellulose membrane at the constant temperature of 2-30 ℃ for later use.
(4) Preparing a sealing treatment soaking solution, and adding purified water with actual production capacity into a liquid preparation tank; respectively weighing buffer solution, sugar, blocking protein and preservative, directly adding the above components into a liquid preparation tank, stirring until the components are completely dissolved, adding purified water to a desired volume, and fully and uniformly stirring for at least 10 minutes for later use; the prepared sealing treatment soaking solution contains 0.1Mol of buffer solution, 0.5 mass percent of sugar, 1 mass percent of sealing protein and 0.05 mass percent of preservative, wherein the buffer solution is phosphate buffer solution, the sugar is cane sugar, the preservative is thimerosal, and the sealing protein is bovine serum albumin.
(5) Placing the membranes coated with the detection lines and the quality control lines in a treatment tank, adding the prepared sealing treatment soaking liquid of the step (4), ensuring that each membrane is completely immersed in the sealing treatment soaking liquid of the step (4) for 30 minutes, ensuring that the membranes do not move and overlap, taking out the membranes from the treatment tank after 30 minutes, and pouring the sealing treatment soaking liquid of the step (4); and (3) placing the membrane on gauze by using tweezers, and airing to be slightly dry to obtain the antibody bearing membrane.
(6) Pasting a board and drying: tearing off white paper in the middle of a cutting line on double-sided adhesive of a rubber plate, putting an antibody bearing film subjected to sealing treatment right at the central blank position of the rubber plate by using tweezers, enabling the right side of the rubber plate to be flush with the right side of the film, avoiding errors in the production process, ensuring that the color development position is relatively accurate, pasting one end of all top quality control lines when pasting the plate, smearing the film surface through the double-sided adhesive paper after the film is pasted on the rubber plate, avoiding bubbles, controlling the temperature in a control room to be 18-28 ℃ and the relative humidity to be less than or equal to 40%, and ensuring that air can circulate in a drying room and air of a dehumidifier can not directly blow on the film surface. The drying time is more than or equal to 4 hours and is reserved.
2. Preparation of colloidal gold adsorption pad
(1) Preparing a colloidal gold complex solution: adding an actual production amount of purified water to the liquor preparation tank; trehalose, bovine serum albumin, trisodium citrate, polyethylene glycol and NaN were weighed using an electronic analytical balance3Directly adding the colloidal gold complex solution into a liquid preparation tank for stirring until the colloidal gold complex solution is completely dissolved, adding purified water to a constant volume to a required volume, fully and uniformly stirring, wherein the stirring time is more than 30 minutes, and the obtained colloidal gold complex solution contains 5 mass percent of trehalose, 2 mass percent of bovine serum albumin, 0.5 mass percent of trisodium citrate, 0.05 mass percent of polyethylene glycol and 0.05 mass percent of NaN3。
(2) Measuring colloidal gold with required labeling amount by using a measuring cylinder, adjusting according to pH7.0-7.3, namely adding 0.2mol/L potassium carbonate solution according to the volume percentage content of 0.50%, stirring for 15 minutes on a magnetic stirrer, taking a novel coronavirus (S-RBD) antigen, diluting the novel coronavirus (S-RBD) antigen by using double distilled water, labeling the colloidal gold according to 4-5 mu g/ml (namely, after adding the colloidal gold into the novel coronavirus (S-RBD) antigen diluent, the final concentration of the novel coronavirus (S-RBD) antigen is 4-5ug/ml, selecting 4 mu g/ml in the embodiment), adding the novel coronavirus (S-RBD) antigen into the colloidal gold, stirring for 30 minutes on the magnetic stirrer, adding stabilizer polyethylene glycol with the volume percentage content of 0.5 thousandth, stirring for 30 minutes, centrifuging, collecting precipitate, redissolving by using the colloidal gold composite solution according to the volume percentage of 3%, stirring on a magnetic stirrer until the mixture is uniformly mixed to obtain the colloidal gold-novel coronavirus (S-RBD) antigen conjugate composite solution with the volume percentage of 3% for later use.
(3) Measuring the colloidal gold with the required labeling amount by using a measuring cylinder, adjusting according to the pH value of 7.0-7.3, namely adding 0.2mol/L potassium carbonate solution according to the volume percentage of 0.50%, stirring for 15 minutes on a magnetic stirrer, taking the rabbit IgG antibody, diluting the rabbit IgG antibody by using double distilled water, labeling the colloidal gold according to the volume percentage of 4-5ug/ml (in the embodiment, 4ug/ml is selected, namely, after the colloidal gold is added into the rabbit IgG antibody diluent, the final concentration of the rabbit IgG antibody is 4ug/ml), adding the rabbit IgG antibody into the colloidal gold, stirring for 30 minutes on the magnetic stirrer, adding 0.5 thousandth of stabilizer polyethylene glycol according to the volume percentage, stirring for 30 minutes, centrifuging, collecting precipitates, re-dissolving the colloidal gold re-solution according to the volume percentage of 3%, stirring on the magnetic stirrer until the mixture is uniform, obtaining the colloidal gold-rabbit IgG antibody conjugate re-solution with the volume percentage of 3%, and (5) standby.
