CN111273008A - Colloidal gold immunochromatography kit for rapidly detecting novel coronavirus IgM antibody and preparation method thereof - Google Patents
Colloidal gold immunochromatography kit for rapidly detecting novel coronavirus IgM antibody and preparation method thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Abstract
The invention discloses a colloidal gold immunochromatographic kit for rapidly detecting novel coronavirus IgM antibody and a preparation method thereof, relating to the field of biological medicine, adopting an antigen-antibody sandwich method and a colloidal gold immunochromatographic method principle to qualitatively detect whether anti-novel coronavirus nucleocapsid protein IgM antibody exists in human serum or plasma, applying colloidal gold labeled novel coronavirus nucleocapsid protein containing 6 xHis label to combine to form gold labeled N protein and then adsorbing the gold labeled N protein on a gold labeled pad, using the novel coronavirus nucleocapsid protein as an indication marker, coating a mouse anti-human u chain monoclonal antibody on a detection line of an NC membrane and coating a mouse anti-6 xHis monoclonal antibody on a quality control line of the NC membrane, realizing qualitative detection of the anti-novel coronavirus nucleocapsid protein IgM antibody in blood by the sandwich method technology, conveniently detecting whether the anti-novel coronavirus nucleocapsid protein IgM antibody exists in the blood, the kit has high sensitivity, short detection time and strong anti-interference so as to be used by a clinician to quickly judge whether to diagnose the novel coronavirus.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to a colloidal gold immunochromatographic kit for rapidly detecting a novel coronavirus IgM antibody and a preparation method thereof, in particular to a kit for qualitatively detecting the novel coronavirus nucleocapsid protein IgM antibody in blood by applying a colloidal gold immunochromatographic method based on the sandwich method principle.
Background
The common signs of the novel coronavirus (2019-nCoV) are fever, hypodynamia, dry cough and gradual dyspnea, while the disease symptoms of part of patients are slight, even asymptomatic patients without clinical symptoms exist. The human-transmissible agent has the characteristic of human transmissible, the incubation period is 1-14 days generally, the incubation period is infectious, asymptomatic infected persons can also become an infection source, and the infection is transmitted mainly through respiratory droplets and close contact, so that the human is generally susceptible.
So far, there are still many suspected cases that have not been diagnosed yet because of reasons such as insufficient detection efficiency, how to rapidly screen suspected cases on a large scale for clinicians to rapidly judge whether the suspected cases are novel coronavirus pneumonia, so as to rapidly isolate and prevent infection to more people, which is a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a colloidal gold immunochromatographic kit for quickly detecting a novel coronavirus IgM antibody, which is quick, simple, reliable, low in cost and high in sensitivity.
The invention also aims to provide a preparation method of the colloidal gold immunochromatographic kit for rapidly detecting the novel coronavirus IgM antibody.
The technical scheme provided by the invention is as follows: a colloidal gold immunochromatographic kit for rapidly detecting a novel coronavirus IgM antibody comprises a box body and a pasting support positioned in the box body, wherein a sample pad, a gold label pad, an NC membrane and a sample sucking pad are sequentially pasted on the pasting support in the direction of the extension degree, a detection line is arranged on the NC membrane close to the gold label pad side, a quality control line is arranged on the NC membrane close to the sample sucking pad side, the gold label pad contains a gold label N protein, and the gold label N protein is a novel coronavirus nucleocapsid protein which is labeled by colloidal gold and contains a 6 XHis label;
the detection line is coated with a mouse anti-human u chain monoclonal antibody;
the quality control line is coated with a mouse anti-6 × His monoclonal antibody.
As a further improvement of the invention, the preparation method of the gold-labeled N protein comprises the following steps:
s101, preparing colloidal gold: adding 2mL of 1% chloroauric acid into 100mL of aqueous solution, heating to boil, adding 2-5mL of 1% trisodium citrate, mixing uniformly, boiling until the color of the colloidal gold solution changes from blue to mauve, and cooling;
s102, preparing a gold-labeled solution: adding 100 mu L0.1M PB (with a pH value of 7.2) into 1mL of colloidal gold, uniformly mixing, adding 0.1M NaOH solution to adjust the pH value to 7.2-7.6, adding 15-25 mu g of novel coronavirus nucleocapsid protein containing 6 XHis tags, fully mixing, adding 100 mu L of bovine serum albumin with a volume of 10%, and uniformly mixing;
s103, purifying the gold standard solution: centrifuging the gold-labeled solution, removing the supernatant, and dissolving in 20% sucrose-containing gold-labeled complex solution.
As a further improvement of the invention, the particle size of the colloidal gold in the gold-labeled N protein is 15-30 nm.
As a further improvement of the invention, the coating concentration of the mouse anti-6 XHis monoclonal antibody on the quality control line is 0.75-1mg/ml, and the coating concentration of the mouse anti-human u-chain monoclonal antibody on the detection line is 1.0-1.5 mg/ml.
As a further improvement of the invention, the colloidal gold in the gold-labeled N protein is subjected to antibody cross-linking with the novel coronavirus nucleocapsid protein containing the 6 XHis tag near the isoelectric point of the protein.
