CN111999507A - Fluorescence immunochromatography test paper for detecting novel coronavirus antibody - Google Patents
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Abstract
The invention discloses a fluorescence immunochromatographic test paper for detecting a novel coronavirus antibody. The combination pad of the test paper is coated with an anti-human IgM antibody marked by time-resolved fluorescent microspheres; the preparation method of the bonding pad comprises the following steps: mixing the time-resolved fluorescent microspheres and the anti-human IgM antibodies, and adding a boric acid buffer solution containing 0.1-0.5% BSA for sealing; centrifuging and collecting precipitate; resuspending the precipitate with a borate buffer containing 0.1-0.5% BSA to obtain a labeled solution; spraying the marking solution on the membrane, and drying to obtain a bonding pad; the boric acid buffer solution is pH7.0-8.5, 10-100mM boric acid buffer solution. Experiments prove that the fluorescence immunochromatographic test paper provided by the invention can be used for detecting whether a sample contains a novel coronavirus antibody, and has the advantages of simple operation, short consumed time, high accuracy, good specificity and high sensitivity. The fluorescence immunochromatographic test paper provided by the invention has important application value in rapidly detecting the novel coronavirus antibody on site.
Description
Technical Field
The invention belongs to the biotechnology and the field, and particularly relates to a fluorescence immunoassay test paper for detecting a novel coronavirus antibody.
Background
Coronavirus is a kind of enveloped single-stranded positive-strand RNA virus, and is named because the virus particle is shaped like a crown under an electron microscope. The host range of coronaviruses mainly includes birds and mammals, and seven kinds of coronaviruses that can infect humans have been identified so far, among which the emergence of SARS virus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2) pose a great threat to world economy and health. The novel coronavirus belongs to the family of coronaviridae and the genus coronavirus, can cause symptoms of different degrees after infecting people, and mainly shows fever, weakness and dry cough, and severe people have pneumonia, acute respiratory distress syndrome and even die. The main modes of transmission of the novel coronaviruses are droplet transmission and contact transmission.
The detection of a novel coronavirus is achieved by detecting whether or not a sample contains a viral nucleic acid, but the positive rate is lowered due to sample collection, reagents, handling, and the like. The suspected cases with the pneumonia imaging characteristics are used as the clinical diagnosis case standard. Similar histological pathological changes may occur with different pneumonia types and there is a risk of cross-infection with CT detection.
Disclosure of Invention
The object of the present invention is to detect novel coronaviruses.
The invention firstly protects a fluorescence immunochromatographic test paper for detecting a novel coronavirus antibody, wherein a binding pad of the fluorescence immunochromatographic test paper is coated with an anti-human IgM antibody marked by a time-resolved fluorescent microsphere;
the preparation method of the bonding pad can be as follows:
(1) mixing the time-resolved fluorescent microspheres with anti-human IgM antibody, adding 0.1-0.5% (such as 0.1-0.3%, 0.3-0.5%, 0.1%, 0.3% or 0.5%) BSA containing boric acid buffer solution, and sealing;
(2) after the step (1) is finished, centrifuging and collecting precipitates;
(3) after step (2) is completed, resuspending the pellet in a borate buffer containing 0.1-0.5% (e.g., 0.1-0.3%, 0.3-0.5%, 0.1%, 0.3%, or 0.5%) BSA to obtain a labeling solution;
(4) spraying the labeling solution on a membrane, and drying to obtain a binding pad coated with the anti-human IgM antibody labeled by the time-resolved fluorescent microspheres;
the boric acid buffer is pH7.0-8.5 (such as pH7.0-7.5, pH7.5-8.0, pH8.0-8.5, pH7.0, pH7.5, pH8.0 or pH8.5), 10-100mM (such as 10-25mM, 25-50mM, 50-100mM, 10mM, 25mM, 50mM or 100mM) boric acid buffer.
