CN116519927B - Kit for qualitative detection of monkey pox virus antigen - Google Patents
Kit for qualitative detection of monkey pox virus antigen Download PDFInfo
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Classifications
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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
- G01N33/569—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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
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- General Physics & Mathematics (AREA)
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- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
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Abstract
The invention discloses a kit for qualitative detection of monkey pox virus antigen. The invention relates to a test strip for the qualitative detection of monkey pox virus antigen, which comprises a sample pad, a nitrocellulose membrane, a water absorption pad and a bottom plate; the sample pad, the gold standard strip, the nitrocellulose membrane and the water absorption pad are sequentially fixed on the bottom plate along the flowing direction of the sample; the gold mark strip is coated with a colloidal gold marked anti-monkey pox specific protein A29L antibody; the nitrocellulose membrane comprises a detection line and a quality control line; the detection line is coated with an anti-monkey poxvirus specific protein M1R antibody; the quality control line is coated with a secondary antibody of goat anti-mouse or goat anti-rabbit IgG. The kit comprises the test strip and a sample diluent; the sample diluent comprises the following components in percentage by mass: 0.6 to 1 percent of Tris,0.5 to 1.0 percent of bovine serum albumin, 8 percent of sodium chloride, 0.8 to 1.5 percent of mixed nonionic surfactant and 0.02 percent of preservative; the pH was 8.0. The kit can detect the monkey poxvirus, and the sample can be whole blood, serum, plasma or poxvirus secretion.
Description
Technical Field
The invention belongs to a detection kit in the field of biotechnology, and relates to a kit for qualitative detection of monkey pox virus antigen.
Background
Colloidal gold labeling technology was a labeling method developed in the last 80 th century, following three labeling technologies of fluorescein, radioisotope and enzyme. In 1971, faulk and Taylor introduced colloidal gold into immune biochemistry, and after that, the labeling method was widely applied in the fields of drug detection, biomedical detection, agricultural and veterinary drug detection, clinical disease detection and the like. There are hundreds of colloidal gold immunochromatography kits commercialized at home and abroad at present, for example, common: early pregnancy kit, drug detection kit, fecal occult blood detection kit, infectious disease detection kit, etc.
The colloidal gold labeling technology is to use colloidal gold as a labeling tracer and initially apply the colloidal gold to an immune electron microscope; along with the development of immunology, the method is gradually applied to animal agglutination experiments, immunoblotting, optical lens staining, immune spots and immunochromatography technologies. Because of the high electron density of the surface of the colloidal gold, the colloidal gold can be firmly combined with most proteins without destroying the activity of the proteins.
Colloidal gold is used in immunodetection, and is mainly used for marking antigen or antibody; the antibody or antigen which is immunologically reactive with the labeled antigen or antibody is then immobilized on a substrate, whereby binding of the antigen or antibody is revealed by a macroscopic color change. The binding degree of the colloidal gold and the protein, the pH of the reaction system, the concentration of the reacted protein and the like can influence the binding of the protein and the colloidal gold.
Monkey pox (monkey pox), also known as smallpox, has been identified as an unknown zoonotic disease. Monkey poxvirus is used as pathogen and originates from tropical rainforest areas in China and Western Africa. According to the World Health Organization (WHO) epidemic record, a pathogen "monkey poxvirus" of cynomolgus monkeys was first identified in the danish copenhagen laboratory in 1958 and named. Outbreaks of monkey pox have occurred in the united states in the middle of 4 nd 2003. Recently, in European countries, there has been a outbreak of the epidemic of the community transmission of monkey pox viruses, with a certain risk of causing global epidemics.
The monkey poxvirus genus poxviridae (poxviridae) chordopoxvirinae (chopoxviridae) orthopoxviridae (orthopoxviridae). Poxviruses that cause human infections are also vaccinia virus (vaccinia virus), vaccinia virus (cowpox virus) and variola virus (variola virus). The poxvirus is the biggest and the most complex double-stranded DNA virus in the virus, the size of the poxvirus is 220 nm-450 nm, and the poxvirus is in a round brick shape or an oval shape. Under electron microscope, the poxvirus has mulberry type (M) and capsule type (C) particles. The monkey poxvirus is stable at low temperature drying and resistant to diethyl ether, but can be inactivated at 56 ℃ for 20 minutes. Formaldehyde, ethanol, sodium dodecyl sulfate, phenol, and chloroform can inactivate viruses.
At present, the detection of the monkey poxvirus is mainly nucleic acid detection and immunodetection, and the main detection methods are as follows: RT-PCR, enzyme-linked immunosorbent assay and the like. In the existing enzyme-linked immunochromatography, the detection kit prepared by the anti-monkey poxvirus protein antibody has poor detection specificity and low sensitivity.
Some products on the market are currently tested mainly on a single sample or two samples. For the detection of different types of samples, different kits are used. Moreover, antigen detection kits for the monkey pox virus (A29L protein) and (M1R protein) double antibody sandwich method are not available on the market.
Therefore, there is a need for an antigen detection kit capable of realizing a double antibody sandwich method for detecting monkey pox virus (A29L protein) and (M1R protein) in various types of samples.
