CN114236120A - Novel background fluorescence immunochromatographic test strip for coronavirus N antigen and preparation method thereof - Google Patents
Novel background fluorescence immunochromatographic test strip for coronavirus N antigen and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a novel coronavirus N antigen background fluorescence immunochromatographic test strip and a preparation method thereof. The test strip comprises a PVC base plate, and a sample pad, a gold-labeled combination pad, a nitrocellulose membrane and absorbent paper are sequentially built on the PVC base plate from left to right. Fluorescent dye is immobilized on the detection line and the quality control line in advance. The gold-labeled combination pad is coated with a mouse anti-human novel coronavirus No. 1 monoclonal antibody labeled by colloidal gold; the nitrocellulose membrane is coated with a mouse anti-human novel coronavirus No. 2 monoclonal antibody as a detection line and a novel coronavirus recombinant antigen as a quality control line. The background fluorescence immunochromatographic test strip provided by the invention adopts a sandwich/competition combination mode to detect the new coronavirus N antigen, has better sensitivity and specificity and higher recovery rate, and provides great convenience for clinical use.
Description
Technical Field
The invention relates to the technical field of immunochromatography, in particular to a novel coronavirus N antigen background fluorescence immunochromatography test strip and a preparation method thereof.
Background
The new coronaviruses are composed of four structural proteins and single-stranded positive-strand RNA, namely Spike Protein (Spike Protein), Nucleocapsid Protein (Nucleocapsid Protein), Envelope Protein (Envelope Protein) and Membrane Protein (Membrane Protein), which play a role in molecular characterization and host cell entry. The nucleocapsid protein (N protein) is combined with the viral genomic RNA to form a spiral ribocapsid, participates in genome protection, viral RNA replication, virosome assembly and immune evasion, and is an important target protein for antigen-antibody detection.
The new coronaviruses are composed of four structural proteins and single-stranded positive-strand RNA, namely Spike Protein (Spike Protein), Nucleocapsid Protein (Nucleocapsid Protein), Envelope Protein (Envelope Protein) and Membrane Protein (Membrane Protein), which play a role in molecular characterization and host cell entry. The nucleocapsid protein (N protein) is combined with the viral genomic RNA to form a spiral ribocapsid, participates in genome protection, viral RNA replication, virosome assembly and immune evasion, and is an important target protein for antigen-antibody detection.
At present, an immunochromatographic test strip is used for detecting the infection of the new coronavirus, and the detection of the IgG/IgM antibody and the total antibody of the new coronavirus is generally carried out. But the test paper for detecting the novel coronavirus antigen is less. China generally detects novel coronavirus through PCR-based nucleic acid detection. Therefore, the invention provides a novel test strip for detecting coronavirus antigens, which is suitable for clinical auxiliary diagnosis of novel coronavirus infection and has the characteristics of rapidness, accuracy and high sensitivity.
Disclosure of Invention
Therefore, the problem to be solved by the invention is to provide a test strip for detecting a novel coronavirus N antigen, which is suitable for clinical auxiliary diagnosis of novel coronavirus infection, has the characteristics of rapidness, accuracy and high sensitivity, and can realize field detection of a sample and provide a quantitative detection result.
The invention provides a novel coronavirus N antigen background fluorescence immunochromatographic test strip and a preparation method thereof, wherein the preparation method comprises the following steps:
the PVC base plate and the base plate are sequentially overlapped and lapped with a sample pad, a gold-labeled combination pad, a nitrocellulose membrane and a water absorption pad. The nitrocellulose membrane is provided with a detection line and a quality control line which are spaced, wherein the two lines are firstly immobilized with background fluorescent dye and then coated with an antibody. The detection line is close to the gold-labeled combination pad, and the quality control line is close to the water absorption pad.
The solid carrier liquid of the background fluorescent dye comprises the following components in percentage by weight:
the balance was 0.01M, pH phosphate buffer solution at 7.4.
The gold-labeled bonding pad is coated with a colloidal gold-labeled mouse anti-human novel coronavirus monoclonal antibody 1.
A mouse anti-human novel coronavirus monoclonal antibody 2 is coated on a detection line of the nitrocellulose membrane.
The quality control line of the nitrocellulose membrane is coated with a novel coronavirus N antigen.
The test strip has the advantages that the coating concentration of the novel coronavirus monoclonal antibody on the detection line is 2 mg/ml.
The test paper strip has the quality control line that the coating concentration of the novel coronavirus N antigen is 0.5 mg/ml.
The test paper strip is combined with the novel coronavirus monoclonal antibody marked on the pad, and the concentration of the novel coronavirus monoclonal antibody is 16.8 ug/ml.