In the above fluorescence immunochromatographic test paper, the step of spraying the labeling solution on the membrane may be a step of spraying the labeling solution on a glass cellulose membrane.
In the above fluorescence immunochromatographic test strip, the boric acid buffer may be specifically a boric acid buffer of ph8.0 and 25 mM.
In the above fluorescence immunochromatographic test paper, the time-resolved fluorescent microspheres may be europium-containing time-resolved fluorescent microspheres.
In the fluorescence immunochromatographic test paper, the anti-human IgM antibody can be coated on the position of the quality control line on the reaction membrane. The position of the detection line can be coated with a novel coronavirus antigen.
Any one of the novel coronavirus antigens described above can be a novel coronavirus NP antigen.
Any of the above anti-human IgM antibodies may be a goat anti-human IgM antibody, a mouse anti-human IgM antibody or a rabbit anti-human IgM antibody.
The antibody of any of the above sheep anti-human IgM antibodies may specifically be a rabbit anti-sheep IgM antibody.
Any one of the reaction membranes described above may be a nitrocellulose membrane.
The preparation method of any of the above sheep anti-human IgM antibodies can be referred to the following documents: preparation of sheep anti-human mu chain serum, Chinese public health, 1985, 4 (4): 38-40.
The preparation method of any one of the rabbit anti-sheep IgM antibodies is referred to the following documents: 1 simple method for the preparation of goat anti-rabbit IgG antibodies, first university of medicine, 1998, 19 (1).
The fluorescence immunochromatographic test paper specifically comprises a sample pad (made of a glass cellulose membrane), a combination pad, a nitrocellulose membrane coating a quality control line and a detection line, and a water absorption pad (such as filter paper), and the specific structural schematic diagram is shown in fig. 1.
In any of the above-mentioned fluorescence immunochromatographic test strips, the preparation method of the conjugate pad may specifically be as follows:
(1-1) adding 800 mu L of sheep anti-human IgM antibody with the concentration of 1mg/ml into 6-10ml of a solution containing europium time-resolved fluorescent microspheres, and uniformly mixing for 60min on a rotary uniform mixer;
(1-2) adding a boric acid buffer solution containing 0.3% BSA, and blocking overnight;
(1-3) centrifuging at 15000rpm for 40min, and collecting the precipitate;
(1-4) resuspending the precipitate with 12mL of 0.1% (m/v) BSA boric acid buffer solution to obtain a solution of the goat anti-human IgM antibody labeled with europium-containing time-resolved fluorescent microspheres;
(1-5) uniformly spraying a solution of the goat anti-human IgM antibody marked by the europium-containing time-resolved fluorescent microspheres on a glass cellulose membrane, and then freeze-drying by a freeze dryer to obtain a binding pad;
the boric acid buffer solution may specifically be a 25mM boric acid buffer solution having a pH of 8.0.
In any of the above-mentioned fluorescence immunochromatographic test paper, the preparation method of the nitrocellulose membrane coating the quality control line and the detection line may specifically be as follows:
(2-1) diluting the novel coronavirus NP antigen with 0.01M PBS (pH7.4) to obtain a detection line solution with the concentration of 3mg/mL, and using the detection line solution to coat the detection line;
(2-2) diluting the rabbit anti-sheep IgM antibody with 0.01M PBS buffer solution (pH7.4) to obtain a quality control line solution with the concentration of 2mg/mL, and coating the quality control line;
(2-3) taking the quality control line solution and the detection line solution, and respectively and uniformly spraying the quality control line solution and the detection line solution on the nitrocellulose membrane (the sample spraying amount is 10 mu L/cm)2) Forming a quality control line (C) and a detection line (T) which are separated from each other, and drying for 3-5h at 37 ℃ to obtain the nitrocellulose membrane coating the quality control line and the detection line. Bag (bag)And on the nitrocellulose membrane of the quality control line and the detection line, the distance between the quality control line and the detection line is 0.5 cm.