As a further improvement of the invention, the content of the novel coronavirus nucleocapsid protein containing the 6 XHis tag on the gold-labeled pad is higher than that of the mouse anti-human u-chain monoclonal antibody on the detection line.
As a further improvement of the invention, the sample pad material is a glass fiber membrane, and is treated overnight by a treatment solution containing Tris, sodium caseinate and PVP-10.
As a further improvement of the invention, the gold-labeled pad is made of a glass fiber membrane and is treated by a treatment solution containing Tris, S-17 and bovine serum albumin.
A preparation method of a colloidal gold immunochromatographic kit for rapidly detecting novel coronavirus IgM antibodies comprises the following steps:
s1, preparing a gold-labeled pad: preparing gold-labeled N protein by labeling a novel coronavirus nucleocapsid protein mouse containing a 6 × His label with colloidal gold, spraying the gold-labeled N protein on a glass fiber membrane, and drying to obtain a gold-labeled pad;
s2, preparation of NC film: coating a mouse anti-human u-chain monoclonal antibody on a nitrocellulose membrane to form a detection line, and coating a mouse anti-6 × His monoclonal antibody on the nitrocellulose membrane to form a quality control line;
s3, cutting and assembling: and (3) sequentially adhering the sample pad, the gold label pad prepared in the step (S1), the NC membrane prepared in the step (S2) and the sample sucking pad in the length direction of the adhering bracket, cutting the sample pad, the NC membrane and the sample sucking pad into test strips with the width of 3.5mm +/-0.5 mm, and filling the test strips into a box body to obtain the reagent strip.
As a further improvement of the invention, in step S1, the spraying amount of the gold-labeled N protein on the gold-labeled pad is 1.4-2.0 μ L/cm.
The main materials of the application are as follows:
sample pad: the material is a glass fiber membrane, the glass fiber membrane is treated overnight by sample pad treatment fluid (mainly containing Tris, casein sodium salt and PVP-10), and the sample pad is used for removing large particles in a sample and buffering a detection system so as to ensure the cross linking of an antigen and a specific antibody in an environmental system;
gold label pad: glass fiber membrane treated by special treatment liquid (containing Tris, S-17, bovine serum albumin and the like) is sprayed with gold-labeled N protein; the exact sample amount of the colloidal gold labeled antibody needs to be strictly tested, a detection line is suitable for color development detection of the anti-novel coronavirus nucleocapsid protein IgM antibody, the colloidal gold labeled N protein and the anti-novel coronavirus nucleocapsid protein IgM antibody are specifically combined to form a colloidal gold labeled compound, redundant colloidal gold labeled antibody is required to enter a quality control line to show color, the color of the quality control line cannot be displayed when the colloidal gold labeled on the colloidal gold labeled N protein contains the 6 XHis labeled novel coronavirus nucleocapsid protein, and the colloidal gold labeled N protein becomes a failure strip, so that the content of the 6 XHis labeled novel coronavirus nucleocapsid protein on the colloidal gold labeled N protein is higher than the content of mouse anti-human u chain monoclonal antibody on the detection line, and the excessive colloidal gold labeled N protein reaches a T region and can generate false positive, and the spraying amount of the colloidal gold labeled N protein on the colloidal gold labeled N protein is 1.4-2.0 muL/cm;
NC membrane (nitrocellulose membrane): the first area scribed on the nitrocellulose membrane is a detection line area, the second area scribed on the nitrocellulose membrane is a quality control line area, the detection line and the quality control line are in a linear shape covered on the nitrocellulose membrane, the detection line is coated with the mouse anti-human u-chain monoclonal antibody to capture a colloidal gold cross-linked complex, the quality control line is coated with the mouse anti-6 XHis monoclonal antibody, when the complex reaches the quality control line from the gold-labeled pad in a capillary motion manner, the complex is captured by the mouse anti-6 XHis monoclonal antibody to form a visible wine red strip, and the quality control line is a control line so as to test the effectiveness of the detection system, if the quality control line does not go out, the test is invalid;
a sample sucking pad: the Whatman filter paper is used for promoting the sample chromatography to pass through the sample pad, the gold label pad and the nitrocellulose membrane and reach the sample sucking pad area so as to absorb redundant samples to finish the detection;
pasting a support: is a PVC backup pad made of thin PVC plastic to support the assembly.