Disclosure of Invention
The invention aims to provide a kit for qualitative detection of monkey pox virus antigens.
The kit is based on the principle of a double antibody sandwich method, namely, a colloidal gold labeled anti-monkey pox virus protein (A29L) antibody, a nitrocellulose membrane is coated with an anti-monkey pox virus protein (M1R) antibody, and a quality control line is coated with a secondary antibody; therefore, the detection of the monkey pox virus antigen in the sample can be ensured, and the validity of the product can be ensured. The kit prepared by the method is a kit for detecting antigens based on antibodies prepared against the monkey poxvirus protein stability sites. Monkey poxvirus antigens in whole blood, serum, plasma and poxvirus secretions can be detected.
The invention provides a test strip for the qualitative detection of monkey pox virus antigen, which comprises a sample pad, a nitrocellulose membrane, a water absorption pad and a bottom plate;
the sample pad, the gold standard strip, the nitrocellulose membrane and the water absorbing pad are sequentially fixed on the bottom plate along the flowing direction of the sample;
the gold standard strip is coated with a colloidal gold-labeled anti-monkey pox specific protein A29L antibody;
the nitrocellulose membrane comprises a detection line and a quality control line; the detection line is coated with an anti-monkey pox virus specific protein M1R antibody; the quality control line is coated with a secondary antibody of goat anti-mouse or goat anti-rabbit IgG.
In the invention, the anti-monkey pox specific protein A29L antibody is an IgG antibody of murine or rabbit origin.
In the test strip, when the anti-monkey pox specific protein A29L antibody is labeled with colloidal gold, the final concentration of the anti-monkey pox specific protein A29L antibody in a labeling system is 7-10 mug/mL, and can be specifically 8 mug/mL;
when the anti-monkey pox virus specific protein M1R antibody is coated at the detection line, the concentration of the anti-monkey pox virus specific protein M1R antibody is 1mg/mL;
when the secondary antibody is coated at the quality control line, the concentration of the secondary antibody is 0.5mg/mL.
In the test strip, the preparation of the anti-monkey pox specific protein A29L antibody marked by the colloidal gold comprises the following steps:
(1) Regulating the pH value of the colloidal gold solution to 8.0 by using a potassium carbonate solution to obtain a regulated colloidal gold solution;
diluting the concentration of the colloidal gold-labeled anti-monkey pox specific protein A29L antibody to 1mg/mL with 2mM Tris buffer pH 7.4;
(2) Adding 1mg/mL of the anti-monkey pox specific protein A29L antibody marked by the colloidal gold obtained after dilution into the adjusted colloidal gold solution, and mixing to obtain a mixed solution;
(3) Adding 2-8.5% of BSA aqueous solution to the mixed solution for mixing, and then adding 1-2% of PEG aggregate for mixing;
(4) Centrifuging the solution system in the step (3), removing the supernatant, and then re-dissolving and precipitating with 2mM Tris buffer solution with pH of 7.4 to obtain the colloidal gold-labeled anti-monkey pox specific protein A29L antibody.
In the test strip, the molecular weight of the PEG aggregate is 15000-30000, and can be 20000;
in the step (3), the mixing time is 30-60 minutes. In the invention, the preparation of the colloidal gold labeled anti-monkey pox specific protein A29L antibody specifically comprises the following steps:
(1) The colloidal gold solution is measured into a beaker according to the preparation amount, such as: taking hundred parts of colloidal gold solution in a beaker;
(2) The pH of the colloidal gold solution was adjusted to 8.0 with 0.1M potassium carbonate solution in an amount of 3 parts potassium carbonate solution per hundred parts solution; and placing the mixture on a magnetic stirrer for stirring;
(3) Diluting the concentration of the anti-monkey pox virus specific protein antibody to 1mg/mL with 2mM Tris buffer pH7.4, then adding 1 part per hundred parts of solution, and adding the diluted antibody solution to the colloidal gold solution; adding the solution while stirring;
preparation of Tris buffer: weighing Tris according to the amount of 2.2 parts of Tris per hundred parts of water, placing the Tris in a beaker, adding 9 parts of water, then adjusting the pH to 7.4 by using 6M hydrochloric acid solution, and then fixing the volume to the required volume by using purified water to obtain the Tris-containing compound preparation;
(4) Stirring on a magnetic stirrer for 25 minutes;
(5) Adding 8.5% BSA aqueous solution to the solution in an amount of 2.5 parts per hundred parts of solution, and stirring for 45 minutes;
(6) Adding 2% PEG20000 into the above solution according to the amount of 2 parts per hundred parts of solution, and continuing stirring for 1 hour;
(7) Transferring the reacted solution into a centrifuge tube;
(8) At 4℃at a centrifugal speed of 8000 to 15000rpm/min, for example: centrifugal speed of 12000rpm/min, centrifugal 35 minutes; removing the supernatant;
(9) The precipitate was reconstituted with 1 part per hundred parts of 2mM Tris buffer, pH7.4, to give a labeled anti-monkey pox virus specific antibody.