The invention also provides a method for detecting whether the liquid to be detected contains the novel coronavirus antibody by using any one of the fluorescence immunochromatographic test paper.
The method for detecting whether the solution to be detected contains the novel coronavirus antibody by adopting the fluorescence immunochromatographic test paper protected by the invention can be specifically the first method, and comprises the following steps: immersing the sample pad of the fluorescence immunochromatographic test paper in a solution to be tested, standing for more than 10min (such as 10min and 15min), irradiating the reaction membrane with fluorescence (specifically, the fluorescence with an excitation wavelength of 365 nm), observing a red reaction line displayed on the reaction membrane, and judging as follows:
if red reaction lines appear at the detection line and the quality control line, the liquid to be detected contains the novel coronavirus antibody;
if a red reaction line appears at the quality control line and a red reaction line does not appear at the detection line, the liquid to be detected does not contain the novel coronavirus antibody;
if the red reaction line is not shown at the quality control line, the result is invalid.
The method for detecting whether the solution to be detected contains the novel coronavirus antibody by adopting the fluorescence immunochromatographic test paper protected by the invention can be specifically a method II, and comprises the following steps: immersing the sample pad of the fluorescence immunochromatographic test paper into a solution to be detected, standing for more than 10min (such as 10min and 15min), detecting fluorescence values at a detection line and a quality control line, and judging as follows:
if the fluorescence values of the detection line and the quality control line are both larger than 50, the liquid to be detected contains a novel coronavirus antibody;
if the fluorescence value at the quality control line is more than 50 and the fluorescence value at the detection line is less than 50, the liquid to be detected does not contain the novel coronavirus antibody;
if the fluorescence value at the control line is 50 or less, it is an invalid result.
In the second method, the fluorescence values at the detection line and the quality control line can be detected by a dry fluorescence detector.
In any of the above methods, the test solution may be serum or a serum dilution.
Any of the methods described above are useful for non-disease diagnosis and treatment.
In any of the above methods, the inclusion of the novel coronavirus antibody may indicate infection with or prior to infection with the novel coronavirus.
The fluorescence immunochromatographic test strip is used for non-disease diagnosis and treatment.
Experiments prove that the fluorescence immunochromatographic test paper provided by the invention can be used for detecting whether a sample contains a novel coronavirus antibody, and has the advantages of simple operation, short consumed time, high accuracy, good specificity and high sensitivity. The fluorescence immunochromatographic test paper provided by the invention has important application value in rapidly detecting whether serum contains a novel coronavirus antibody on site.
Drawings
FIG. 1 is a schematic structural diagram of a fluorescence immunochromatographic test strip for detecting novel coronavirus antibodies.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from conventional biochemical reagents, unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 preparation of fluorescent immunochromatographic test strip for detecting novel coronavirus antibody
Preparation of first, combined pad
1. BSA was dissolved in a boric acid buffer to obtain working solution 1 having a concentration of 0.3% (m/v) BSA and working solution 2 having a concentration of 0.1% (m/v) BSA.
2. To 6-10ml europium-containing time-resolved fluorescent microspheres (Thermo Scientific)TMCompany product, cat # 93470520010150; particle size of 200nm) was added to 800. mu.L of goat anti-human IgM antibody (see the following references for a method for preparing goat anti-human IgM antibody): preparation of sheep anti-human mu chain serum, Chinese public health, 1985, 4 (4): 38-40), and uniformly mixing on a rotary mixer for 60 min.
3. After completion of step 2, add working solution 1 and block overnight.
4. After completion of step 3, the precipitate was collected by centrifugation at 15000rpm for 40 min.
5. And (4) after the step 4 is finished, resuspending and precipitating by using 12mL of working solution 2 to obtain a solution of the sheep anti-human IgM antibody marked by the europium-containing time-resolved fluorescent microspheres.