The detection principle of the application is as follows: during detection, a serum or plasma sample to be detected is dripped on a sample pad, and moves forwards to a gold-labeled pad through capillary effect chromatography, because the uninfected normal human blood does not contain anti-novel coronavirus nucleocapsid protein IgM antibody, and the blood of a patient infected with novel coronavirus has anti-novel coronavirus nucleocapsid protein IgM antibody (the IgM antibody is an antibody appearing in an acute infection stage, the patient can be positive about 3 days after the disease occurs, and can disappear after 1 to 2 months after the infection and the hair is dyed), if the detected sample contains the anti-novel coronavirus nucleocapsid protein IgM antibody, the gold-labeled N protein and the anti-novel coronavirus nucleocapsid protein IgM antibody can be specifically combined to form a gold-labeled compound, and are combined with the mouse anti-human u-chain monoclonal antibody fixed on a detection line in the chromatography process to form an Au-sandwich antigen-antibody compound of 'N protein-anti-IgM antibody-mouse anti-human u-chain monoclonal antibody containing 6 xHIS mark', the sandwich antigen-antibody complex is gathered on a detection line to form a wine red strip, then whether the anti-novel coronavirus nucleocapsid protein IgM antibody exists in blood or not is detected on the detection line through visual inspection, the fact that the wine red strip does not appear in a detection line area indicates that the anti-novel coronavirus nucleocapsid protein IgM antibody does not exist in a sample, no matter whether the antibody exists in the detected sample or not, N protein with a gold mark containing a 6 XHIS mark is subjected to upward chromatography to a quality control line and reacts with a mouse anti-6 XHis monoclonal antibody to form a wine red strip, the wine red strip presented by the quality control line is a standard for judging whether a chromatography process is normal or not, and is also used as an internal control standard of a reagent.
Compared with the prior art, the invention has the following advantages:
the invention relates to a colloidal gold immunochromatographic kit for rapidly detecting a novel coronavirus IgM antibody, which is characterized by adopting an antigen-antibody sandwich method and a colloidal gold immunochromatographic method to qualitatively detect whether an anti-novel coronavirus nucleocapsid protein IgM antibody exists in human serum or plasma, applying colloidal gold labeled novel coronavirus nucleocapsid protein containing 6 XHis labeled to combine to form gold labeled N protein, then adsorbing the gold labeled N protein on a gold labeled pad, using the novel coronavirus nucleocapsid protein containing 6 XHis labeled as an indication marker, coating a mouse anti-human u chain monoclonal antibody on a detection line of an NC membrane and coating a mouse anti-6 XHis monoclonal antibody on a quality control line of the NC membrane, and realizing qualitative detection of the anti-novel coronavirus nucleocapsid protein IgM antibody in blood by the sandwich method technology, and the invention is a dynamic monitoring device for household or clinic, which can conveniently and rapidly detect whether the anti-novel coronavirus nucleocapsid protein IgM antibody exists in the blood, the method is used for clinicians to quickly judge whether to diagnose the novel coronavirus, has short detection time, convenient use, high detection sensitivity, strong anti-interference capability and low cost, and is suitable for large-scale quick screening of the novel coronavirus initial-stage infection patients.
The preparation method of the colloidal gold immunochromatographic kit for rapidly detecting the novel coronavirus IgM antibody has the advantages of simple preparation process, low cost, simple and convenient product use and suitability for popularization.
Drawings
FIG. 1 is a schematic structural diagram of the colloidal gold immunochromatographic kit for rapidly detecting novel coronavirus IgM antibodies of the present invention.
FIG. 2 is a graph showing the absorbance of colloidal gold bound to a novel coronavirus nucleocapsid protein containing a 6 XHis tag in solutions of paragraph 1.2.2 at different pH values prepared for the assay of the present application.
Detailed Description
The following describes a specific embodiment of the present invention with reference to the drawings and the detailed description.
Test preparation for the present application:
1. screening of preparation conditions for gold-labeled N protein
1.1 determination of the size of the gold colloidal particles
The preparation principle is as follows: the colloidal gold is produced by a trisodium citrate reduction method, the gold particle size is greatly influenced by factors such as trisodium citrate concentration, and the colloidal gold with different particle sizes can be produced by changing the trisodium citrate concentration.
The test method comprises the following steps: adding 2mL of 1% chloroauric acid (HAuCl 4) solution into 100mL of water, heating to boil, adding 1-8 mL of 1% trisodium citrate, mixing and boiling for 5 minutes until the color is not changed.
The marking effect experiment of the colloidal gold particles with different particle sizes is as follows: colloidal gold particles with different diameters are prepared by the method, novel coronavirus nucleocapsid protein containing 6 XHis labels are labeled to prepare a reagent, the biological activity of gold-labeled binders with different particle sizes is compared by using the lowest detection quantity of a quality control product (Cut-Off, 10 copies of virus/ml) and requiring that a detection line appears within 2 minutes and the same below) and negative serum sample adding (requiring that no detection line appears within 20 minutes), and the results are shown in the following table 1.
Experimental results and analysis:
table 1: comparison of the effect of gold particle labeling of different sizes:
| amount of trisodium citrate | Diameter of gold particle | Cut-Off results | Negative serum results | Stability of |
| 8mL | 18nm | With an outgoing line (++) within 3 minutes | ― | In general |
| 4mL | 25nm | With an outgoing line (++) within 3 minutes | ― | Good taste |
| 1mL | 50nm | No line (-) was formed in 3 minutes | ― | Difference (D) |
(Note "-" indicates a negative result, i.e., a mauve band appears on the quality inspection line and no mauve band appears on the inspection line; and "+" indicates a positive result, i.e., a mauve band appears on each of the quality inspection line and the inspection line.)
