CN114590789B - Preparation method of monodisperse carboxyl colloidal carbon and application of monodisperse carboxyl colloidal carbon in novel coronavirus antibody detection test paper - Google Patents
Preparation method of monodisperse carboxyl colloidal carbon and application of monodisperse carboxyl colloidal carbon in novel coronavirus antibody detection test paper Download PDFInfo
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- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B32/00—Carbon; Compounds thereof
- C01B32/05—Preparation or purification of carbon not covered by groups C01B32/15, C01B32/20, C01B32/25, C01B32/30
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- G—PHYSICS
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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
- G01N33/531—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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract
The invention discloses a preparation method of monodisperse carboxyl colloidal carbon, which comprises the steps of preparing carbon black suspension, condensing and refluxing with oxidant solution at a certain temperature, centrifuging for several times to separate carboxyl colloidal carbon precipitate, and drying to prepare a carboxyl colloidal carbon powder finished product. The carboxyl colloid carbon powder has a plurality of oxygen-containing polar groups on the surface, has good dispersibility and markability in a matrix, uniform particle size and good biocompatibility, and is very suitable for immunochromatography. The invention also discloses a novel coronavirus antibody detection test paper, wherein a colloidal carbon-novel coronavirus recombinant antigen complex and a colloidal carbon-chicken IgY antibody complex are coated on a binding pad of the test paper. The test paper has higher accuracy, lower detection limit and higher specificity for the novel coronavirus antibody.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of monodisperse carboxyl colloidal carbon and novel coronavirus antibody detection test paper using the colloidal carbon as a carrier.
Background
The formal classification of the novel coronavirus (2019-nCoV, hereinafter new coronavirus) is named severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus, sars-CoV-2), a new strain of coronavirus that has never been found in humans before. After the human is infected with the novel coronavirus for the first time, the immune system of the human body can perform immune defense on the virus and generate specific antibodies. IgM antibodies generally appear between 1 and 2 weeks, with IgG antibodies produced about 4 weeks. The use of the novel coronavirus IgM/IgG antibody reagent provides serological evidence for clinic, and can be used in combination with a nucleic acid detection method, thereby providing more comprehensive and accurate information for diagnosis of the novel coronavirus. The antibody detection card can also assist the development of a novel coronavirus (SARS-CoV-2) vaccine for detecting the condition of generating antibodies in vivo after vaccination and the effect of the vaccine; the method can also be used for epidemiological investigation, and can be used for knowing the number of individuals in the crowd contacted with the corresponding novel viruses and helping the state to judge and control the overall epidemic situation.
Raw carbon black is amorphous carbon, and is widely used as an important petrochemical raw material in the fields of color paste, paint, printing and dyeing and the like. Carbon black has low surface oxygen content, so that the hydrophilicity is not strong enough, the dispersibility in water is limited, and the carbon black cannot be directly used as a carrier material for a detection test strip of the novel coronavirus, and the carbon black needs to be treated.
In the prior art, technical literature (Lin Xianghua, university of cisco and paramedical university, 2020 (07), 16-18 and 31) discloses that hydroxyl groups and carboxyl groups on the surface of colloidal carbon nanoparticles can increase the adsorption capacity of the nanoparticles, but the void structure and the functionalization of colloidal carbon which is not subjected to post-modification are limited, and the hydroxyl groups and carbonyl groups rich in the surface are beneficial to the post-surface modification; the method for oxidizing in the air at 300 ℃ can improve the carboxyl content of the surface of the colloidal carbon, but can be high in cost, low in carboxyl content and poor in stability and biocompatibility. The method provided by the technical literature comprises the steps of dissolving glucose and sodium gluconate in water, performing ultrasonic dispersion, transferring into a polytetrafluoroethylene lining, putting into a reaction kettle, reacting for 4 hours at 160 ℃, taking out brown or black solution, centrifuging to remove supernatant, and drying the rest product at 70 ℃ to obtain the finished product. The monodisperse carboxylated colloidal carbon nano-particles prepared by the method have good monodispersity, the carboxyl content on the surfaces of the nano-particles reaches 7.2mmol/g, and the biocompatibility is good, but the nano-particles cannot be used as a raw material of a test strip for detecting a new coronavirus.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of monodisperse carboxyl colloidal carbon, which is realized by the following technology.
The preparation method of the monodisperse carboxyl colloidal carbon comprises the following steps:
s1, ultrasonic treatment: mixing carbon black and ultrapure water according to a weight ratio of 1:30, and setting power of 500-1500W for ultrasonic treatment for 10-60min to obtain carbon black suspension;
s2, oxidation reaction: dropwise adding an oxidant solution into the carbon black suspension, uniformly stirring, and condensing and refluxing for reaction;
s3, centrifuging: centrifuging the solution obtained in the step S2 for a plurality of times, wherein the primary centrifugation parameter is 3000rpm for 5min;
removing supernatant after each centrifugation except the last centrifugation, taking out the lower sediment, adding the ultrapure water with the same volume as that in the step S1, and setting power of 500-1500W for ultrasonic treatment for 10-60min; until the pH value of the supernatant after the last centrifugation is between 6 and 7; removing supernatant after the last centrifugation, and taking out the lower sediment for later use;
s4, drying: and (3) drying the lower precipitate obtained in the step (S3) at 60 ℃ to obtain the powder of the carboxyl colloidal carbon.