6. And (3) uniformly spraying a solution of the goat anti-human IgM antibody marked by the europium-containing time-resolved fluorescent microspheres on a glass cellulose membrane, and then freeze-drying by a freeze dryer to obtain the bonding pad.
Preparation of nitrocellulose membrane coating quality control line and detection line
1. The novel coronavirus NP antigen (product of eastern Marine medicine institute, cat # B35-Ag1) was diluted with 0.01M PBS buffer (pH7.4) to obtain a test line solution with a concentration of 3mg/mL for coating the test line.
2. Rabbit anti-sheep IgM antibody (the preparation method of rabbit anti-sheep IgM antibody is described in the following literature: 1 simple method for preparing goat anti-rabbit IgG antibody, proceedings of the university of capital medicine, 1998, 19(1)) was diluted with 0.01M PBS buffer (pH7.4) to obtain a quality control line solution having a concentration of 2mg/mL for coating the quality control line.
3. Respectively and uniformly spraying the quality control line solution and the detection line solution on the nitrocellulose membrane (the sample spraying amount is 10 muL/cm)2) Forming a quality control line (C) and a detection line (T) which are separated from each other, and drying for 3-5h at 37 ℃ to obtain the nitrocellulose membrane coating the quality control line and the detection line. And the nitrocellulose membrane coating the quality control line and the detection line has a distance of 0.5 cm.
Assembly of fluorescent immune chromatography test paper for detecting novel coronavirus antibody
The fluorescent immunochromatographic test paper for detecting the novel coronavirus antibody consists of a sample pad (made of a glass cellulose membrane), a combination pad, a nitrocellulose membrane coating a quality control line and a detection line, and a water absorption pad (such as filter paper).
The assembly steps of the fluorescence immunochromatographic test paper for detecting the novel coronavirus antibody are as follows: the method comprises the following steps of pasting a water absorption pad on one end (the overlapping length is 0.2cm) of a nitrocellulose membrane covering a quality control line and a detection line by using a double-faced adhesive tape, pasting one end (the overlapping length is 0.2cm) of a combination pad on the other end of the nitrocellulose membrane covering the quality control line and the detection line by using the double-faced adhesive tape, pasting a sample pad (the overlapping length is 0.2cm) on the other end of the combination pad by using the double-faced adhesive tape, finally pasting the sample pad and the combination pad on a plastic back plate (such as a PVC (polyvinyl chloride) bottom plate) by using the double-faced adhesive tape, and cutting the sample pad into the fluorescent immunochromatographic test paper with the width. The fluorescence immunochromatographic test paper for detecting the novel coronavirus antibody is sealed by a plastic package and then is stored in a drying box for later use.
The structural schematic diagram of the fluorescence immunochromatographic test paper for detecting the novel coronavirus antibody is shown in figure 1. According to the requirement, a sample adding hole can be arranged on the sample pad.