From the experimental results, the colloidal gold of 18nm and 25nm can simultaneously meet the requirements of the detection time and the detection result of the minimum detection quantity quality control product and the negative serum. Therefore, 15-30nm of colloidal gold is selected for labeling, and the preparation process of the colloidal gold particles is determined as follows: adding 1% chloroauric acid 2mL into 100mL of water solution, heating to boil, adding trisodium citrate 1% 2-5mL, mixing, boiling for 5 min, and cooling for use after the color of the colloidal gold solution changes from blue to purple. The colloidal gold particles produced by the process meet the detection requirements of reagents.
1.2 Condition optimization of colloidal gold-tagged 6 × His-tagged novel coronavirus nucleocapsid proteins
1.2.1 determination of the binding concentration of the novel coronavirus nucleocapsid protein containing 6 × His tag to colloidal gold
Materials and test methods: taking 1mL of colloidal gold solution, adjusting the pH value to about 7.0, respectively adding 0 mug, 5 mug, 10 mug, 15 mug, 20 mug and 25 mug of novel coronavirus nucleocapsid protein containing 6 XHis labels which are respectively labeled as 1#, 2#, 3#, 4#, 5# and 6#, standing for 10min, adding 100 mug L of 10% NaCl, mixing uniformly, standing for 2h, and observing the result. (Note: the novel coronavirus nucleocapsid protein containing 6 XHis tag is a product of Darri Biotechnology Ltd.)
Results and analysis: the colloidal gold is a negatively charged hydrophobic particle, and when no protein or antigen is added or the amount of the added antigen is not enough to adsorb the complete colloidal gold, the property of the colloid is changed and the coagulation phenomenon from red to blue appears when NaCl is added due to the salt ion effect. We observed that tubes # 4, # 5 and # 6 did not change color, tubes # 1 and # 2 turned blue, and tubes # 3 did not change color, so that the amount of protein required to stabilize 1mL of colloidal gold was 15-25. mu.g.
1.2.2 determination of the pH Range of colloidal gold when binding to the novel coronavirus nucleocapsid protein containing the 6 × His tag
Materials and test methods: to a 1mL volume of colloidal gold solution (to which 100. mu.L of 0.1M, pH7.2 PB had been added), 1M NaOH solution was added to adjust the pH to 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, and then 20. mu.g of the novel coronavirus nucleocapsid protein containing the 6 XHis tag was added, the mixture was left to stand for 5 minutes, 100. mu.L of 10% NaCl solution was added, and after standing for 10 minutes, the absorbance at 520nm and 580nm was measured. The differential absorbance (A520-A580) was plotted on the ordinate and the pH on the abscissa, and the results are shown in FIG. 2.
Results and analysis: as shown in FIG. 2, colloidal gold was labeled in solutions of different pH values, and the A520-A580 difference peaked at pH 7.2-7.6, which indicates that the adsorption of colloidal gold and protein was good in this pH range, and thus pH 7.2-7.6 was selected as the pH range in which the colloidal gold was labeled with the novel coronavirus nucleocapsid protein containing 6 XHis tag.
1.3 determination of amount of gold-labeled N protein sprayed
The principle is as follows: the amount of gold on the gold pad greatly affects the sensitivity of the final reagent.
Materials and test methods: 1.0 muL, 1.2 muL, 1.4 muL, 1.6 muL, 1.8 muL and 2.0 muL of gold-labeled N protein are uniformly sprayed on the gold-labeled pad according to each 1cm, dried to prepare a reagent, and the reagent is detected by Cut-Off and negative serum and observed, and the result is shown in Table 2.
Results and analysis:
table 2: test results for different coating weights
| Spray amount (μ L/cm) | Cut-Off results | Negative serum results |
| 1.0 | No line (-) was formed in 2 minutes | － |
| 1.2 | No line (-) was formed in 2 minutes | － |
| 1.4 | 2 min with out line (+) | － |
| 1.6 | 2 min with out line (+) | － |
| 1.8 | 2 min with out line (+) | － |
| 2.0 | 2 min with out line (+) | － |
(Note "-" indicates a negative result, i.e., a mauve band appears on the quality inspection line and no mauve band appears on the inspection line; and "+" indicates a positive result, i.e., a mauve band appears on each of the quality inspection line and the inspection line.)
From the experimental results, when the spraying amount of the gold-labeled N protein is 1.4-2.0 muL/cm, the detection results of Cut-Off and negative serum meet the design requirements.
2. Determination of NC membranous control line coated mouse anti-6 XHis monoclonal antibody concentration
The main materials are as follows: murine anti-6 × His mab: product of Metammune, USA; NC film: millipore hf135NC membranes; preparing a coating solution: PBS +0.5% BSA +3% sucrose, pH 7.4.
The test method comprises the following steps: the mouse anti-6 XHis monoclonal antibody is diluted into 1.5mg/ml, 1mg/ml, 0.75mg/ml and 0.65mg/ml by PBS, coated on an NC membrane to be used as a quality control line, the gold-labeled mouse anti-human u chain monoclonal antibody is used for forming a test strip after being dried, the PBS is used as a sample for testing, the outlet condition of the quality control line is observed within 12 minutes, and the experimental result is shown in Table 3.