In the preparation method of the monodisperse carboxyl colloidal carbon, the carbon black is commercially available material with the particle size smaller than 1 mu m, and the concentrated nitric acid and the potassium permanganate are commercially available materials. Oxidizing the surface of carbon black powder by using an oxidant solution, wherein the reaction conditions are matched with the reaction mode of heating reflux of the oxidant, so that the oxygen-containing polar groups on the surface are obviously increased, and the dispersibility and the labelling property of the carbon black powder in a matrix are improved, so that the monodisperse carboxyl colloidal carbon with uniform particle size and good biocompatibility is obtained, and is suitable for immunochromatography; the monodisperse carboxyl colloid carbon is rich in negative charge, and the biomolecules such as proteins and the like are marked by means of charge adsorption or amide bond formation between carboxyl and amino; the immune labeling method is simple, and the immune complex with biological activity can be obtained by only mixing and incubating at normal temperature, sealing, centrifugal purification, re-dissolving and the like, thus being very suitable for preparing products such as novel coronavirus antibody detection test paper and the like.
Preferably, the oxidant solution used in step S2 is concentrated nitric acid, potassium permanganate or hydrogen peroxide solution.
More preferably, the oxidation reaction of step S2 is specifically: taking concentrated nitric acid according to the weight of the carbon black as a reference, dripping the concentrated nitric acid into the carbon black suspension according to the proportion of 2.5mL/g, adding ultrapure water to the volume of twice the volume of the carbon black suspension in the step S1, and heating, condensing and refluxing for 2-8h in an oil bath at the temperature of 105 ℃.
Further preferably, in step S2, the time of the condensation reflux is 5 hours.
More preferably, step S2 is specifically: heating the carbon black suspension to boil, taking 0.05M potassium permanganate solution according to the weight of the carbon black as a proportion of 15mL/g, dripping the potassium permanganate solution into the carbon black suspension, and heating and condensing the solution for 0.5-4h in an oil bath at 105 ℃.
Further preferably, in step S2, the time of the condensation reflux is 1h.
Preferably, in the centrifugation of step S3, the parameter of each centrifugation starting from the second centrifugation is increased by 3000rpm from the last time, and the centrifugation time is increased by 5min.
The invention also provides novel coronavirus antibody detection test paper, which comprises a PVC plate, wherein a sample pad, a combination pad, a blood filtering film, a nitrocellulose film and an absorption pad are sequentially stuck on the surface of the PVC plate, a detection line and a quality control line are arranged on the nitrocellulose film, the combination pad is coated with a colloidal carbon-novel coronavirus recombinant antigen compound and a colloidal carbon-chicken IgY antibody compound, the detection line is coated with a mouse anti-human IgG secondary antibody and a mouse anti-human IgM secondary antibody, and the quality control line is coated with a rabbit anti-chicken IgY secondary antibody;
the colloidal carbon-novel coronavirus recombinant antigen complex and the colloidal carbon-chicken IgY antibody complex are respectively formed by respectively labeling and combining the novel coronavirus recombinant antigen and the chicken IgY antibody by taking the monodisperse carboxyl colloidal carbon as a carrier; the colloidal carbon-novel coronavirus recombinant antigen complex comprises a colloidal carbon-novel coronavirus recombinant N antigen complex and a colloidal carbon-novel coronavirus recombinant S antigen complex.