In the test strip, the base pad of the gold mark strip is a glass fiber pad.
In the test strip, the sample pad is prepared by soaking in a buffer system;
the buffer system is prepared by the following steps:
1) Weighing borax according to the amount of adding 3-6 parts of borax per hundred parts of water by volume; adding 80 parts of water, placing the beaker on a magnetic stirrer, and stirring;
2) Adding 0.5 part of ethylenediamine tetraacetate into the step 1), and continuously stirring;
3) Adding 0.2 part of casein salt into the step 2), and continuously stirring;
4) Adding 1 part of propylene oxide-ethylene oxide-vinyl diamine copolymer into the step 3), and continuously stirring;
5) Adding 2 parts by volume of Proclin300 into the step 4) according to the volume of water of every ten thousand parts, and continuously stirring;
6) Adding 0.8-2 parts (1 part is good) of Tween20 into the step 5) according to the volume of every ten thousand parts of water, and continuously stirring.
7) After the solution in step 6) is completely dissolved and clarified, the pH of the solution is adjusted to 8.2 with hydrochloric acid or sodium hydroxide, and then the volume is fixed to the desired volume with purified water.
The invention also provides a kit for the qualitative detection of the monkey pox virus antigen, which comprises the test strip for the qualitative detection of the monkey pox virus antigen and sample diluent required by the detection of a sample;
the sample diluent comprises the following components in percentage by mass: 0.6 to 1 percent of Tris,0.5 to 1.0 percent of bovine serum albumin, 0.8 percent of sodium chloride, 0.8 to 1.5 percent of mixed nonionic surfactant and 0.02 percent of preservative; the pH is 8.0; the solvent is water.
In the invention, the sample diluent specifically comprises the following components in percentage by mass: 0.6% Tris,0.7% bovine serum albumin, 0.8% sodium chloride, 1.2% mixed nonionic surfactant, 0.02% preservative; the pH is 8.0; the solvent is water.
In the kit, the mixed nonionic surfactant comprises polyoxyethylene castor oil and propylene oxide-ethylene oxide-vinyl diamine copolymer which are mixed according to the mass ratio of 1:1-3, and preferably polyoxyethylene castor oil and propylene oxide-ethylene oxide-vinyl diamine copolymer which can be 1:2;
the preservative is at least one selected from Proclin300, sodium azide and krovin 300.
In the above kit, the kit for qualitative detection of monkey pox virus antigen further comprises a cassette; the test strip for the qualitative detection of the monkey pox virus antigen is arranged in the clamping shell, the clamping shell is provided with a sample adding hole at the position of the sample pad corresponding to the test strip for the qualitative detection of the monkey pox virus antigen, and the clamping shell is provided with an opening or a visible window in the region corresponding to the detection line and the quality control line, so that when the kit is used, the color change of lines of the detection line and the quality control line can be observed through the observation window to judge the result.
The kit for the qualitative detection of the monkey pox virus antigen is applied to the detection of the monkey pox virus.
Further, the application is a non-disease diagnostic application, such as for quality control of blood products.
Further, the sample when the detection is performed may be: whole blood, serum, plasma or epidermoid monkey poxvirus secretion.
The invention further provides a method for detecting the monkey pox virus by using the kit for qualitatively detecting the monkey pox virus antigen, which comprises the following steps:
placing a sample to be tested on the sample pad of the kit for the qualitative detection of the monkey pox virus antigen; then adding the sample diluent, and detecting the following results:
1) When the detection area is colored and the quality control area is also colored, the detection result of the sample to be detected is positive;
2) When the detection area does not develop color and the quality control area develops color, the detection result of the sample to be detected is negative;
3) When the quality control zone is not developed, the kit is not effective and a new one is needed for re-measurement.
In the invention, when the kit for the qualitative detection of the monkey pox virus antigen is used for detecting a sample, the detection result is observed 15 minutes after sample addition, and after the detection result exceeds 30 minutes, the result is judged to be invalid; therefore, the result determination range is 15 minutes to 30 minutes.
In the above method, the sample to be tested comprises whole blood, serum, plasma or epidermoid monkey pox virus secretion.
Further, the method is a non-disease diagnostic application.
The invention has the following beneficial effects:
1. the invention adopts a double-antibody sandwich colloidal gold immunochromatography method, and the prepared monkey pox virus antigen detection kit can qualitatively detect the monkey pox virus antigen in the skin surface of a human body in addition to the monkey pox virus antigen in blood, blood plasma or serum. Is a highly sensitive product compared with the similar immune products in the market. Antibodies against monkey poxvirus-specific proteins were used to avoid crossing with structurally similar protein analogs.
2. The buffer system of the invention is added with the surface active substance propylene oxide-ethylene oxide-vinyl diamine copolymer, thereby effectively improving the sensitivity and specificity of the product. The sample loading diluent uses the mixed nonionic surfactant according to a certain proportion, and simultaneously, the product can be used for detecting four samples of the monkey pox virus secretion at the rash positions of the whole blood, serum, plasma and human skin surface.