Results and analysis:
TABLE 3 detection results of different concentrations of mouse anti-6 × His monoclonal antibody coating
| Concentration (mg/ml) | 1.5 | 1 | 0.75 | 0.65 |
| Results | Reddish brown, fuzzy and dense lines | Bright red, uniform and compact lines | Bright red, uniform and compact lines | Light red, blurred lines |
The experimental result shows that when the concentration of the mouse anti-6 XHis monoclonal antibody is coated by 0.75-1mg/ml, the color band is obvious, the thickness is proper, the uniformity is uniform and the density is compact.
3. Determination of concentration of anti-human u chain monoclonal antibody of detection line coated mouse on NC membrane
The main materials are as follows: mouse anti-human u-chain monoclonal antibody: is a product of American Metammune company or Beijing Aibitui biotechnology company; NC film: millipore hf135 membranes; preparing a coating solution: PBS +0.5% BSA +3% sucrose, ph 7.4; quality control product: the 2019-nCoV patient serum is a quality control product, is obtained from the Guangdong province disease control center, and is prepared into corresponding concentration after being inactivated at 56 ℃ for 30 minutes.
The test method comprises the following steps: the mouse u chain monoclonal antibody of anti-human is diluted to different concentrations of 2mg/ml, 1.5mg/ml, 1mg/ml and 0.75mg/ml by PBS, parameters are adjusted to be coated on an NC membrane and a quality control line is coated at the same time, internal control products of patient serum are used for testing, and the results are observed and are shown in Table 4.
Results and analysis:
TABLE 4 detection results of mouse anti-human u chain monoclonal antibody coating with different concentrations
| Concentration (mg/ml) | 2 | 1.5 | 1 | 0.75 |
| Low abundance serum | ＋＋ | ＋ | ＋/－ | ＋/－ |
| 50ng/mlCRP | － | － | － | － |
| 1IU/lIL-6 | ＋/－ | － | － | － |
| 60ng/ml calcitonin (calcein) | － | － | － | － |
The results showed that the sensitivity and specificity of detection were good at a coating protein concentration of 1.0-1.5 mg/ml, with specificity decreasing at too high a concentration and sensitivity decreasing at lower a concentration.
4. The test results are summarized as follows:
the main production process and the reaction system are researched by the test preparation, and the kit has the following process parameters: preparing colloidal gold solution with colloidal gold particle diameter of 15-30nm by adopting a chloroauric acid-trisodium citrate method; the labeling amount is 15-25 mug, and the novel coronavirus nucleocapsid protein containing 6 XHis labels is per ml of colloidal gold; the pH range of the colloidal gold label containing the 6 × His-labeled novel coronavirus nucleocapsid protein is 7.2-7.6; the spraying amount of the gold-labeled N protein is 1.4-2.0 mu L/cm; the concentration of the NC membranous control line coated mouse anti-6 × His monoclonal antibody is 0.75-1 mg/ml; the concentration of the mouse anti-human u chain monoclonal antibody coated on the detection line on the NC membrane is 1.0-1.5 mg/ml.
Example 1, a method for preparing a colloidal gold immunochromatographic kit for rapidly detecting a novel coronavirus IgM antibody:
s1, manufacturing a gold label pad 3: adding 2mL of 1% chloroauric acid into 100mL of aqueous solution, heating to boil, adding 2mL of 1% trisodium citrate, mixing uniformly, boiling until the color of the colloidal gold solution changes from blue to purple, and cooling to obtain colloidal gold; adding 1mL of colloidal gold into PB with the volume of 100 mu L0.1M and the pH value of 7.2, uniformly mixing, adding 0.1M NaOH solution to adjust the pH value to 7.2, adding 15 mu g of novel coronavirus nucleocapsid protein containing 6 XHis labels, fully and uniformly mixing, adding 100 mu L of bovine serum albumin with the volume of 10%, and uniformly mixing to obtain a gold standard solution; centrifuging the gold-labeled solution, removing supernatant, adding gold-labeled complex solution (0.01M PB + 1% BSA) containing 20% sucrose, and dissolving to obtain gold-labeled N protein; spraying the gold-labeled N protein on the gold-labeled pad 3 at a coating weight of 1.4 mu L/cm, and drying for 8h in a drying environment;
s2, preparation of NC film 4: coating a mouse anti-human u-chain monoclonal antibody on a nitrocellulose membrane at the concentration of 1mg/ml to form a detection line 41, coating a mouse anti-6 XHis monoclonal antibody on an NC membrane 4 at the concentration of 1mg/ml to form a quality control line 42, and drying for 8 hours in a drying environment;
s3, cutting and assembling: as shown in fig. 1, taking a pasting bracket 1 in a dry environment (temperature 35 ℃, humidity less than 30%), pasting an NC film 4 coated with an antibody at the center of the pasting bracket 1, pasting a sample sucking pad 5 on the pasting bracket 1, laminating the upper edge of the NC film 4, pasting a gold label pad 3 on the lower edge of the NC film 4, pasting a sample pad 2 on the lower edge of the gold label pad 3, and completing the assembly of a reagent large plate; cutting the pasted pasting bracket 1 into test strips with proper width of 3.5mm by a cutting machine; the cut test strips are correctly placed in a box (not shown).