When the novel coronavirus antibody detection test paper is used, the novel coronavirus antibody in the sample is specifically combined with the colloidal carbon-novel coronavirus recombinant antigen complex on the detection test paper; detecting the specific binding of the mouse anti-human IgG secondary antibody and the mouse anti-human IgM secondary antibody coated on the line with the novel coronavirus antibody in the sample; the quality control line is coated with an anti-chicken IgY secondary antibody which is specifically combined with the chicken IgY antibody on the colloidal carbon-chicken IgY antibody compound;
the invention also provides a preparation method of the novel coronavirus antibody detection test paper, which comprises the following steps:
p1, labeling of novel coronavirus recombinant antigen and colloidal carbon:
p11, preparing a colloidal carbon-novel coronavirus recombinant N antigen complex: 1mL of the monodisperse carboxyl colloidal carbon solution was prepared with 5mM boric acid buffer solution having pH=8.8 and 0.01%; 60 mug of new coronavirus recombinant N protein is added, and the mixture is shaken for 60 minutes at room temperature; then 100. Mu.L of 10% BSA solution is added, and the mixture is shaken at room temperature for 10min, and then 10. Mu.L of 5% PEG20000 solution is added, and the mixture is shaken at room temperature for 10min; centrifuging the mixed solution at 4deg.C for 30min with a centrifugation parameter of 10000rpm; taking the lower layer of sediment, fixing the volume to 100 mu L by using 0.3 percent of Tris buffer solution with pH value of 8.0, namely the colloidal carbon-novel coronavirus recombinant N antigen complex, and placing the colloidal carbon-novel coronavirus recombinant N antigen complex at the temperature of 4 ℃ for standby;
p12, preparing a colloidal carbon-novel coronavirus recombinant S antigen complex: preparing 1mL of the monodisperse carboxyl colloidal carbon solution with 5mM carbonate buffer solution and pH=9.6, adding 20 mug of novel coronavirus recombinant S protein, shaking at room temperature for 60min, adding 100 mug of 10% BSA solution, shaking at room temperature for 10min, adding 10 mug of 5% PEG20000 solution, and shaking at room temperature for 10min; centrifuging the mixed solution at 4deg.C for 30min with a centrifugation parameter of 10000rpm; taking the lower layer of sediment, fixing the volume to 100 mu L by using 0.3 percent of Tris buffer solution with pH value of 8.0, namely the colloidal carbon-novel coronavirus recombinant S antigen complex, and placing the colloidal carbon-novel coronavirus recombinant S antigen complex at the temperature of 4 ℃ for standby;
p2, preparing a colloidal carbon-chicken IgY antibody complex: preparing 1mL of 0.01% of the monodisperse carboxyl colloidal carbon solution by using 5mM boric acid buffer solution with pH=8.8, adding 10 mug of chicken IgY monoclonal antibody, shaking at room temperature for 60min, adding 100 mug of 10% BSA solution, shaking at room temperature for 10min, adding 10 mug of 5% PEG20000 solution, and shaking at room temperature for 10min; centrifuging the mixed solution at 4 ℃ for 30min, and setting the centrifugal parameter to 10000rpm; taking the lower layer of sediment, fixing the volume to 100 mu L by using 0.3 percent of Tris buffer solution with pH value of 8.0, namely the colloidal carbon-chicken IgY antibody compound, and placing the solution at 4 ℃ for standby;
p3, preparing new type coronavirus antibody test paper
P31, preparing a bonding pad: mixing the colloidal carbon-novel coronavirus recombinant N antigen complex, the colloidal carbon-novel coronavirus recombinant S antigen complex and the colloidal carbon-chicken IgY monoclonal antibody complex according to the proportion of 5:1:5, and spraying the mixture on a treated bonding pad, wherein the spraying amount is 2 mu L/cm; drying in a baking oven at 37 ℃ for 16-24 hours, adding a drying agent, and sealing and preserving for later use;
p32, scribing: pasting a nitrocellulose membrane on a PVC bottom plate, marking 1.0mg/mL rabbit anti-chicken IgY monoclonal antibody on a quality control line on the nitrocellulose membrane, marking 0.8mg/mL mouse anti-human IgG antibody and 1.0mg/mL mouse anti-human IgM antibody on a detection line on the nitrocellulose membrane, drying in a baking oven at 37 ℃ for 16-24h, adding a drying agent aluminum foil bag, sealing and preserving for later use;
p33, assembling: and (3) sequentially adhering the bonding pad, the blood filtering film, the sample pad and the absorption pad obtained in the step (P31) on a PVC bottom plate on which the nitrocellulose film is adhered, cutting, assembling and sealing to obtain the novel coronavirus antibody detection test paper, and preserving at room temperature for later use.
The using method of the test paper comprises the following steps:
(1) Taking out the test paper from the plastic package, placing the test paper on a dry tabletop, and displaying a quality control area (C) and a test area (G, M) on the test paper;
(2) Collecting fingertip blood by a hemostix, stopping bleeding after blood collection, and adding the collected blood sample into a sample adding hole;
(3) Unscrewing the bottle cap of the diluent (phosphate buffer solution, pH value is 6.5-8.0), and dripping 2-3 drops of diluent into the sample adding hole for dripping the blood sample; reading the result within 10min, and reading the result after not more than 15min;
(4) Interpretation of the results:
positive IgG, a black strip appears in the test area (G), and a black strip appears in the quality control area (C);
positive IgM, a black strip appears in the test area (M), and a black strip appears in the quality control area (C);
IgG positive and IgM positive, wherein a black strip appears in the test area (G, M) and a black strip appears in the quality control area (C);
negative: only one black stripe appears in the quality control area (C), and no stripe appears in the test area (G, M);
invalid results: as long as no black stripes appear in the quality control region (C).