Fourth, use method and judgment standard of fluorescence immunochromatographic test paper for detecting novel coronavirus antibody
Taking the fluorescence immunochromatographic test paper for detecting the novel coronavirus antibody, adding 100 mu L of a solution to be detected (serum of a person to be detected or serum diluent of the person to be detected) into a sample pad (or a sample adding hole), continuously moving the solution to be detected to the upper end after being absorbed by the sample pad, reacting with a sheep antihuman IgM antibody marked by europium-containing time-resolved fluorescent microspheres when flowing through a combination pad, then migrating to a nitrocellulose membrane covering a quality control line and a detection line, and reacting for 15 min. The nitrocellulose membrane was illuminated with a fluorescent flashlight with an excitation wavelength of 365 nm. The following judgment is carried out according to the condition of the red reaction line shown on the film:
1. if red reaction lines are displayed on the detection line and the quality control line on the nitrocellulose membrane, the solution to be detected contains a novel coronavirus antibody, namely a person to be detected is infected with a novel coronavirus; the principle is that a novel coronavirus antibody in a liquid to be detected is combined with a goat anti-human IgM antibody marked by europium-containing time-resolved fluorescent microspheres on a combination pad to form an immune complex, the complex can be combined with a novel coronavirus NP antigen through a detection line and can bridge europium-containing time-resolved fluorescent microspheres, so that a red reaction line appears at the detection line, and the complex can be combined with a rabbit anti-goat IgM antibody through a quality control line and can bridge europium-containing time-resolved fluorescent microspheres, so that a red reaction line appears at the quality control line;
2. if only a red reaction line appears at the quality control line on the nitrocellulose membrane, the solution to be tested does not contain the novel coronavirus antibody, namely the person to be tested is not infected with the novel coronavirus; the principle is that the solution to be detected does not contain a novel coronavirus antibody, so the solution cannot be combined with a goat anti-human IgM antibody marked by europium-containing time-resolved fluorescent microspheres on a combination pad to form an immune complex, the goat anti-human IgM antibody marked by the europium-containing time-resolved fluorescent microspheres cannot be combined with a novel coronavirus NP antigen through a detection line, so a red reaction line is not shown at the detection line, and the goat anti-human IgM antibody marked by the europium-containing time-resolved fluorescent microspheres can be combined with a rabbit anti-goat IgM antibody through a quality control line, so the red reaction line is shown at the quality control line;
3. if the quality control line on the nitrocellulose membrane does not show a red reaction line, the result of the experiment is invalid and needs to be determined again.
It should be noted that, the interpretation result of the nitrocellulose membrane irradiated by the fluorescent flashlight with the excitation wavelength of 365nm, or the interpretation result of the fluorescence values at the detection line and the quality control line detected by the dry fluorescent detector may be specifically as follows: if the fluorescence values of the detection line and the quality control line are both larger than 50, the liquid to be detected contains a novel coronavirus antibody, namely the person to be detected is infected with the novel coronavirus; if the fluorescence value at the quality control line is more than 50 and the fluorescence value at the detection line is less than 50, the liquid to be detected does not contain the novel coronavirus antibody, namely the person to be detected is not infected with the novel coronavirus; if the fluorescence value at the quality control line is below 50, the result is an invalid experiment result and needs to be determined again.
Example 2 and example 1 optimization of the fluorescent immunochromatographic test strip for detecting novel coronavirus antibodies prepared in example 1
The inventors of the present invention optimized the conjugate pad, specifically, the boric acid buffer solution used in the preparation of the conjugate pad.
Optimization of pH value of boric acid buffer solution
1. 25mM boric acid buffer (pH6.0), 25mM boric acid buffer (pH6.5), 25mM boric acid buffer (pH7.0), 25mM boric acid buffer (pH7.5), 25mM boric acid buffer (pH8.0), 25mM boric acid buffer (pH8.5), 25mM boric acid buffer (pH9.0), and 25mM boric acid buffer (pH9.5) were prepared, respectively.
2. According to the method of steps one to three of example 1, the boric acid buffer solution of step 1 in step one was replaced with 25mM boric acid buffer solution (pH6.0), 25mM boric acid buffer solution (pH6.5), 25mM boric acid buffer solution (pH7.0), 25mM boric acid buffer solution (pH7.5), 25mM boric acid buffer solution (pH8.0), 25mM boric acid buffer solution (pH8.5), 25mM boric acid buffer solution (pH9.0) and 25mM boric acid buffer solution (pH9.5), respectively, and test papers 1 to 8 were obtained in this order without changing the other steps.
3. Serum of a patient infected with the novel coronavirus was diluted 10-fold with PBS buffer to obtain a diluted serum solution.
4. Adding 100 mu L of serum diluent into sample pads (or sample adding holes) of the test paper 1-8 respectively, reacting for 15min, then irradiating the nitrocellulose membrane by using a fluorescent flashlight with an excitation wavelength of 365nm, and observing the condition of a red reaction line displayed at a detection line.