The particle size of the colloidal gold particles on the gold-labeled bonding pad is 20-40 nm.
The distance between the detection line and the quality control line is 0.8-1cm, and the detection line and the quality control line are not interfered with each other.
The sample pad and the gold label pad are both glass cellulose membranes.
In the invention, the background fluorescence immunochromatographic test strip adopts a mode of combining double antibody sandwich and competition, and is combined with the quenching effect of colloidal gold on fluorescence. If the sample is positive, the novel coronavirus antigen in the sample is combined with the gold-labeled monoclonal antibody, chromatography is carried out on absorbent paper under the capillary action, the sample is combined with the immobilized capture monoclonal antibody when passing through the detection line so as to generate quenching effect on the fluorescence, and the rest of the gold-labeled antibody which is not combined with the sample antigen is combined with the novel coronavirus antigen at the quality control line. And if the sample is negative, the gold-labeled antibody is not combined with the capture antibody when the sample flows to the detection line in the chromatography, so that fluorescence quenching cannot be generated, more combination is performed when the sample flows to the control line, and the quenching effect is stronger. The quantitative detection of the novel coronavirus antigen is realized through the fluorescence intensity ratio of the detection line and the quality control line.
The invention provides a preparation method of a test strip for detecting a novel coronavirus N antigen, which comprises the following steps:
1) preparing a gold-labeled conjugate pad, and coating the gold-labeled conjugate pad with the novel coronavirus monoclonal antibody 1 labeled by colloidal gold.
2) Preparing a nitrocellulose membrane, coating the novel coronavirus monoclonal antibody 2 on the nitrocellulose membrane as a detection line, and coating the novel coronavirus antigen as a quality control line.
3) And sequentially overlapping the sample pad, the gold label combination pad, the nitrocellulose membrane and the absorbent filter paper on the PVC bottom plate from left to right.
In the preparation method, the antibody antigen is purchased from Wuhan Fengceng company, and has no special regulation, and the selection and preparation can be carried out by a person skilled in the art according to actual needs.
In the preparation method, the antibody coating buffer solution is 0.01M, the PBS buffer solution with the pH value of 7.4D, and the Tris-HCL buffer solution with the antigen coating buffer solution is 0.01M and the pH value of 8.0.
In the preparation method, the preparation of the gold-labeled bonding pad comprises the following steps:
1) a1.5 mL centrifuge tube is taken, 1mL of colloidal gold solution is added, the pH of the solution is adjusted by potassium carbonate solution, 16.8uL of 1mg/mL monoclonal antibody 1 is added, the mixture is placed in a refrigerator at 4 ℃ for bonding for half an hour and then taken out, 50uL of 10% BSA solution is added for sealing for 1 hour, and then the mixture is taken out and centrifuged at 12000r/min and 4 ℃ for 20 min.
2) After removal, the mixture was reconstituted with 0.01M PBS containing 1% BSA, 5% sucrose at pH 7.4.
3) The re-solution was coated on the pre-treated bonding pad.
4) And drying the bonding pad in a 37 ℃ oven for 1 hour, taking out, sealing and storing to obtain the gold-labeled bonding pad.
In the preparation method, the invention provides a test paper strip for quantitatively detecting novel coronavirus, and the specific use method is as follows:
1) and sucking 70uL of a sample to be detected, and slowly dripping the sample to be detected on a sample pad of the test strip.
2) The sample was taken out about 15 minutes at the time of measurement, and the result was read with a self-assembled fluorometer.
The technical scheme of the invention has the following advantages:
1) the invention provides a novel coronavirus N antigen background fluorescence immunochromatographic test strip and a preparation method thereof, wherein the preparation method comprises the following steps: the PVC bottom plate and the bottom plate are sequentially overlapped and lapped with a sample pad, a gold-labeled combination pad, a nitrocellulose membrane and a water absorption pad. The nitrocellulose membrane is provided with a detection line and a quality control line which are spaced from each other, the detection line is close to the gold-labeled combination pad, and the quality control line is close to the water absorption pad. The gold-labeled binding pad is coated with a novel coronavirus monoclonal antibody 1 labeled by colloidal gold, the detection line is coated with a novel coronavirus monoclonal antibody 2, and the quality control line is coated with a novel coronavirus antigen. By pretreating the sample and the conjugate pad, the flow of the sample is smooth without being retarded, and nonspecific adsorption is prevented. The fluorescent dye is immobilized on the detection line and the quality control line in advance, so that the antibody is prevented from being directly marked by the fluorescent dye, and the common colloidal gold is used for marking the antibody, so that the method is simple and convenient to operate, easy to couple, cost-saving and free of damaging the activity of the antibody.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings used in the embodiments will be briefly described below.