Preferably, the blood collection process of step (2) is specifically as follows: the blood taking device comprises a blood taking needle, a blood taking suction tube and alcohol cotton; firstly disinfecting a blood sampling part by using alcohol cotton, then puncturing the blood sampling part by using a blood sampling needle, then sucking a drop of blood sample by using a blood sampling straw, and adding a sample for detection; directly dripping the blood drop into the sample adding hole, and then adding 2-3 drops of diluent; after blood collection, the blood collection part is pressed by alcohol cotton to stop bleeding.
In general, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
compared with the prior art, the invention has the following advantages: the preparation method of the monodisperse carboxyl colloidal carbon provided by the invention is simple, raw materials are easy to obtain, the particle size of the product is stable, the monodispersity is good, a large amount of negative charges are carried, the protein can be directly marked, and the monodisperse carboxyl colloidal carbon has stronger contrast ratio than that of colloidal gold and lower cost when being used for immunochromatography.
Drawings
FIG. 1 is a transmission electron microscope image of monodisperse carboxyl colloidal carbon prepared by a concentrated nitric acid reaction in example 1;
FIG. 2 is a transmission electron microscope image of monodisperse carboxyl colloidal carbon prepared by the concentrated nitric acid reaction in example 2;
FIG. 3 is a schematic diagram of the structure of a novel coronavirus antibody test strip.
In the figure: 1. a PVC bottom plate; 2. a sample pad; 3. a bonding pad; 4. a blood filtering membrane; 5. detecting a line M; 6. a detection line G line; 7. a quality control line C line; 8. a nitrocellulose membrane; 9. an absorbent pad.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
Example 1
The monodisperse carboxyl colloidal carbon of the embodiment is prepared by the following preparation method:
s1, ultrasonic treatment: mixing 1g of carbon black with 30mL of ultrapure water, and performing ultrasonic treatment for 30min at 800W power to obtain 30mL of carbon black suspension;
s2, oxidation reaction: diluting the carbon black suspension obtained in the step S1 by 1 time, slowly adding 2.5mL of 69.8% concentrated nitric acid into the diluted carbon black suspension, and uniformly stirring; adding ultrapure water to a constant volume of 60mL, transferring the reaction system into a 100mL single-neck flask, connecting a condensing reflux pipe, heating in an oil bath at 105 ℃ and condensing reflux for 5h;
s3, centrifuging: centrifuging the solution obtained in the step S2 for five times;
the parameters of the first centrifugation are 3000rpm for 5min; the parameters of the second centrifugation are 6000rpm for 10min; the third centrifugation is performed at 9000rpm for 15min; the fourth centrifugation is performed at 12000rpm for 20min; the fifth centrifugation is performed at 15000rpm for 25min;
and (3) centrifuging for the first four times, removing supernatant after the last centrifugation is finished, adding 30mL of ultrapure water into the lower sediment for ultrasonic dispersion, and then centrifuging for the next time.
After the fifth centrifugation, the pH value of the supernatant is between 6 and 7, the supernatant is removed, and the lower layer of sediment is taken for standby;
s4, drying: and (3) drying the lower precipitate obtained in the step (S3) at 60 ℃ to obtain the powder of the carboxyl colloidal carbon.
Example 2
This embodiment is substantially the same as embodiment 1, with the only difference that: the oxidation reaction in step S2 is: heating the carbon black suspension obtained in the step S1 to boiling, slowly dripping 15mL of 0.05M potassium permanganate solution into the diluted carbon black suspension, uniformly stirring, transferring the reaction system into a 100mL single-neck flask, connecting a condensing reflux pipe, heating in an oil bath at 105 ℃ and condensing and refluxing for 1h; ,
example 3: preparation of novel coronavirus antibody test paper
The novel coronavirus antibody detection test paper comprises a PVC plate 1, wherein a sample pad 2, a bonding pad 3, a blood filtering film 4, a nitrocellulose film 8 and an absorption pad 9 are sequentially stuck on the surface of the PVC plate 1, a detection line G line 6, a detection line M line 5 and a quality control line C line 7 are arranged on the nitrocellulose film 8, the bonding pad 3 is coated with a colloidal carbon-novel coronavirus recombinant antigen compound and a colloidal carbon-chicken IgY antibody compound, the detection line G line 6 is coated with a mouse anti-human IgG secondary antibody, the detection line M line 5 is coated with a mouse anti-human IgM secondary antibody, and the quality control line is coated with a rabbit anti-chicken IgY secondary antibody;
the colloidal carbon-novel coronavirus recombinant antigen complex and the colloidal carbon-chicken IgY antibody complex are respectively formed by respectively using the monodisperse carboxyl colloidal carbon prepared in the example 1 and the example 2 as a carrier and respectively labeling and combining the novel coronavirus recombinant antigen and the chicken IgY antibody; the colloidal carbon-novel coronavirus recombinant antigen complex comprises a colloidal carbon-novel coronavirus recombinant N antigen complex and a colloidal carbon-novel coronavirus recombinant S antigen complex.