(4) Taking the colloidal gold-novel coronavirus (S-RBD) antigen conjugate complex solution with the volume percentage of 3% and the colloidal gold-rabbit IgG antibody conjugate complex solution with the volume percentage of 3% in the above (2) and (3), re-dissolving the colloidal gold complex solution with the volume percentage of 50%, uniformly mixing on a magnetic stirrer, and uniformly mixing according to the volume percentage of 50 mul/cm2Pouring the colloidal gold onto a prepared colloidal gold adsorption pad, placing the colloidal gold adsorption pad in a drying chamber to be dried for more than or equal to 4 hours, controlling the temperature of the drying chamber to be 18-28 ℃, controlling the relative humidity to be less than or equal to 40%, ensuring smooth air and preventing airflow from directly blowing on the colloidal gold adsorption pad, placing the dried colloidal gold adsorption pad into an aluminum foil bag filled with a drying agent, sealing and storing, and making a labeled colloidal gold adsorption pad for later use.
Note: the colloidal gold is cast because the cast colloidal gold can be freely cut in width, thereby being convenient for adjusting the color development of the product.
3. Assembling and cutting:
(1) taking a semi-finished product of a transparent substrate adhered with an antibody bearing film, cutting a test strip colloidal gold adsorption pad into strips of 0.5cm multiplied by 30cm according to the width of 0.5cm, adhering the strips to the transparent substrate close to one side of a detection line, keeping the strips to be overlapped with the antibody bearing film for about 1mm, compounding a test strip water absorption pad on the transparent substrate close to one side of a quality control line and overlapped with the antibody bearing film for about 1mm, compounding a test strip sample loading pad on one end, far away from the antibody bearing film, of the test strip colloidal gold adsorption pad and overlapped with the antibody bearing film for about 1mm, and marking for later use;
(2) and cutting the assembled substrate into strip test paper for later use.
Example 3
This example provides a novel coronavirus neutralizing antibody detection device, as shown in fig. 2, comprising a housing 7, wherein two novel coronavirus neutralizing antibody detection test strips of example 1 are arranged in parallel inside the housing 7. Along the chromatography direction of the novel coronavirus neutralizing antibody detection test paper, a sample hole 8 is formed in the position, corresponding to the sample pad, of the shell 7, and an observation port 9 is formed in the positions of a detection line T2, a detection line T1 and a quality control line C.
Example 4
This example provides a novel coronavirus neutralizing antibody detection kit, which includes the novel coronavirus neutralizing antibody detection test strip of example 1 or example 2, or the novel coronavirus neutralizing antibody detection device of example 3.
Example 5
This example provides a non-disease diagnostic test for a novel coronavirus neutralizing antibody, comprising the steps of:
the test strips (without opening the foil bags) were returned to room temperature.
And opening the aluminum foil packaging bag, taking out the detection device and flatly placing on the table top.
2 drops of diluent (in this example, the diluent contains 0.01M PBS, 0.3% (w/v) complexing protein salt, 0.05% (w/v) preservative, pH7.0) are vertically dropped into the well B, which is used as a negative control, the test paper is marked as a test paper B, 20ul of sample is sucked and added into the well A, and 2 drops of diluent are dropped into the well A, and the test paper is marked as a test paper A.
The results were observed and recorded after 15 minutes, and the results were not valid after 20 minutes.
After the test is finished, the used detection test paper and the dropper are treated as biomedical wastes.
Note that the test paper should be removed from the original package as soon as possible within 1 hour, especially at room temperature above 30 ℃ or in a high humidity environment. At ambient temperature, the samples were processed within 1 hour after collection. If the treated sample cannot be detected in time, the treated sample is refrigerated for 2-8 ℃ and is detected within 12 hours after treatment. Samples were strictly prohibited from being subjected to freeze-thaw processing.
2. And (4) judging a result:
2.1 negative:
and B, test paper color development: purple red bands appear on the quality control line C and the detection line T2, and no purple red band appears on the detection line T1, and the results show that: no novel coronavirus neutralizing antibody could be detected in the specimen. The color development result of the test paper B is consistent with that of the test paper A, and the test paper B is negative.