Fig. 1 is a structural schematic diagram of a novel coronavirus N antigen background fluorescence immunoassay chromatography test strip according to an embodiment of the invention.
In the figure, the lower part is a PVC bottom plate, and the upper part is a sample pad, a gold-labeled combination pad, a nitrocellulose membrane and a water absorption pad in sequence from left to right.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited thereto.
The experimental procedures in the following examples were carried out in the conventional manner unless otherwise specified, and the experimental materials used were purchased from conventional biochemical reagent manufacturers unless otherwise specified.
Example 1:
a novel coronavirus N antigen background fluorescence immunochromatographic test strip is shown in figure 1, wherein a sample pad, a gold-labeled binding pad, a nitrocellulose membrane and a water absorption pad are sequentially lapped on a bottom plate.
The nitrocellulose membrane is provided with a detection line T and a quality control line C which are spaced from each other, the detection line is close to the gold-labeled combination pad, and the quality control line is close to the water absorption pad.
The gold-labeled bonding pad is coated with a colloidal gold-labeled mouse anti-human novel coronavirus monoclonal antibody 1.
A mouse anti-human novel coronavirus monoclonal antibody 2 is coated on a detection line of the nitrocellulose membrane.
The quality control line of the nitrocellulose membrane is coated with a novel coronavirus N antigen.
Example 2:
this example provides the composition of the background fluorescence solid carrier solution on the nitrocellulose membrane of example 1, including the following:
the balance was 0.01M, pH phosphate buffer solution at 7.4.
Example 3:
this example provides the preparation of dilutions of antibody antigen and nitrocellulose membrane in example 1.
1) The nitrocellulose membrane was cut into a strip having a width of 3mm and a length of 10cm for use.
2) Both monoclonal antibodies were diluted to the desired concentration with 0.01M PBS buffer at pH 7.4 (1 mg/ml for antibody 1 and 2mg/ml for antibody 2) and the antigen was diluted to 0.5mg/ml with 0.01M Tris-HCL buffer at pH 8.0.
3) The resulting diluted solution was scribed on nitrocellulose using a gold-spraying film scribing apparatus, and scribing was performed at a speed of 0.4 ul/cm.
4) And (3) placing the cellulose nitrate membrane with the marked line in an oven to be dried for 0.5 hour at 37 ℃, taking out after drying, and sealing and storing.
Example 4:
this example provides the preparation of the colloidal gold of example 1.
1) Adding 0.01 percent of chloroauric acid solution calculated by the total amount under heating.
2) After boiling under heating for 3 minutes, 1% by weight of trisodium citrate, calculated as the total amount, is added.
3) The reaction is carried out under the heating condition, thereby preparing the colloidal gold solution with the required particle size.
This example provides the preparation of the gold labeled conjugate pad of example 1.
1) A1.5 mL centrifuge tube is taken, 1mL of colloidal gold solution is added, the pH of the solution is adjusted by potassium carbonate solution, 16.8uL of 1mg/mL monoclonal antibody 1 is added, the mixture is placed in a refrigerator at 4 ℃ for bonding for half an hour and then taken out, 50uL of 10% BSA solution is added for sealing for 1 hour, and then the mixture is taken out and centrifuged at 12000r/min and 4 ℃ for 20 min.
2) After removal, the mixture was reconstituted with 0.01M PBS containing 1% BSA, 5% sucrose at pH 7.4.
3) The re-solution was coated on the pre-treated bonding pad.
4) And drying the bonding pad in a 37 ℃ oven for 1 hour, taking out, sealing and storing to obtain the gold-labeled bonding pad.
As shown in fig. 1, the detection principle of the novel coronavirus N antigen background fluorescence immunoassay test strip of the present invention is as follows:
in conclusion, the background fluorescence immunochromatographic test strip adopts a mode of combining double antibody sandwich and competition, and realizes the detection of the target sample by combining the quenching effect of colloidal gold on fluorescence. If the sample is positive, the novel coronavirus antigen in the sample is combined with the gold-labeled monoclonal antibody, chromatography is carried out on the sample under the capillary action, the sample is combined with the immobilized capture monoclonal antibody when passing through the detection line, so that the fluorescence on the sample is quenched, and the rest of the gold-labeled antibody which is not combined with the sample antigen is combined with the novel coronavirus antigen at the quality control line. And if the sample is negative, the gold-labeled antibody is not combined with the capture antibody when the sample flows to the detection line by chromatography, so that fluorescence quenching cannot be generated, more binding is realized when chromatography is carried out to the control line, and the quenching effect is stronger. The quantitative detection of the novel coronavirus antigen is realized through the fluorescence intensity ratio of the detection line and the quality control line.