3. The kit prepared by the invention avoids the phenomenon that the product is invalid and cannot be monitored due to the independent quality control line. The detection line of the invention is coated with an anti-monkey poxvirus specific protein (M1R site protein) antibody. The quality control line is coated with a secondary antibody of the anti-monkey pox specific protein A29L antibody marked by the colloidal gold. The quality control line can be specifically combined with the gold mark, and the effectiveness of the product can be effectively monitored. If the detection gold marks out a problem, the quality control line cannot develop color, and the quality of the product can be effectively monitored. Even in the detection of samples in the presence of very high concentrations of monkey poxvirus, the color development of the quality control line may be relatively shallow; because most of the gold marks are gathered at the detection line position, the rest gold marks are fewer, so that the color development of the quality control line is shallower, but under the condition that the product is effective, the condition that the quality control line cannot observe is avoided.
4. In the sample diluent, the laurinol polyethylene oxide (9) ether is added, so that the color development of the product is obviously improved. When the product is detected, the red line at the detection line position is positive, the red-free line is negative, and the quality control line position always displays the red line. The result is convenient and visual to observe.
Drawings
FIG. 1 is a schematic representation of a test strip for the qualitative detection of monkey pox virus antigens according to the invention.
1 sample pad; 2, gold mark strips; 3 nitrocellulose membrane; 4, a water absorption pad; 5, detecting lines; and 6, quality control line.
FIG. 2 is a graph showing the negative test results of the kit of the present invention on whole blood samples.
FIG. 3 is a graph showing the positive test results of the kit of the present invention on whole blood samples.
FIG. 4 is a graph showing the negative test results of the kit of the present invention on serum samples.
FIG. 5 is a graph showing the results of a positive test of a serum sample with the kit of the present invention.
FIG. 6 is a graph showing the negative test results of the kit of the present invention on a plasma sample.
FIG. 7 is a graph showing the positive test results of the kit of the present invention on a plasma sample.
FIG. 8 shows the results of a negative test for the secretion of the epidermoid papulopoxvirus by the kit of the invention.
FIG. 9 shows the positive test results of the kit of the invention for the secretion of the epidermoid rash monkey poxvirus.
FIG. 10 shows the negative test results of the kit of the present invention on a negative solution.
FIG. 11 shows the positive test results of the kit of the invention on 10ng/mL recombinant monkey poxvirus surface protein antigen.
FIG. 12 shows the positive test results of the kit of the invention on 5ng/mL recombinant monkey poxvirus surface protein antigen.
FIG. 13 shows the positive test results of the kit of the invention on 2ng/mL recombinant monkey poxvirus surface protein antigen.
FIG. 14 shows the positive test results of the kit of the invention on 1ng/mL recombinant monkey poxvirus surface protein antigen.
FIG. 15 shows the negative test results of the kit of the invention on 0.5ng/mL recombinant monkey poxvirus surface protein antigen.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
As shown in the figure, the test strip for the qualitative detection of the monkey pox virus antigen is schematically shown. It comprises a sample pad 1, a nitrocellulose membrane 3, a water absorbing pad 4 and a bottom plate;
along the flowing direction of the sample, the sample pad 1, the gold standard strip 2, the nitrocellulose membrane 3 and the water absorbing pad 4 are sequentially fixed on a bottom plate;
the gold mark strip 2 is coated with a colloidal gold marked anti-monkey pox specific protein A29L antibody;
the nitrocellulose membrane 3 comprises a detection line 5 and a quality control line 6; the detection line 5 is coated with an anti-monkey poxvirus specific protein M1R antibody; the quality control line 6 is coated with goat anti-mouse IgG.
Example 1,
The preparation process of the product specifically comprises the following steps:
1 preparation of colloidal gold
The colloidal gold prepared by the technology is colloidal gold sol prepared by a trisodium citrate reduction method, and the particle diameter is 60nm. The colloidal gold and the antibody against the specific protein of the monkey pox virus can be effectively combined with each other with uniform size.
(1) 2mL of chloroauric acid aqueous solution with the concentration of 0.1g/mL is prepared and stored in a dark place for later use.
(2) 10mL of trisodium citrate aqueous solution with the concentration of 0.1g/mL is prepared and stored in a dark place for standby.
(3) Adding water with the volume of colloidal gold solution to be prepared into a beaker, such as: 1L of water; the beaker was then placed on a magnetic stirrer and heated to boiling.
(4) Adding 1.6mL of aqueous chloroauric acid solution into the beaker while heating; then, 0.15mL of the trisodium citrate solution was added to a beaker containing the chlorine Jin Suanshui solution, and stirring was continued for 5 minutes to obtain a 60nm colloidal gold solution.
(5) Stopping heating, and then cooling the colloidal gold solution after the reaction to the room temperature under the stirring condition, and keeping the colloidal gold solution for standby at 2-8 ℃ in a dark place.