2.2 Positive:
(1) a light purple red strip appears on the test paper A at the detection line T1, a purple red strip appears on the detection line T2, the color development of the detection line T2 is darker than that of the detection line T1, but the color development of the test paper A detection line T2 is lighter than that of the test paper B detection line T2. The results show that: samples contained low titers of neutralizing antibodies to the novel coronavirus.
(2) A light purple red strip appears on the test paper A at the detection line T1 and the detection line T2, the color development depths of the detection lines T1 and T2 are equivalent, and the color development of the test paper A detection line T2 is lighter than that of the test paper B detection line T2. The results show that: the samples contained the neutralizing antibody titers of the novel coronavirus.
(3) The test paper A has a purple-red strip at a detection line T1, and has no purple-red strip at a detection line T2. The results show that: the specimen contains the novel coronavirus neutralizing antibody with high titer.
And (3) failure: the absence of a purple-red band on control line C indicates an improper procedure or kit failure. In this case the instructions should be read again and retested with a new test card.
The presence of a purplish red band at the control line is used as a criterion for the internal control of the kit to determine if there is enough sample, if the chromatographic process is normal, if the correct procedure is used and the correct test results are obtained.
Experimental example 1
A commercially available recombinant neutralizing antibody of the new coronavirus (purchased from Catalog # REGN-10933, am source medical technology, ltd.) was selected to prepare a solution having a concentration as shown in table 1 below as a sample to be tested, and the detection was performed according to the non-disease diagnosis detection method of the novel neutralizing antibody of coronavirus of example 5, and the chromogenic result was compared with the colorimetric card of fig. 3, and the detection result is shown in table 1 below.
TABLE 1 test results
Note: the color development of G1-G14 was gradually increased, and G1 was not developed.
From the above results, the novel test paper for detecting coronavirus neutralizing antibodies of the present invention can detect the concentration of the novel coronavirus neutralizing antibodies, can detect the concentration of the neutralizing antibodies as low as 0.625 μ g/mL, has high specificity and high sensitivity during detection, and has the advantages of simple detection operation and short time.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A novel coronavirus neutralizing antibody detection test paper is characterized by comprising:
the sample loading device comprises a substrate and a sample loading pad, a colloidal gold adsorption pad, an antibody bearing film and a water absorption pad which are sequentially overlapped and lapped on the substrate; the antibody bearing film is provided with a detection line T1, a detection line T2 and a quality control line C which are spaced, the detection line T2 is close to the colloidal gold adsorption pad, and the quality control line C is close to the water absorption pad;
the colloidal gold adsorption pad is coated with a receptor binding domain RBD antigen of the colloidal gold labeled novel coronavirus S protein and a colloidal gold labeled antibody unrelated to the novel coronavirus;
the detection line T1 is coated with a secondary antibody of a novel coronavirus antibody IgG;
the detection line T2 is coated with angiotensin converting enzyme 2;
the control line C coats a secondary antibody that specifically binds to an antibody unrelated to the colloidal gold-labeled neocoronavirus.
2. The novel coronavirus neutralizing antibody test strip according to claim 1, wherein the secondary antibody of the novel coronavirus antibody IgG is a mouse anti-human IgG monoclonal antibody; or
The colloidal gold labeled antibody irrelevant to the new coronavirus is a rabbit IgG antibody; or
The secondary antibody which is labeled by the colloidal gold and is specifically combined with the antibody irrelevant to the new coronavirus is goat anti-rabbit IgG polyclonal antibody.
3. A method for preparing a novel test strip for detecting neutralizing antibodies against coronavirus according to any one of claims 1 to 2, comprising the steps of:
sequentially overlapping the sample loading pad, the colloidal gold adsorption pad, the antibody bearing film and the water absorption pad on the substrate;
coating a receptor binding domain RBD antigen of the colloidal gold-labeled novel coronavirus S protein and a colloidal gold-labeled antibody unrelated to the novel coronavirus on the colloidal gold adsorption pad;
a secondary antibody of a novel coronavirus antibody IgG, angiotensin converting enzyme 2 and a secondary antibody specifically combined with the antibody irrelevant to the novel coronavirus labeled by colloidal gold are respectively coated on a detection line T1, a detection line T2 and a quality control line C on an antibody bearing film.
4. The method of claim 3, wherein the secondary antibody of the novel coronavirus antibody IgG is a murine anti-human IgG monoclonal antibody; or
The colloidal gold labeled antibody irrelevant to the new coronavirus is a rabbit IgG antibody; or
The secondary antibody which is labeled by the colloidal gold and is specifically combined with the antibody irrelevant to the new coronavirus is goat anti-rabbit IgG polyclonal antibody.