Example 2, a method for preparing a colloidal gold immunochromatographic kit for rapidly detecting a novel coronavirus IgM antibody:
s1, manufacturing a gold label pad 3: adding 2mL of 1% chloroauric acid into 100mL of aqueous solution, heating to boil, adding 3.5mL of 1% trisodium citrate, mixing uniformly, boiling until the color of the colloidal gold solution changes from blue to purple, and cooling to obtain colloidal gold; adding 1mL of colloidal gold into PB with the volume of 100 mu L0.1M and the pH value of 7.2, uniformly mixing, adding 0.1M NaOH solution to adjust the pH value to 7.6, adding 20 mu g of novel coronavirus nucleocapsid protein containing 6 XHis labels, fully and uniformly mixing, adding 100 mu L of bovine serum albumin with the volume of 10%, and uniformly mixing to obtain a gold standard solution; centrifuging the gold-labeled solution, removing supernatant, adding gold-labeled complex solution (0.01M PB + 1% BSA) containing 20% sucrose, and dissolving to obtain gold-labeled N protein; spraying the gold-labeled N protein on a gold-labeled pad 3 in a coating amount of 2.0 mu L/cm, and drying for 16h in a drying environment;
s2, preparation of NC film 4: coating a mouse anti-human u-chain monoclonal antibody on a nitrocellulose membrane at the concentration of 1.5mg/ml to form a detection line 41, coating a mouse anti-6 XHis monoclonal antibody on the nitrocellulose membrane at the concentration of 0.8mg/ml to form a quality control line 42, and drying for 12 hours in a drying environment;
s3, cutting and assembling: as shown in fig. 1, taking a pasting bracket 1 in a dry environment (temperature 35 ℃, humidity less than 30%), pasting an NC film 4 coated with an antibody at the center of the pasting bracket 1, pasting a sample sucking pad 5 on the pasting bracket 1, laminating the upper edge of the NC film 4, pasting a gold label pad 3 on the lower edge of the NC film 4, pasting a sample pad 2 on the lower edge of the gold label pad 3, and completing the assembly of a reagent large plate; cutting the pasted pasting bracket 1 into test strips with proper width of 3.0mm by a cutting machine; the cut test strips are correctly placed in a box (not shown).
Example 3, a method for preparing a colloidal gold immunochromatographic kit for rapidly detecting a novel coronavirus IgM antibody:
s1, manufacturing a gold label pad 3: adding 2mL of 1% chloroauric acid into 100mL of aqueous solution, heating to boil, adding 5mL of 1% trisodium citrate, mixing uniformly, boiling until the color of the colloidal gold solution changes from blue to purple, and cooling to obtain colloidal gold; adding 1mL of colloidal gold into PB with the volume of 100 mu L0.1M and the pH value of 7.2, uniformly mixing, adding 0.1M NaOH solution to adjust the pH value to 7.4, adding 25 mu g of novel coronavirus nucleocapsid protein containing 6 XHis labels, fully and uniformly mixing, adding 100 mu L of bovine serum albumin with the volume of 10%, and uniformly mixing to obtain a gold standard solution; centrifuging the gold-labeled solution, removing supernatant, adding gold-labeled complex solution (0.01M PB + 1% BSA) containing 20% sucrose, and dissolving to obtain gold-labeled N protein; spraying the gold-labeled N protein on the gold-labeled pad 3 at a coating weight of 1.6 mu L/cm, and drying for 16h in a drying environment;
s2, preparation of NC film 4: coating a mouse anti-human u-chain monoclonal antibody on a nitrocellulose membrane at the concentration of 1.3mg/ml to form a detection line 41, coating a mouse anti-6 XHis monoclonal antibody on the nitrocellulose membrane at the concentration of 0.75mg/ml to form a quality control line 42, and drying for 12 hours in a drying environment;
s3, cutting and assembling: as shown in fig. 1, taking a pasting bracket 1 in a dry environment (temperature 35 ℃, humidity less than 30%), pasting an NC film 4 coated with an antibody at the center of the pasting bracket 1, pasting a sample sucking pad 5 on the pasting bracket 1, laminating the upper edge of the NC film 4, pasting a gold label pad 3 on the lower edge of the NC film 4, pasting a sample pad 2 on the lower edge of the gold label pad 3, and completing the assembly of a reagent large plate; cutting the pasted pasting bracket 1 into test strips with proper width of 4.0mm by a cutting machine; the cut test strips are correctly placed in a box (not shown).
Preclinical self-test tests were performed on the kits prepared in examples 1-3 above
Test samples:
(1) experimental samples: novel coronavirus weakly-positive serum (a sample provided by the disease control center in Guangdong province, which is a blood sample labeled with IgM antibody detection amount); negative serum of normal person, wherein the weak positive serum and the negative serum sample are respectively 10 parts, and virus is inactivated at 56 ℃ for 30 minutes;
(2) interference samples: CRP, IL-6, calcein, were prepared to the concentrations of reagents supplied by Albicin Biotechnology, Inc. of Beijing, ProSpec, Israel, Fitzgerald, USA, and Metammune, respectively, and added to normal human serum.