2 labeling of anti-monkey pox specific protein A29L antibodies
The technology adopts a direct marking method. The prepared anti-monkey pox specific protein A29L antibody is directly reacted with colloidal gold solution at room temperature, and then centrifuged to obtain the required gold marker. The specific method comprises the following steps:
2.1 determination of the pH of the label:
(1) The colloidal gold solutions prepared in 1 were placed in EP tubes for a total of 10 tubes, and then the pH of the above colloidal gold solutions was adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 with 0.1M potassium carbonate, respectively, and labeled. Then, antibodies specific for anti-monkey poxvirus protein A29L were added in an amount of 10. Mu.g/mL, respectively, and vortexed and mixed.
(2) Each of the above solutions was allowed to stand for 10 minutes, 10% sodium chloride was added in an amount of 100. Mu.L/tube to dissolve in each EP tube, and the mixture was vortexed and mixed well.
(3) Standing for 15 minutes, and observing color change.
(4) Selecting the pH of the tube of colloidal gold solution without precipitation, purple or black to be the optimal labeling pH; i.e. the pH of the solution which is just not changed in colour and still has a reddish wine-like colour is not optimal, i.e. pH8.0.
2.2 determination of the amount of marking
(1) The colloidal gold solutions prepared in 1 were placed in EP tubes for a total of 10 tubes, and then the pH of the above colloidal gold solutions was adjusted to 8.0 with 0.1M potassium carbonate, respectively, and labeled. Then, the anti-monkey pox virus specific protein antibodies were added in an amount of 2. Mu.L/mL, 4. Mu.L/mL, 6. Mu.L/mL, 8. Mu.L/mL, 10. Mu.L/mL, 12. Mu.L/mL, 14. Mu.L/mL, 16. Mu.L/mL, 18. Mu.L/mL, and 20. Mu.L/mL, respectively, and vortexed and mixed.
(2) After standing for 10 minutes, an aqueous BSA solution (0.85 parts BSA per ten parts water) was added to each tube in an amount of 24. Mu.L/mL, and vortexed and mixed well.
(3) An aqueous solution of PEG20000 (0.2 parts of PEG2000 per ten parts of water) was added in an amount of 20. Mu.L/mL and vortexed.
(4) After standing for 15 minutes, an aqueous sodium chloride solution (1.5 parts of sodium chloride per ten parts of water) was added to each of the above tubes in an amount of 100. Mu.L/mL, and the mixture was vortexed and homogenized.
(5) Standing for 2 hours, and observing sedimentation and color change in each tube. Selecting the amount of protein in the tube of colloidal gold solution which is not precipitated, purple or black as the optimal labeling amount; i.e. the amount of protein in the solution which is just not changed in colour but is still reddish in wine is the optimum labelling amount, i.e. 8. Mu.g/mL.
2.3 colloidal gold labelling of anti-monkey poxvirus specific protein A29L antibodies
(1) The colloidal gold solution is measured into a beaker according to the preparation amount, such as: taking hundred parts of colloidal gold solution and placing the colloidal gold solution into a beaker.
(2) The pH of the colloidal gold solution was adjusted to 8.0 with 0.1M potassium carbonate solution in an amount of 2 parts potassium carbonate solution per hundred parts colloidal gold solution volume. And placed on a magnetic stirrer for stirring.
(3) The concentration of the anti-monkey pox virus specific protein A29L antibody was diluted to 1mg/mL with 2mM Tris buffer pH7.4, and then the diluted antibody solution was added to the colloidal gold solution in an amount of 1 part per hundred parts of solution. The above solution was added while stirring.
Preparation of Tris buffer: weighing Tris according to the amount of 2.2 parts per hundred parts of water, placing the Tris in a beaker, adding 9 parts of water, then adjusting the pH to 7.4 by using 6M hydrochloric acid solution, and then fixing the volume to the required volume by using purified water to obtain the Tris-containing compound.
(4) Stirring on a magnetic stirrer for 25 minutes;
(5) To the above solution was added a 5% aqueous solution of BSA in an amount of 2.5 parts per hundred parts of solution, and stirring was continued for 45 minutes.
(6) 2% PEG20000 was added to the above solution in an amount of 2 parts per hundred parts of solution and stirring was continued for 1 hour.
(7) Transferring the reacted solution into a centrifuge tube;
(8) At 4℃at a centrifugal speed of 8000 to 15000rpm/min, for example: centrifugal speed of 12000rpm/min, centrifugal 35 minutes; removing the supernatant.
(9) And (3) re-dissolving and precipitating the antibody by using a Tris buffer solution with the pH of 7.4 and containing 1 part per hundred parts of 2mM to obtain the marked anti-monkey pox virus specific protein A29L antibody.
2.4 preparation of buffer System for Whole blood, serum, plasma and epidermoid rash Poxvirus secretion Loading
The method is to prepare a buffer system simultaneously applicable to whole blood, serum, plasma and epidermoid rash poxvirus secretion samples, and prepare the buffer system into a sample pad.
2.4.1 formulation of buffer systems
(1) Borax was weighed in an amount of 4 parts per hundred parts of borax, placed in a beaker, 80 parts of water was added to the beaker, and the beaker was placed on a magnetic stirrer and stirred.