5. The method of claim 4, wherein the coating concentration of goat anti-rabbit IgG polyclonal antibody is: 2.0-2.5 mg/ml; or
The coating concentration of the mouse anti-human IgG monoclonal antibody is as follows: 0.5-0.6 mg/ml; or
Angiotensin converting enzyme 2 coating concentration: 1.5-2.0 mg/ml; or
Colloidal gold labeling concentration of receptor binding domain RBD antigen of novel coronavirus S protein: 4-5 mug/ml; or
Colloidal gold labeling concentration of rabbit IgG: 4-5 μ g/ml.
6. A novel coronavirus neutralizing antibody detection device, comprising a housing, and a novel coronavirus neutralizing antibody detection test strip according to any one of claims 1 to 2 or a novel coronavirus neutralizing antibody detection test strip prepared by the preparation method according to any one of claims 3 to 5 arranged inside the housing;
along the chromatography direction of the novel coronavirus neutralizing antibody detection test paper, a sample hole is formed in the position, corresponding to the sample pad, of the shell, and observation ports are formed in the positions of the detection line T2, the detection line T1 and the quality control line C.
7. A novel reagent kit for detecting neutralizing antibodies against coronavirus, comprising the novel test strip for detecting neutralizing antibodies against coronavirus according to any one of claims 1 to 2, the novel test strip for detecting neutralizing antibodies against coronavirus prepared by the preparation method according to any one of claims 3 to 5, or the novel device for detecting neutralizing antibodies against coronavirus according to claim 6.
8. Use of the novel test strip for detecting neutralizing antibodies against coronavirus according to any one of claims 1 to 2, the novel test strip for detecting neutralizing antibodies against coronavirus prepared by the method according to any one of claims 3 to 5, or the novel device for detecting neutralizing antibodies against coronavirus according to claim 6 for the preparation of a product for detecting neutralizing antibodies against novel coronavirus.
9. A method for detecting a novel coronavirus neutralizing antibody in a non-disease diagnosis, which comprises using the novel coronavirus neutralizing antibody test strip according to any one of claims 1 to 2, the novel coronavirus neutralizing antibody test strip prepared by the preparation method according to any one of claims 3 to 5, or the novel coronavirus neutralizing antibody detection device according to claim 6.
10. The assay of claim 9 wherein the sample is applied to a pad of test strips, followed by the application of a diluent to the pad, wherein the test strips are designated as test strips a; meanwhile, adding a diluent to another test paper as a negative control, wherein the test paper is marked as test paper B; observing the test paper A and the test paper B after 15 minutes, and obtaining an invalid result after 20 minutes;
optionally, when a mauve strip appears at the position of the quality control line C of the test paper A and the detection line T2, the mauve strip does not appear in the detection line T1, the color development result of the test paper A is consistent with that of the test paper B, and the detection result is negative;
when the test paper A has a light purple red strip at the detection line T1 and a purple red strip at the detection line T2, the detection line T2 has a darker color than the detection line T1, the test paper A detection line T2 has a lighter color than the test paper B detection line T2, and the detection result shows that the sample contains the novel coronavirus neutralizing antibody with low titer;
when the test paper A has light purple red strips at the detection lines T1 and T2, the color development depths of the detection lines T1 and T2 are equivalent, the color development of the test paper A detection line T2 is lighter than that of the test paper B detection line T2, and the detection result shows that the sample contains the titer of the novel coronavirus neutralizing antibody;
when the A test paper has a purple red strip at a T1 detection line and does not have a purple red strip at a T2 detection line, the detection result shows that the sample contains the novel coronavirus neutralizing antibody with high titer;
and if the quality control line C of the test paper A and the test paper B does not have a purple red strip, indicating that the operation process is incorrect or fails.
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| WO2021244629A1 (en) * | 2020-06-05 | 2021-12-09 | 南京金斯瑞生物科技有限公司 | Immunochromatographic device for detecting sars-cov-2 virus neutralizing antibody and application thereof |
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| CN211697830U (en) * | 2019-09-04 | 2020-10-16 | 南通伊仕生物技术股份有限公司 | Luteinizing hormone detection test paper and test paper card |
| CN111337669A (en) * | 2020-03-03 | 2020-06-26 | 南通大学 | Novel coronavirus detection test paper and detection method for rapid detection |
| CN111879951A (en) * | 2020-08-17 | 2020-11-03 | 南通伊仕生物技术股份有限公司 | Novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip |
| CN211905393U (en) * | 2020-09-22 | 2020-11-10 | 正元盛邦(天津)生物科技有限公司 | Novel coronavirus specific antibody and neutralizing antibody combined detection test paper and device |
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| WO2021244629A1 (en) * | 2020-06-05 | 2021-12-09 | 南京金斯瑞生物科技有限公司 | Immunochromatographic device for detecting sars-cov-2 virus neutralizing antibody and application thereof |
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