The specific preparation method comprises the following steps:
p1, labeling of novel coronavirus recombinant antigen and colloidal carbon:
p11, preparing a colloidal carbon-novel coronavirus recombinant N antigen complex: 1mL of the monodisperse carboxyl colloidal carbon solution was prepared with 5mM boric acid buffer solution having pH=8.8 and 0.01%; 60 mug of new coronavirus recombinant N protein is added, and the mixture is shaken for 60 minutes at room temperature; then 100. Mu.L of 10% BSA solution is added, and the mixture is shaken at room temperature for 10min, and then 10. Mu.L of 5% PEG20000 solution is added, and the mixture is shaken at room temperature for 10min; centrifuging the mixed solution at 4deg.C for 30min with a centrifugation parameter of 10000rpm; taking the lower layer of sediment, fixing the volume to 100 mu L by using 0.3 percent of Tris buffer solution with pH value of 8.0, namely the colloidal carbon-novel coronavirus recombinant N antigen complex, and placing the colloidal carbon-novel coronavirus recombinant N antigen complex at the temperature of 4 ℃ for standby;
p12, preparing a colloidal carbon-novel coronavirus recombinant S antigen complex: preparing 1mL of the monodisperse carboxyl colloidal carbon solution with 5mM carbonate buffer solution and pH=9.6, adding 20 mug of novel coronavirus recombinant S protein, shaking at room temperature for 60min, adding 100 mug of 10% BSA solution, shaking at room temperature for 10min, adding 10 mug of 5% PEG20000 solution, and shaking at room temperature for 10min; centrifuging the mixed solution at 4deg.C for 30min with a centrifugation parameter of 10000rpm; taking the lower layer of sediment, fixing the volume to 100 mu L by using 0.3 percent of Tris buffer solution with pH value of 8.0, namely the colloidal carbon-novel coronavirus recombinant S antigen complex, and placing the colloidal carbon-novel coronavirus recombinant S antigen complex at the temperature of 4 ℃ for standby;
p2, preparing a colloidal carbon-chicken IgY antibody complex: preparing 1mL of 0.01% of the monodisperse carboxyl colloidal carbon solution by using 5mM boric acid buffer solution with pH=8.8, adding 10 mug of chicken IgY monoclonal antibody, shaking at room temperature for 60min, adding 100 mug of 10% BSA solution, shaking at room temperature for 10min, adding 10 mug of 5% PEG20000 solution, and shaking at room temperature for 10min; centrifuging the mixed solution at 4 ℃ for 30min, and setting the centrifugal parameter to 10000rpm; taking the lower layer of sediment, fixing the volume to 100 mu L by using 0.3 percent of Tris buffer solution with pH value of 8.0, namely the colloidal carbon-chicken IgY antibody compound, and placing the solution at 4 ℃ for standby;
p3, preparing new type coronavirus antibody test paper
P31, preparing the bonding pad 3: mixing the colloidal carbon-novel coronavirus recombinant N antigen complex, the colloidal carbon-novel coronavirus recombinant S antigen complex and the colloidal carbon-chicken IgY monoclonal antibody complex according to the proportion of 5:1:5, and spraying the mixture on the treated bonding pad 3, wherein the spraying amount is 2 mu L/cm; drying in a baking oven at 37 ℃ for 16-24 hours, adding a drying agent, and sealing and preserving for later use;
p32, scribing: pasting a nitrocellulose membrane on a PVC base plate, marking 1.0mg/mL rabbit anti-chicken IgY monoclonal antibody on a quality control line C line 7 on the nitrocellulose membrane 8, marking 0.8mg/mL mouse anti-human IgG antibody and 1.0mg/mL mouse anti-human IgM antibody on a detection line G line 6 and a detection line G line 5 on the nitrocellulose membrane respectively, drying in a baking oven at 37 ℃ for 16-24h, and sealing and preserving in an aluminum foil bag with a drying agent for standby;
p33, assembling: as shown in fig. 3, the binding pad 3, the blood filtering membrane 4, the sample pad 2 and the absorption pad 9 obtained in the step P31 are sequentially adhered to the PVC base plate 1 to which the nitrocellulose membrane 8 is adhered, and then the novel coronavirus antibody detection test paper is obtained by slitting, assembling and sealing and is preserved at room temperature for standby. The assembled novel coronavirus antibody test paper is shown in fig. 3.