The test method comprises the following steps:
(1) adding 100uL of sample by using a sample adding gun or adding 3 drops of sample to the sample pad by using a burette;
(2) timing by a timer, and horizontally placing and standing for 2-15 minutes;
(3) the color development results of the kit were observed and recorded, and the detection results are shown in table 5.
Table 5: detection result of preclinical self-test
| Test sample | Example 1 testing Results | Example 1 detection of line appearance Colour time | Example 2 testing Results | Example 2 detection of line display Colour time | Example 3 testing Results | Example 3 detection of line appearance Colour time |
| Positive sample 1 | ＋ | 2'24" | ＋ | 2'01" | ＋ | 2'46" |
| Positive sample 2 | ＋ | 2'55" | ＋ | 2'42" | ＋ | 3'02" |
| Positive sample 3 | ＋ | 3'13" | ＋ | 2'56" | ＋ | 3'26" |
| Positive sample 4 | ＋ | 2'07" | ＋ | 2'01" | ＋ | 2'19" |
| Positive sample 5 | ＋ | 2'58" | ＋ | 2'45" | ＋ | 3'10" |
| Positive sample 6 | ＋ | 2'29" | ＋ | 2'20" | ＋ | 2'34" |
| Positive sample 7 | ＋ | 2'10" | ＋ | 2'08" | ＋ | 2'30" |
| Positive sample 8 | ＋ | 3'15" | ＋ | 3'01" | ＋ | 3'21" |
| Positive sample 9 | ＋ | 3'12" | ＋ | 2'46" | ＋ | 3'20" |
| Positive sample 10 | ＋ | 2'43" | ＋ | 2'33" | ＋ | 2'55" |
| Negative sample 1 | － | 0 | － | 0 | － | 0 |
| Negative sample 2 | － | 0 | － | 0 | － | 0 |
| Negative sample 3 | － | 0 | － | 0 | － | 0 |
| Negative sample 4 | － | 0 | － | 0 | － | 0 |
| Negative sample 5 | － | 0 | － | 0 | － | 0 |
| Negative sample 6 | － | 0 | － | 0 | － | 0 |
| Negative sample 7 | － | 0 | － | 0 | － | 0 |
| Negative sample 8 | － | 0 | － | 0 | － | 0 |
| Negative sample 9 | － | 0 | － | 0 | － | 0 |
| Negative sample 10 | － | 0 | － | 0 | － | 0 |
| 50ng/ml CRP/Normal human serum | － | 0 | － | 0 | － | 0 |
| 1000 mIU/lIL-6/normal person Serum | － | 0 | － | 0 | － | 0 |
| 60ng/ml Calcitonin/Zheng Serum of normal person | － | 0 | － | 0 | － | 0 |
| 10ng/ml Katacalcin/zheng Serum of normal person | － | 0 | － | 0 | － | 0 |
(Note "-" indicates a negative result, i.e., a mauve band appears on the quality inspection line and no mauve band appears on the inspection line; and "+" indicates a positive result, i.e., a mauve band appears on each of the quality inspection line and the inspection line.)
As can be seen from the detection results in table 5, when the kit prepared in embodiments 1 to 3 of the present application is subjected to preclinical self-test, the detectable rate of positive IgM samples reaches 100%, novel coronavirus IgM antibodies contained in the samples can be accurately detected, and negative samples of normal persons are not detected, which indicates that the sensitivity of the present application is as high as 100%, and meanwhile, CRP, IL-6, and calceinin are used as specific interference samples to verify the anti-interference capability of the kit, and after normal human serum is added, the test results show that: the normal human serum treated by the C-reactive protein, the interleukin-6 and the calcitonin has no influence on a test result, the anti-interference rate is up to 100 percent consistent with the test result of the normal human serum, the internal quality control product detection is in accordance with the standard, the performance of developing a kit is in accordance with the requirement, the detection time of the application to a positive sample is 2'01 to 3'26 ', the application is short in detection time, whether the anti-novel coronavirus nucleocapsid protein IgM antibody exists in the blood can be rapidly detected, and the method is suitable for rapidly screening the novel coronavirus initial-stage infection patients on a large scale.
The concentration of the anti-novel coronavirus nucleocapsid protein IgM antibody in the sample is reflected by the color of the detection line, if the content of the anti-novel coronavirus nucleocapsid protein IgM antibody in the sample is low, the combined colloidal gold-antibody reaching a detection line area is also low, and the combined mouse anti-human u-chain monoclonal antibody which can be combined in the detection line area is low, so that the color of a colloidal gold wine red strip formed in the detection line area is light, and the anti-novel coronavirus nucleocapsid protein IgM antibody in the sample is reflected to be low; conversely, the darker the red colloidal gold band formed in the detection line region, the more IgM antibodies reflecting the novel coronavirus nucleocapsid protein in the sample. Thus, the presence or absence of IgM antibodies against the novel coronavirus nucleocapsid protein in the sample can be determined by comparing the color of the test T-line.