(2) Adding 0.5 part of ethylenediamine tetraacetate into the beaker, and continuously stirring;
(3) Adding 0.2 part of casein salt into the solution, and continuously stirring;
(4) Adding 1 part of propylene oxide-ethylene oxide-vinyl diamine copolymer (product number D132872) into the solution, and continuously stirring;
(5) 2 parts per ten thousand of Proclin300 are added into the solution, and stirring is continued;
(6) To the above solution, 1 part of Tween20 per ten thousand parts was added, and stirring was continued.
(7) After the solution is completely dissolved and clarified, the pH of the solution is adjusted to 8.2 with hydrochloric acid or sodium hydroxide, and then the volume is fixed to the desired volume with purified water.
(8) And (5) standing by at room temperature.
2.4.2 preparation of sample pad
(1) A commercially available glass fiber mat (length. Times. Width: 300 mm. Times. 254 mm) was prepared;
(2) Soaking the glass fiber mat according to the amount of 1 piece per hundred parts; the glass fiber mat was first soaked in the solution for 10 minutes, then taken out, and excess water was gently filtered off with a glass rod. Turning over, soaking the glass fiber mat into the solution again for 10 minutes, and lightly filtering out excessive water by using a glass rod. Obtaining the sample pad.
(3) The resulting sample pad was placed in an oven at 37℃and dried overnight (time 17-20 hours).
(4) After drying, the sample pad is stored in a sealed condition and placed in a drying environment.
2.5 preparation of sheet materials
(1) Sticking a nitrocellulose membrane on a PVC bottom plate;
(2) Preparing a coating liquid:
and (3) detecting lines: the anti-monkey poxvirus-specific protein M1R antibody was diluted to 1mg/mL with 0.01M PB, and the amount of detection line was prepared in an amount of 50. Mu.L per tablet.
Quality control line: the secondary antibody (i.e., goat anti-mouse IgG) was diluted to 0.5mg/mL with 0.01M PB, and the amount of the quality control line was prepared in an amount of 50. Mu.L per tablet.
Coating: the solution was coated on nitrocellulose membrane with a film-drawing agent.
(3) The coated sheet was placed in a 37 ℃ oven and dried overnight.
(4) And (5) hermetically preserving the dried sheet and storing the sheet in a dry place.
2.6 preparation of gold Mark strip
(1) The gold standard solution prepared in 2.3 was diluted with 0.01M PB buffer containing 1 part of bovine serum albumin and 20 parts of sucrose per hundred parts. The dilution ratio was adjusted to 46 parts of the gold standard solution and 54 parts of the above-mentioned 0.01M PB solution.
(2) The diluted solution was sprayed with gold marks in an amount of 45. Mu.L each using a spot gold marking machine. The sprayed base mat was a glass fiber mat (length. Times. Width: 300 mm. Times.84 mm).
(3) After spraying, the gold bar is put into a 37 ℃ oven for overnight drying.
(4) The dried gold label strip is sealed in a self-sealing bag, and then is preserved in a dry environment in a dark place.
2.7 assembly of reagent cards
(1) Cutting the prepared gold standard strip into strips with the length of 300mm and the width of 8mm, pasting the strips on the lower edge of a nitrocellulose membrane of a sheet, and pressing the nitrocellulose membrane for 1-2mm;
(2) Cutting the sample pad into strips with the length of 300mm and the width of 17mm, and pasting the strips at the lower end of the sheet; the lower edge of the sample pad is aligned with the lower edge of the sheet, and the upper edge of the sample pad is finished with the white edge of the gold standard strip.
(3) The absorbent filter paper was cut into small strips 300mm long and 17mm wide with the upper edge of the absorbent filter paper aligned with the upper edge of the sheet and the lower edge pressed about 2mm across the nitrocellulose membrane.
(4) And compacting the stuck reagent card. Then cut into small strips 3.0mm wide by a cutter.
2.8 Assembly of the kit
And (5) filling the cut strips with the diameter of 3.0mm into a clamping shell of the kit to obtain the monkey pox virus antigen detection kit.
Preparation of sample dilution
0.6 part of Tris,0.7 part of bovine serum albumin, 0.8 part of sodium chloride and 1.2% of mixed nonionic surfactant are added into each hundred parts of water, wherein the mixing ratio of the polyoxyethylene castor oil to the propylene oxide-ethylene oxide-vinyl diamine copolymer is 1:2; and 2 parts Proclin300 per ten thousand parts by volume of water was added to prepare a solution and the pH of the solution was adjusted to 8.0.
4 test
1 drop (about 20-25 mu L) of whole blood/serum/plasma is taken and added into a sample adding hole of the kit, then 2-3 drops (about 75-120 mu L) of sample diluent is added into the sample adding hole, and the detection result can be observed after 15 minutes of time.
Or soaking the monkey pox virus secretion at the position of the epidermis rash with a cotton stick/swab, and then inserting the cotton stick into a dropper containing 0.3-0.4 mL of sample diluent; after the secretion dipped by the cotton stick is dissolved into the solution by rotating, the cotton stick/swab is extracted, and the cotton stick or swab head is extruded while the cotton stick/swab is extracted, so that the secretion is released. Then, the dropper was capped, 3 drops (about 80. Mu.L to 120. Mu.L) were added to the well of the kit, and the result was observed by counting 15 minutes.