Application example 1: determination of the Mass of novel coronavirus antibody test strips
1. Accuracy of novel coronavirus antibody detection test paper
(1) Novel coronavirus IgM antibody detection accuracy
The accuracy of the novel coronavirus IgM antibodies was determined by test strips. The novel coronavirus IgM antibodies are used as national reference samples, namely negative reference products numbered N1-N25 and positive reference products numbered P1-P10. The coincidence rate of the negative reference sample should be not lower than 24/25, and the coincidence rate of the positive reference sample should be 10/10. The specific detection using method is as follows:
(1) taking out the test paper from the plastic package, placing the test paper on a dry tabletop, and displaying a quality control area (C) and a test area (G, M) on the test paper;
(2) collecting fingertip blood by a hemostix, stopping bleeding after blood collection, and adding the collected blood sample into a sample adding hole;
(3) unscrewing the bottle cap of the diluent (phosphate buffer solution, pH value is 6.5-8.0), and dripping 2-3 drops of diluent into the sample adding hole for dripping the blood sample; the results were read within 10min, and not after 15 min. The method comprises the steps of carrying out a first treatment on the surface of the
(4) Interpretation of the results:
positive IgG, a black strip appears in the test area (G), and a black strip appears in the quality control area (C);
positive IgM, a black strip appears in the test area (M), and a black strip appears in the quality control area (C);
IgG positive and IgM positive, wherein a black strip appears in the test area (G, M) and a black strip appears in the quality control area (C);
negative: only one black stripe appears in the quality control area (C), and no stripe appears in the test area (G, M);
invalid results: as long as no black stripes appear in the quality control region (C).
The test results of the test strips prepared using the monodisperse carboxyl colloidal carbon of example 1 are shown in table 1 below:
table 1 test paper accuracy test results corresponding to example 1
From table 1 above, it can be seen that the detection test paper prepared by using the monodisperse carboxyl colloidal carbon of example 1 meets the requirements of the coincidence rate of the negative reference sample and the positive reference sample on the detection result of the novel coronavirus IgM antibody reference sample.
The test results of the test strips prepared using the monodisperse carboxyl colloidal carbon of example 2 are shown in table 2 below:
table 2 test paper accuracy test results corresponding to example 2
From table 2 above, it can be seen that the test paper prepared by using the monodisperse carboxyl colloidal carbon of example 2 also meets the requirements of the coincidence rate of the negative reference sample and the positive reference sample on the detection result of the novel coronavirus IgM antibody reference sample.
(2) Novel coronavirus IgG antibody detection accuracy
The accuracy of the novel coronavirus IgG antibodies was determined in the same manner as described above for the detection of novel coronavirus IgM antibodies. The novel coronavirus IgG antibody is used as a national reference sample, and is respectively a negative reference sample numbered N1-N25 and a positive reference sample numbered P1-P10. The coincidence rate of the negative reference sample should be not lower than 24/25, and the coincidence rate of the positive reference sample should be 9/10. The specific detection method is the same as the detection method of the novel coronavirus IgM antibody.
The test results of the test strips prepared using the monodisperse carboxyl colloidal carbon of example 1 are shown in table 3 below:
TABLE 3 accuracy test results for test strips corresponding to example 1
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From table 3 above, it can be seen that the detection results of the test paper prepared from the monodisperse carboxyl colloidal carbon of example 1 on the novel coronavirus IgG antibody reference sample meet the requirements of the coincidence rate of the negative reference sample and the positive reference sample.
The test results of the test strips prepared using the monodisperse carboxyl colloidal carbon of example 2 are shown in table 4 below:
TABLE 4 accuracy test results for test strips corresponding to example 2
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From table 4 above, it can be seen that the detection results of the test paper prepared from the monodisperse carboxyl colloidal carbon of example 2 on the novel coronavirus IgG antibody reference sample meet the requirements of the coincidence rate of the negative reference sample and the positive reference sample.
2. Detection limit of novel coronavirus antibody detection test paper
(1) Detection limit for novel coronavirus IgM antibody detection
The lowest limit of detection of novel coronavirus IgM antibodies was determined by test paper according to the detection method used in the above detection accuracy. The adopted novel coronavirus IgM antibody is the national reference sample with the lowest detection limit, and the detection result meets the requirements that L1-L2 is positive and L3-L10 can be positive or negative.
The lowest limit of detection of the test paper prepared using the monodisperse carboxyl colloidal carbon of example 1 is shown in table 5 below:
TABLE 5 minimum detection limit detection results of the test strips corresponding to example 1
| Sample numbering | Results: yin (-) yang (+) |
| L1 | + |
| L2 | + |
| L3 | + |
| L4 | + |
| L5 | - |
| L6 | - |
| L7 | - |
| L8 | - |
| L9 | - |
| L10 | - |
As is clear from Table 5, the test strips prepared by using the monodisperse carboxyl colloidal carbon of example 1 showed positive samples L1 to L4 and negative samples L5 to L10, satisfying the above requirements.