The results are shown in Table 1. The result shows that when the solution of the goat anti-human IgM antibody marked by the europium-containing time-resolved fluorescent microspheres is prepared, the optimal pH value of the boric acid buffer solution is 8.0.
TABLE 1
| Test paper | pH value | Red reaction line condition displayed at detection line |
| Test paper 1 | 6.0 | - |
| Test paper 2 | 6.5 | + |
| Test paper 3 | 7.0 | ++ |
| Test paper 4 | 7.5 | +++ |
| Test paper 5 | 8.0 | ++++ |
| Test paper 6 | 8.5 | ++ |
| Test paper 7 | 9.0 | - |
| Test paper 8 | 9.5 | - |
Note: -no red reaction line is shown, + a faint, + a visible, clear, and +++ a bright line.
Optimization of concentration of boric acid buffer solution
1. 5mM boric acid buffer (pH8.0), 10mM boric acid buffer (pH8.0), 25mM boric acid buffer (pH8.0), 50mM boric acid buffer (pH8.0), 100mM boric acid buffer (pH8.0), 200mM boric acid buffer (pH8.0), and 400mM boric acid buffer (pH8.0) were prepared, respectively.
2. According to the method of steps one to three of example 1, test paper a-test paper g were obtained in this order by replacing the boric acid buffer solution of step 1 in step one with 5mM boric acid buffer solution (pH8.0), 10mM boric acid buffer solution (pH8.0), 25mM boric acid buffer solution (pH8.0), 50mM boric acid buffer solution (pH8.0), 100mM boric acid buffer solution (pH8.0), 200mM boric acid buffer solution (pH8.0) and 400mM boric acid buffer solution (pH8.0), respectively, and the other steps were not changed.
3. Serum of a patient infected with the novel coronavirus was diluted 10-fold with PBS buffer to obtain a diluted serum solution.
4. Adding 100 mu L of serum diluent into sample pads (or sample adding holes) of the test paper a-test paper g respectively, reacting for 15min, then irradiating the nitrocellulose membrane by using a fluorescent flashlight with an excitation wavelength of 365nm, and observing the condition of a red reaction line displayed at a detection line.
The results are shown in Table 2. The result shows that when the solution containing the europium time-resolved fluorescent microsphere labeled goat anti-human IgM antibody is prepared, the optimal concentration of the boric acid buffer solution is 25 mM.
TABLE 2
Note: -no red reaction line is shown, + a faint, + a visible, clear, and +++ a bright line.
The above results indicate that the optimum borate buffer was 25mM borate buffer (pH8.0) when the fluorescence immunochromatographic test strip for detecting a novel coronavirus antibody was prepared by the method of example 1.
Example 3 accuracy test of fluorescent immunochromatographic test strip for detecting novel coronavirus antibody
1. According to the method of the first step to the third step of the example 1, the boric acid buffer solution in the first step 1 is replaced by 25mM boric acid buffer solution (pH8.0), and other steps are not changed, so that the optimal fluorescence immunoassay chromatography test paper for detecting the novel coronavirus antibody is obtained.
2. Serum of 16 clinically confirmed patients (named G1-G16) infected with the novel coronavirus was randomly taken and diluted to 10-fold with PBS buffer to obtain serum dilutions of infected patients.
3. Serum of 15 healthy persons (named J1-J15) was randomly selected and diluted to 10 times with PBS buffer to obtain dilutions of the serum of the healthy persons.
4. And (2) adding 100 mu L of serum diluent (infected person serum diluent or healthy human serum diluent) into the sample pad (or the sample adding hole) of the fluorescence immunochromatographic test paper prepared in the step (1) for reaction for 15min, and then detecting the fluorescence values at the detection line and the quality control line by using a dry fluorescence detector. The following judgment is made according to the fluorescence value: if the fluorescence values at the detection line and the quality control line are both larger than 50, the serum diluent contains a novel coronavirus antibody, namely a person to be detected is infected with the novel coronavirus; if the fluorescence value at the quality control line is more than 50 and the fluorescence value at the detection line is less than 50, the serum diluted solution does not contain the novel coronavirus antibody, namely the person to be detected is not infected with the novel coronavirus; if the fluorescence value at the control line is 50 or less, the result is invalid and needs to be determined again.