Claims (10)
1. A background fluorescence immunochromatographic test strip based on a novel coronavirus N antigen is characterized in that:
a sample pad, a gold-labeled combination pad, a nitrocellulose membrane and absorbent paper are sequentially arranged on the PVC base plate from left to right;
the gold-labeled combination pad is coated with a mouse anti-human novel coronavirus No. 1 monoclonal antibody labeled by colloidal gold;
the nitrocellulose membrane is coated with a mouse anti-human novel coronavirus No. 2 monoclonal antibody as a detection line and a novel coronavirus recombinant N antigen as a quality control line (a competition line);
the solid carrier liquid of the background fluorescent dye comprises the following components in percentage by weight:
the balance was 0.01M, pH phosphate buffer solution at 7.4.
2. The novel coronavirus N-antigen background fluorescence immunochromatographic test strip according to claim 1, which is characterized in that: the amount of 1mg/ml of the novel coronavirus N protein monoclonal antibody 1 on the conjugate pad was 16.8uL, and the amount of coating on the conjugate pad was 400 uL: the concentration of the mouse anti-human novel coronavirus No. 2 monoclonal antibody coated on the nitrocellulose membrane is 2mg/ml, and the coating amount is 6 uL; the concentration of the novel coronavirus recombinant N antigen is 0.5mg/ml, and the coating amount is 6 uL.
3. The novel coronavirus N-antigen background fluorescence immunochromatographic test strip according to claim 1, which is characterized in that: the combination pad is treated by combination pad pretreatment liquid; the combined pad treatment liquid comprises the following raw materials: 5% sucrose, 1% tween-20, 1% BSA in 0.01M phosphate buffered saline at pH 7.4.
4. The novel coronavirus N-antigen background fluorescence immunochromatographic test strip according to claim 1, which is characterized in that: the sample pad is treated with a sample pad pretreatment solution: the sample pad treatment solution comprises the following raw materials: tween-20 1%, BSA 1% in 0.01M phosphate buffered saline at pH 7.4.
5. The novel coronavirus N-antigen background fluorescence immunochromatographic test strip according to claim 1, which is characterized in that: the grain size of the colloidal gold is 20-40 nm.
6. The novel coronavirus N-antigen background fluorescence immunochromatographic test strip according to claim 1, which is characterized in that: the sample pad and the conjugate pad are both glass cellulose membranes.
7. The novel coronavirus N-antigen background fluorescence immunochromatographic test strip according to claim 1, which is characterized in that: the preparation of the colloidal gold comprises the following steps:
1) adding 0.01 percent of chloroauric acid solution calculated according to the total amount under the heating condition;
2) boiling for 3 minutes under heating condition, and adding 1 percent of trisodium citrate calculated by the total amount;
3) the reaction is carried out under the heating condition, so as to prepare the colloidal gold solution with the required particle size.
8. The novel coronavirus N-antigen background fluorescence immunochromatographic test strip according to claims 1 to 7, which is characterized by comprising the following steps:
1) preparing a gold-labeled binding pad, and coating a colloidal gold-labeled mouse anti-human novel coronavirus No. 1 monoclonal antibody on the binding pad;
2) preparing a nitrocellulose membrane, coating a mouse anti-human novel coronavirus No. 2 monoclonal antibody on the nitrocellulose membrane as a detection line, and coating a novel coronavirus recombinant N antigen as a quality control line (competition line);
3) and sequentially building a sample pad, a gold-labeled combination pad, a nitrocellulose membrane and absorbent paper on the PVC bottom plate from left to right.
9. The novel coronavirus N-antigen background fluorescence immunochromatographic test strip according to claim 8, wherein the preparation of the gold-labeled conjugate pad comprises the following steps:
1) taking a 1.5mL centrifuge tube, adding 1mL of colloidal gold solution, adjusting the pH of the solution by using potassium carbonate solution, adding 16.8uL of 1mg/mL monoclonal antibody 1, placing the solution in a refrigerator at 4 ℃, combining for half an hour, taking out the solution, adding 50uL of 10% BSA solution, sealing for 1 hour, taking out the solution, and centrifuging for 20min at 12000r/min and 4 ℃;
2) taking out, and re-dissolving with PBS (0.01M) containing 1% BSA and 5% sucrose and having pH of 7.4;
3) coating the re-solution on the pretreated bonding pad;
4) and drying the bonding pad in a 37 ℃ oven for 1 hour, taking out, sealing and storing to obtain the gold-labeled bonding pad.
10. A novel coronavirus N antigen background fluorescence immunochromatographic test strip and a preparation method thereof are characterized in that: the test strip comprises the background fluorescence immunochromatographic test strip of any one of claims 1 to 9.
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2021
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