The lowest limit of detection of the test paper prepared using the monodisperse carboxyl colloidal carbon of example 2 is shown in table 6 below:
TABLE 6 minimum detection limit detection results of the test strips corresponding to example 2
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As is clear from Table 6, the test strips prepared by using the monodisperse carboxyl colloidal carbon of example 2 showed positive samples L1 to L4 and negative samples L5 to L10, satisfying the above requirements.
(2) Novel detection limit for coronavirus IgG antibody detection
The lowest limit of detection of the novel coronavirus IgG antibody was determined by the test paper according to the detection method used in the detection accuracy described above. The adopted novel coronavirus IgG antibody is the national reference sample with the lowest detection limit, the detection result accords with that L1 is positive, and L2-L10 can be negative or positive.
The lowest limit of detection of the test paper prepared using the monodisperse carboxyl colloidal carbon of example 1 is shown in table 7 below:
TABLE 7 minimum detection limit detection results of the test strips corresponding to example 1
| Sample numbering | Results: yin (-) yang (+) |
| L1 | + |
| L2 | + |
| L3 | + |
| L4 | - |
| L5 | - |
| L6 | - |
| L7 | - |
| L8 | - |
| L9 | - |
| L10 | - |
As is clear from Table 7, the test paper prepared from the monodisperse carboxyl colloidal carbon of example 1 showed positive samples L1 to L3 and negative samples L4 to L10, satisfying the above requirements.
The lowest limit of detection of the test paper prepared using the monodisperse carboxyl colloidal carbon of example 2 is shown in table 8 below:
TABLE 8 minimum detection limit detection results of the test strips corresponding to example 2
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As is clear from Table 6, the test paper prepared from the monodisperse carboxyl colloidal carbon of example 2 showed positive samples L1 to L3 and negative samples L4 to L10, satisfying the above requirements.
The above examples describe the practice of the invention in detail, however, the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.
Claims (8)
1. The preparation method of the monodisperse carboxyl colloidal carbon is characterized by comprising the following steps:
s1, ultrasonic treatment: mixing carbon black and ultrapure water according to a weight ratio of 1:30, and setting power of 500-1500W for ultrasonic treatment for 10-60min to obtain carbon black suspension;
s2, oxidation reaction: dropwise adding an oxidant solution into the carbon black suspension, uniformly stirring, and condensing and refluxing for reaction;
s3, centrifuging: centrifuging the solution obtained in the step S2 for a plurality of times, wherein the primary centrifugation parameter is 3000rpm for 5min; the parameters of each centrifugation from the second centrifugation were increased by 3000rpm compared to the last centrifugation, and the centrifugation time was increased by 5min;
removing supernatant after each centrifugation except the last centrifugation, taking out the sediment at the lower layer, adding ultrapure water with the same volume as that of the step S1, and setting power of 500-1500W for ultrasonic treatment for 10-60min; until the pH value of the supernatant after the last centrifugation is between 6 and 7; removing supernatant after the last centrifugation, and taking out the lower sediment for later use;
s4, drying: and (3) drying the lower precipitate obtained in the step (S3) at 60 ℃ to obtain the powder of the carboxyl colloidal carbon.
2. The method for preparing monodisperse carboxyl colloidal carbon according to claim 1, wherein the oxidizing agent solution used in step S2 is concentrated nitric acid, potassium permanganate or hydrogen peroxide solution.
3. The method for preparing monodisperse carboxyl colloidal carbon according to claim 2, wherein the oxidation reaction in step S2 is specifically: taking concentrated nitric acid according to the weight of the carbon black as a reference, dripping the concentrated nitric acid into the carbon black suspension according to the proportion of 2.5mL/g, adding ultrapure water to the volume of twice the volume of the carbon black suspension in the step S1, and heating, condensing and refluxing for 2-8h in an oil bath at the temperature of 105 ℃.
4. The method for preparing monodisperse carboxyl colloidal carbon according to claim 3, wherein in the step S2, the time of condensing reflux is 5 hours.
5. The method for preparing monodisperse carboxyl colloidal carbon according to claim 2, wherein step S2 is specifically: heating the carbon black suspension to boil, taking 0.05M potassium permanganate solution according to the weight of the carbon black as a proportion of 15mL/g, dripping the potassium permanganate solution into the carbon black suspension, and heating and condensing the solution for 0.5-4h in an oil bath at 105 ℃.
6. The method for preparing monodisperse carboxyl colloidal carbon according to claim 3, wherein the time of condensing reflux in step S2 is 1h.