The results are shown in Table 3. The result shows that the fluorescence immunochromatographic test paper prepared in the step 1 can detect whether the person to be detected is a person infected by the novel coronavirus, and the accuracy rate reaches 100%.
TABLE 3
Example 4 sensitivity test of the fluorescent immunochromatographic test strip for detecting novel coronavirus antibodies
1. Taking serum of a patient clinically diagnosed as a novel coronavirus infected person, and diluting the serum by using a PBS (phosphate buffer solution) to obtain a serum diluted solution; the dilution of the serum dilution is 20-fold, 40-fold, 80-fold, 160-fold, 320-fold, or 640-fold.
2. To the sample pad (or well) of the immunofluorometric chromatography strip prepared in step 1 of example 3, 100. mu.L of serum diluent was added, reacted for 15min, and then the fluorescence values at the detection line and the quality control line were detected using a dry fluorescence detector. The following judgment is made according to the fluorescence value: if the fluorescence values at the detection line and the quality control line are both larger than 50, the serum diluent contains the novel coronavirus antibody, namely the person to be detected is infected with the novel coronavirus; if the fluorescence value at the quality control line is more than 50 and the fluorescence value at the detection line is less than 50, the serum diluent does not contain the novel coronavirus antibody, namely the person to be detected is not infected with the novel coronavirus; if the fluorescence value at the quality control line is below 50, the result is an ineffective experiment and needs to be determined again.
The results are shown in Table 4. The results show that when the serum dilution multiple of the novel coronavirus infected person is 640 times, the fluorescence values at the detection line and the quality control line are still more than 50. Therefore, the fluorescent immunochromatographic test paper for detecting the novel coronavirus antibody has higher sensitivity.
TABLE 4
Example 5 specificity test of the fluorescent immunochromatographic test strip for detecting novel coronavirus antibodies
1. Serum of 5 clinically confirmed influenza A virus infected persons (named L1-L5) was randomly collected and diluted to 10-fold with PBS buffer to obtain serum dilutions of influenza A infected persons.
2. Serum of 5 clinically confirmed influenza B virus infected persons (named as Y1-Y5) is randomly taken and diluted to 10 times by PBS buffer respectively to obtain the influenza B infected person serum diluent.
3. Serum of 10 healthy persons (named J21-J30) was randomly selected and diluted to 10 times with PBS buffer to obtain dilutions of the serum of the healthy persons.
4. Serum of 5 clinically confirmed new coronavirus infected persons (named as X1-X5) was randomly taken and diluted to 10 times with PBS buffer solution to obtain serum dilutions of new coronavirus infected persons.
5. To the sample pad (or well) of the fluorescence immunochromatographic test paper prepared in step 1 of example 3, 100 μ L of serum diluent (influenza a patient serum diluent, influenza b patient serum diluent, healthy human serum diluent, or new crown infection patient serum diluent) was added, and the reaction was carried out for 15min, after which the fluorescence values at the detection line and the quality control line were detected using a dry fluorescence detector. The following judgment is made according to the fluorescence value: if the fluorescence values at the detection line and the quality control line are both larger than 50, the serum diluent contains the novel coronavirus antibody, namely the person to be detected is infected with the novel coronavirus; if the fluorescence value at the quality control line is more than 50 and the fluorescence value at the detection line is less than 50, the serum diluent does not contain the novel coronavirus antibody, namely the person to be detected is not infected with the novel coronavirus; if the fluorescence value at the control line is less than 50, the result is invalid and needs to be determined again.