Through the above detection, the results are as follows:
the negative and positive test results of the whole blood sample detected by the kit are shown in figures 2-3 respectively.
The negative and positive test results of the serum sample detected by the kit are shown in figures 4-5 respectively.
The negative and positive test results of the plasma sample detected by the kit are shown in fig. 6-7 respectively.
The negative and positive test results of the monkey pox virus secretion samples for detecting the location of the epidermis rash by the kit of the present invention are shown in FIGS. 8-9, respectively.
From the above results, the present invention can be used for the detection of four samples of monkey pox virus secretions at the rash site on the surface of whole blood, serum and plasma, and human skin.
The method for detecting the sample detection sensitivity of the kit for the monkey pox virus antigen qualitative detection has the following results:
1 test of the lowest detection limit of the kit
1.1 detection method
The recombinant monkey poxvirus surface protein antigen was diluted to five concentration gradients of 10ng/mL, 5ng/mL, 2ng/mL, 1ng/mL, 0.5ng/mL, respectively, with the formulated sample dilutions. Each concentration was tested for 5 kits, numbered A-E, respectively.
The five concentration gradient solutions and the negative solution prepared above were dropped 3 drops (about 100. Mu.L) into the wells of the kit, and the observation results were timed for 15 minutes, as shown in FIGS. 10 to 15.
1.2 detection results
TABLE 1 sensitivity test results
In the above results, "+" indicates that the detection result is positive, and "-" indicates that the detection result is negative.
As is clear from the above-described experimental results, when the concentration of the detection positive solution was 1ng/mL or more, all the detection results were positive. When the concentration of the positive solution is 0.5ng/mL, the detection result is negative. The test result of the negative solution is negative. Therefore, the minimum detection of the kit of the present invention is limited to 1ng/mL.
1.2 detection of Whole blood/serum/plasma samples
1.2.1 test methods
1.2.1.1 Whole blood sample
50 mu L of 10ng/mL positive solution is taken in a test tube, 450 mu L of whole blood is added, and after the mixture is fully mixed, a positive whole blood sample containing 1ng/mL monkey pox recombinant antigen is obtained.
1 drop (about 20. Mu.L) of whole blood was taken and added to the well of the kit, and then 3 drops (about 100. Mu.L) of a sample diluent was added to the well, and the test result was observed by counting 15 minutes.
Negative whole blood and positive whole blood samples were tested for 5 kits, respectively. The kit is numbered F1-F5.
1.2.1.2 serum/plasma samples
The collected blood samples are centrifuged according to the routine serum and plasma preparation procedures of hospitals to prepare serum or plasma.
50 mu L of 10ng/mL positive solution is taken in a test tube, 450 mu L of serum/plasma is added, and after complete mixing, a positive serum/plasma sample containing 1ng/mL monkey pox recombinant antigen is obtained.
1 drop (about 25. Mu.L) of serum/plasma sample was taken and added to the well of the kit, and then 3 drops (about 100. Mu.L) of sample diluent was added to the well, and the test results were observed by counting 15 minutes.
Negative serum/plasma and positive serum/plasma samples were tested in 5 kits, respectively. The kit is numbered as E1-E5/G1-G5.
1.2.2 test results
TABLE 2 Whole blood test results
TABLE 3 serum test results
Table 4 plasma test results
1.3 sample of monkey poxvirus secretion at the skin rash site on the surface of human skin
1.3.1 test methods
The monkey pox virus secretion at the site of the epidermis rash is dipped with a cotton stick/swab and then the cotton stick is inserted into a dropper containing 0.3mL of sample dilution; after the secretion dipped by the cotton stick is dissolved into the solution by rotating, the cotton stick/swab is extracted, and the cotton stick or swab head is extruded while the cotton stick/swab is extracted, so that the secretion is released. Then, the dropper was capped, 3 drops (about 100. Mu.L) were dropped into the well of the kit, and the result was observed by counting 15 minutes.
According to the above procedure, 0.45mL of secretion solution was collected, and then 10ng/mL of monkey pox recombinant antigen was added to the secretion solution to obtain 1ng/mL of positive monkey pox secretion solution.
The negative monkey pox secretion solution and the positive monkey pox secretion solution were tested separately for 5-minute kits, numbered H1-H5.
1.3.2 test results
TABLE 5 results of the Pithecellobium intestinum Lithospermi secretion test
In conclusion, the detection sensitivity (minimum detection limit) of the kit prepared by the invention for the secretion of the monkey pox virus on the surfaces of whole blood, serum, plasma and rash is 1ng/mL (recombinant monkey pox virus surface envelope protein).