Claims (10)
1. The utility model provides a colloidal gold immunochromatographic assay kit of short-term test novel coronavirus IgM antibody, includes the box body, is located paste support (1) in the box body, paste support (1) and go up the extension degree direction and paste in proper order and have sample pad (2), gold mark pad (3), NC membrane (4), inhale appearance pad (5), be close to on NC membrane (4) gold mark pad (3) side is equipped with detection line (41), is close to inhale appearance pad (5) side and be equipped with quality control line (42), its characterized in that: the gold-labeled pad (3) contains gold-labeled N protein, and the gold-labeled N protein is novel coronavirus nucleocapsid protein which is labeled by colloidal gold and contains 6 × His label;
the detection line (41) is coated with a mouse anti-human u chain monoclonal antibody;
the quality control line (42) is coated with a mouse anti-6 × His monoclonal antibody.
2. The kit for rapidly detecting the colloidal gold immunochromatographic assay for the novel coronavirus IgM antibodies according to claim 1, wherein the preparation method of the gold-labeled N protein comprises the following steps:
s101, preparing colloidal gold: adding 2mL of 1% chloroauric acid into 100mL of aqueous solution, heating to boil, adding 2-5mL of 1% trisodium citrate, mixing uniformly, boiling until the color of the colloidal gold solution changes from blue to mauve, and cooling;
s102, preparing a gold-labeled solution: adding 100 mu L of 0.1M PB (phosphate buffer) with the pH value of 7.2 into 1mL of colloidal gold, uniformly mixing, adding 0.1M NaOH solution to adjust the pH value to 7.2-7.6, adding 15-25 mu g of novel coronavirus nucleocapsid protein containing 6 XHis tags, fully and uniformly mixing, adding 100 mu L of bovine serum albumin with the volume of 10%, and uniformly mixing;
s103, purifying the gold standard solution: centrifuging the gold-labeled solution, removing the supernatant, and dissolving in 20% sucrose-containing gold-labeled complex solution.
3. The kit for rapidly detecting the colloidal gold immunochromatographic assay for novel coronavirus IgM antibodies according to claim 1, which is characterized in that: the particle size of colloidal gold in the gold-labeled N protein is 15-30 nm.
4. The kit for rapidly detecting the colloidal gold immunochromatographic assay for novel coronavirus IgM antibodies according to claim 1, which is characterized in that: the coating concentration of the mouse anti-6 XHis monoclonal antibody on the quality control line (42) is 0.75-1mg/ml, and the coating concentration of the mouse anti-human u-chain monoclonal antibody on the detection line (41) is 1.0-1.5 mg/ml.
5. The kit for rapidly detecting the colloidal gold immunochromatographic assay for novel coronavirus IgM antibodies according to claim 1, which is characterized in that: and in the gold-labeled N protein, colloidal gold and the novel coronavirus nucleocapsid protein containing the 6 XHis tag are subjected to antibody crosslinking near the isoelectric point of the protein.
6. The kit for rapidly detecting the colloidal gold immunochromatographic assay for novel coronavirus IgM antibodies according to claim 1, which is characterized in that: the content of the novel coronavirus nucleocapsid protein with 6 × His marks on the gold-labeled pad (3) is higher than that of the mouse anti-human u-chain monoclonal antibody on the detection line (41).
7. The kit for rapidly detecting the colloidal gold immunochromatographic assay for novel coronavirus IgM antibodies according to claim 1, which is characterized in that: the sample pad (2) is made of a glass fiber membrane and is treated overnight by a treatment solution containing Tris, casein sodium salt and PVP-10.
8. The kit for rapidly detecting the colloidal gold immunochromatographic assay for novel coronavirus IgM antibodies according to claim 1, which is characterized in that: the gold mark pad (3) is made of a glass fiber membrane and is treated by a treatment solution containing Tris, S-17 and bovine serum albumin.
9. The method for preparing the colloidal gold immunochromatographic kit for rapidly detecting the novel coronavirus IgM antibodies according to any one of claims 1 to 8, comprising the steps of:
s1, preparing a gold-labeled pad (3): adopting colloidal gold to mark novel coronavirus nucleocapsid protein containing 6 × His mark to prepare gold-labeled N protein, spraying the gold-labeled N protein on a glass fiber membrane, and drying to obtain a gold-labeled pad (3);
s2, preparation of NC film (4): coating a mouse anti-human u-chain monoclonal antibody on a nitrocellulose membrane to form a detection line (41), and coating a mouse anti-6 × His monoclonal antibody on the nitrocellulose membrane to form a quality control line (42);
s3, cutting and assembling: and (3) sequentially adhering the sample pad (2), the gold label pad (3) prepared in the step S1, the NC film (4) prepared in the step S2 and the sample sucking pad (5) in the length direction of the adhering bracket (1), cutting into test strips with the width of 3.5mm +/-0.5 mm, and filling into a box body to obtain the reagent strip.
10. The method for preparing the colloidal gold immunochromatographic kit for rapidly detecting the novel coronavirus IgM antibody according to claim 9, which is characterized in that: in step S1, the spraying amount of the gold-labeled N protein on the gold-labeled pad (3) is 1.4-2.0 muL/cm.
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