7. The novel coronavirus antibody detection test paper is characterized by comprising a PVC plate, wherein a sample pad, a binding pad, a blood filtering membrane, a nitrocellulose membrane and an absorption pad are sequentially stuck on the surface of the PVC plate, a detection line and a quality control line are arranged on the nitrocellulose membrane, the binding pad is coated with a colloidal carbon-novel coronavirus recombinant antigen compound and a colloidal carbon-chicken IgY antibody compound, the detection line is coated with a mouse anti-human IgG secondary antibody and a mouse anti-human IgM secondary antibody, and the quality control line is coated with a rabbit anti-chicken IgY secondary antibody;
the colloidal carbon-novel coronavirus recombinant antigen complex and the colloidal carbon-chicken IgY antibody complex are respectively formed by respectively labeling and combining the novel coronavirus recombinant antigen and the chicken IgY antibody by taking the monodisperse carboxyl colloidal carbon as a carrier; the colloidal carbon-novel coronavirus recombinant antigen complex comprises a colloidal carbon-novel coronavirus recombinant N antigen complex and a colloidal carbon-novel coronavirus recombinant S antigen complex.
8. The method for preparing the novel coronavirus antibody test paper as claimed in claim 7, which is characterized by comprising the following steps:
p1, labeling of novel coronavirus recombinant antigen and colloidal carbon:
p11, preparing a colloidal carbon-novel coronavirus recombinant N antigen complex: 1mL of the monodisperse carboxyl colloidal carbon solution was prepared with 5mM boric acid buffer solution having pH=8.8 and 0.01%; 60 mug of new coronavirus recombinant N protein is added, and the mixture is shaken for 60 minutes at room temperature; then 100. Mu.L of 10% BSA solution is added, and the mixture is shaken at room temperature for 10min, and then 10. Mu.L of 5% PEG20000 solution is added, and the mixture is shaken at room temperature for 10min; centrifuging the mixed solution at 4deg.C for 30min with a centrifugation parameter of 10000rpm; taking the lower layer of sediment, fixing the volume to 100 mu L by using 0.3 percent of Tris buffer solution with pH value of 8.0, namely the colloidal carbon-novel coronavirus recombinant N antigen complex, and placing the colloidal carbon-novel coronavirus recombinant N antigen complex at the temperature of 4 ℃ for standby;
p12, preparing a colloidal carbon-novel coronavirus recombinant S antigen complex: preparing 1mL of the monodisperse carboxyl colloidal carbon solution with 5mM carbonate buffer solution and pH=9.6, adding 20 mug of novel coronavirus recombinant S protein, shaking at room temperature for 60min, adding 100 mug of 10% BSA solution, shaking at room temperature for 10min, adding 10 mug of 5% PEG20000 solution, and shaking at room temperature for 10min; centrifuging the mixed solution at 4deg.C for 30min with a centrifugation parameter of 10000rpm; taking the lower layer of sediment, fixing the volume to 100 mu L by using 0.3 percent of Tris buffer solution with pH value of 8.0, namely the colloidal carbon-novel coronavirus recombinant S antigen complex, and placing the colloidal carbon-novel coronavirus recombinant S antigen complex at the temperature of 4 ℃ for standby;
p2, preparing a colloidal carbon-chicken IgY antibody complex: preparing 1mL of 0.01% of the monodisperse carboxyl colloidal carbon solution by using 5mM boric acid buffer solution with pH=8.8, adding 10 mug of chicken IgY monoclonal antibody, shaking at room temperature for 60min, adding 100 mug of 10% BSA solution, shaking at room temperature for 10min, adding 10 mug of 5% PEG20000 solution, and shaking at room temperature for 10min; centrifuging the mixed solution at 4 ℃ for 30min, and setting the centrifugal parameter to 10000rpm; taking the lower layer of sediment, fixing the volume to 100 mu L by using 0.3 percent of Tris buffer solution with pH value of 8.0, namely the colloidal carbon-chicken IgY antibody compound, and placing the solution at 4 ℃ for standby;
p3, preparing new type coronavirus antibody test paper
P31, preparing a bonding pad: mixing the colloidal carbon-novel coronavirus recombinant N antigen complex, the colloidal carbon-novel coronavirus recombinant S antigen complex and the colloidal carbon-chicken IgY monoclonal antibody complex according to the proportion of 5:1:5, and spraying the mixture on a treated bonding pad, wherein the spraying amount is 2 mu L/cm; drying in a baking oven at 37 ℃ for 16-24 hours, adding a drying agent, and sealing and preserving for later use;
p32, scribing: pasting a nitrocellulose membrane on a PVC bottom plate, marking 1.0mg/mL rabbit anti-chicken IgY monoclonal antibody on a quality control line on the nitrocellulose membrane, marking 0.8mg/mL mouse anti-human IgG antibody and 1.0mg/mL mouse anti-human IgM antibody on a detection line on the nitrocellulose membrane, drying in a baking oven at 37 ℃ for 16-24h, adding a drying agent aluminum foil bag, sealing and preserving for later use;
p33, assembling: and (3) sequentially adhering the bonding pad, the blood filtering film, the sample pad and the absorption pad obtained in the step (P31) on a PVC bottom plate on which the nitrocellulose film is adhered, cutting, assembling and sealing to obtain the novel coronavirus antibody detection test paper, and preserving at room temperature for later use.
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