Claims (9)
1. The test strip for the qualitative detection of the monkey pox virus antigen is characterized by comprising a sample pad, a gold mark strip, a nitrocellulose membrane, a water absorption pad and a bottom plate;
the sample pad, the gold standard strip, the nitrocellulose membrane and the water absorption pad are sequentially fixed on the bottom plate along the flowing direction of the sample;
the gold standard strip is coated with a colloidal gold-labeled anti-monkey pox specific protein A29L antibody;
the nitrocellulose membrane comprises a detection line and a quality control line; the detection line is coated with an anti-monkey pox virus specific protein M1R antibody; the quality control line is coated with a secondary antibody of goat anti-mouse or goat anti-rabbit IgG;
the preparation of the colloidal gold labeled anti-monkey pox specific protein A29L antibody comprises the following steps:
(1) Regulating the pH value of the colloidal gold solution to 8.0 by using a potassium carbonate solution to obtain a regulated colloidal gold solution;
the concentration of anti-monkey pox specific protein A29L antibody was diluted to 1mg/mL with 2mM Tris buffer pH 7.4;
(2) Adding 1mg/mL of the anti-monkey pox specific protein A29L antibody obtained after dilution into the regulated colloidal gold solution, and mixing to obtain a mixed solution;
(3) Adding 2-8.5% of BSA aqueous solution to the mixed solution for mixing, and then adding 1-2% of PEG aggregate for mixing;
(4) Centrifuging the solution system in the step (3), removing the supernatant, and then re-dissolving and precipitating with 2mM Tris buffer solution with pH of 7.4 to obtain the colloidal gold-labeled anti-monkey pox specific protein A29L antibody;
the sample pad is prepared by soaking in a buffer system;
the buffer system is prepared by the following steps:
1) Weighing borax according to the amount of adding 3-6 parts of borax per hundred parts of water, adding 80 parts of water, and placing a beaker on a magnetic stirrer for stirring;
2) Adding 0.5 part of ethylenediamine tetraacetate into the step 1), and continuously stirring;
3) Adding 0.2 part of casein salt into the step 2), and continuously stirring;
4) Adding 1 part of propylene oxide-ethylene oxide-vinyl diamine copolymer into the step 3), and continuously stirring;
5) Adding 2 parts by volume of Proclin300 per million parts of water to step 4), and continuing to stir;
6) Adding 0.8-2 parts of Tween20 into the step 5) according to the volume of every ten thousand parts of water, and continuously stirring;
7) After the solution in step 6) is completely dissolved and clarified, the pH of the solution is adjusted to 8.2 with hydrochloric acid or sodium hydroxide, and then the volume is fixed to the desired volume with purified water.
2. The test strip of claim 1, wherein when the anti-monkey pox specific protein a29L antibody is labeled with colloidal gold, the final concentration of the anti-monkey pox specific protein a29L antibody in the labeling system is 7-10 μg/mL;
when the anti-monkey pox virus specific protein M1R antibody is coated at the detection line, the concentration of the anti-monkey pox virus specific protein M1R antibody is 1mg/mL;
when the secondary antibody is coated at the quality control line, the concentration of the secondary antibody is 0.5mg/mL.
3. The test strip of claim 1, wherein the PEG aggregate has a molecular weight of 15000-30000;
in the step (3), the mixing time is 30-60 minutes.
4. The test strip of claim 1 or 2, wherein the base pad of the gold strip is a glass fiber pad.
5. A kit for the qualitative detection of a monkey pox virus antigen comprising the test strip for the detection of a monkey pox virus antigen according to any one of claims 1 to 4 and a sample diluent;
the sample diluent comprises the following components in percentage by mass: 0.6% -1% of Tris,0.5% -1.0% of bovine serum albumin, 8% of sodium chloride, 0.8% -1.5% of mixed nonionic surfactant and 0.02% of preservative; the pH is 8.0; the solvent is water;
the mixed nonionic surfactant is prepared by mixing polyoxyethylene castor oil and epoxypropane-epoxyethane-vinyl diamine copolymer in a mass ratio of 1:1-3.
6. The kit of claim 5, wherein the preservative is selected from at least one of Proclin300, sodium azide, and krovin 300;
the kit for the qualitative detection of the monkey pox virus antigen also comprises a cassette; the test strip for detecting the monkey pox virus antigen is arranged in the clamping shell, the clamping shell is provided with a sample adding hole at the upper sample pad of the test strip for detecting the monkey pox virus antigen, and the clamping shell is provided with an opening or visual window in a region corresponding to the detection line and the quality control line.
7. Use of the kit for the qualitative detection of monkey pox virus antigens according to claim 5 or 6 for the detection of monkey pox viruses for the purpose of non-disease diagnosis and treatment.
8. A method for detecting monkey poxvirus using the kit for qualitative detection of monkey poxvirus antigen according to claim 5 or 6, said method being for the purpose of non-disease diagnosis and treatment, comprising the steps of:
placing a sample to be tested on the sample pad of the kit for qualitative detection of monkey pox virus antigen according to claim 5 or 6; then adding sample diluent, and the detection result is as follows:
1) When the detection area develops color and the quality control area also develops color, the detection result of the sample to be detected is positive;
2) When the detection area does not develop color and the quality control area develops color, the detection result of the sample to be detected is negative;
3) When the quality control region is not developed, the kit is ineffective and a new kit is needed for re-measurement.
9. The method of claim 8, wherein the sample to be tested comprises whole blood, serum, plasma, or epidermoid monkey pox virus secretion.
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