The results are shown in Table 5. The results show that the fluorescence values at the detection line and the quality control line are still more than 50 only when the serum of a person infected by the novel coronavirus is added. Therefore, the fluorescence immunochromatographic test paper for detecting the novel coronavirus antibody has high specificity.
TABLE 5
Claims (10)
1. A fluorescence immunochromatographic test paper for detecting novel coronavirus antibodies, wherein a binding pad of the fluorescence immunochromatographic test paper is coated with an anti-human IgM antibody marked by a time-resolved fluorescent microsphere; the method is characterized in that:
the preparation method of the combined pad comprises the following steps:
(1) mixing the time-resolved fluorescent microspheres and the anti-human IgM antibodies, and adding a boric acid buffer solution containing 0.1-0.5% BSA for sealing;
(2) after the step (1) is finished, centrifuging and collecting precipitates;
(3) after the step (2) is finished, resuspending the precipitate by using a boric acid buffer solution containing 0.1-0.5% BSA to obtain a labeling solution;
(4) spraying the labeling solution on a membrane, and drying to obtain a binding pad coated with the anti-human IgM antibody labeled by the time-resolved fluorescent microspheres;
the boric acid buffer solution is pH7.0-8.5 and 10-100mM boric acid buffer solution.
2. The fluorescence immunochromatographic test strip according to claim 1, characterized in that: the boric acid buffer solution is pH8.0 and 25mM boric acid buffer solution.
3. The fluorescence immunochromatographic test strip according to claim 1, characterized in that: the time-resolved fluorescent microspheres are europium-containing time-resolved fluorescent microspheres.
4. The fluorescence immunochromatographic test strip according to claim 1, characterized in that: the anti-human IgM antibody is coated at the position of the quality control line on the reaction membrane; the detection line position is coated with a novel coronavirus antigen.
5. The fluorescence immunochromatographic test strip according to any one of claims 1 to 4, characterized in that: the anti-human IgM antibody is a goat anti-human IgM antibody, a mouse anti-human IgM antibody or a rabbit anti-human IgM antibody.
6. The fluorescence immunochromatographic test strip according to claim 4, characterized in that: the novel coronavirus antigen is a novel coronavirus NP antigen.
7. The fluorescence immunochromatographic test strip according to claim 4, characterized in that: the reaction membrane is a nitrocellulose membrane.
8. A method for detecting whether a test solution contains a novel coronavirus antibody by using the fluorescent immunochromatographic test strip of any one of claims 1 to 7, comprising the steps of: immersing the sample pad of the fluorescence immunochromatographic test paper of any one of claims 1 to 7 in a solution to be tested, standing for more than 10min, irradiating the reaction membrane with fluorescence, observing a red reaction line appearing on the reaction membrane, and judging as follows:
if red reaction lines appear at the detection line and the quality control line, the liquid to be detected contains the novel coronavirus antibody;
if a red reaction line appears at the quality control line and a red reaction line does not appear at the detection line, the liquid to be detected does not contain the novel coronavirus antibody;
if the red reaction line is not shown at the quality control line, the result is invalid.
9. A method for detecting whether a test solution contains a novel coronavirus antibody by using the fluorescent immunochromatographic test strip of any one of claims 1 to 7, comprising the steps of: immersing the sample pad of the fluorescence immunochromatographic test strip of any one of claims 1 to 7 in a test solution, standing for 10min or more, detecting fluorescence values at a detection line and a quality control line, and judging as follows:
if the fluorescence values of the detection line and the quality control line are both larger than 50, the liquid to be detected contains a novel coronavirus antibody;
if the fluorescence value at the quality control line is more than 50 and the fluorescence value at the detection line is less than 50, the solution to be detected does not contain the novel coronavirus antibody;
if the fluorescence value at the control line is 50 or less, it is an invalid result.
10. The method of claim 8 or 9, wherein: the liquid to be detected is serum or serum diluent.
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