CN115951063A

CN115951063A - Kit for detecting novel coronavirus neutralizing antibody in saliva and application

Info

Publication number: CN115951063A
Application number: CN202210888670.6A
Authority: CN
Inventors: 胡小龙; 屈武斐; 周丽
Original assignee: Venture Biotechnology Co ltd
Current assignee: Venture Biotechnology Co ltd
Priority date: 2022-07-26
Filing date: 2022-07-26
Publication date: 2023-04-11

Abstract

The invention discloses a simple and rapid detection kit for a saliva new crown neutralizing antibody, which adopts a nano-carbon immunochromatography technology, a double-antigen sandwich method to detect the content of the new crown neutralizing antibody in saliva, a reagent strip adopts an NC membrane as a matrix, a reagent pad adopts S-RBD and mouse IgG which are marked by nano-carbon, S-RBD protein and goat anti-mouse IgG as a detection line T and a quality control line C, after the reaction is finished, the color development condition is directly observed by naked eyes for testing, the content of the neutralizing antibody can be judged through the strength of the nano-carbon color development on the detection line, and the detection method is simple and convenient. The detection kit obtained by the application has high sensitivity, intuitive result, low infection risk, safety and no wound, and is a simple, accurate and economic field rapid detection method.

Description

Kit for detecting novel coronavirus neutralizing antibody in saliva and application

Technical Field

The invention relates to the field of novel coronavirus detection kits, G01N33/569, and particularly relates to a kit for detecting a novel coronavirus neutralizing antibody in saliva and application thereof.

Background

The new type coronavirus (SARS-CoV-2) belongs to the family coronaviridae and genus coronavirus, is a single strand positive strand RNA virus, and is also a ubiquitous human and animal comorbid disease. The virus has an envelope structure, and the main structural proteins of the virus consist of spike protein (S), matrix protein (M), small envelope protein (E) and nucleocapsid protein (N). SARS CoV 2 binds to the human angiotensin converting enzyme 2 (ACE 2) receptor of the host cell via the Receptor Binding Domain (RBD) of its surface spike protein, allowing the virus to enter the host cell and replicate the virus. Specific antibodies generated against S-RBD, i.e., neutralizing antibodies, can block binding of new coronaviruses to ACE2, thereby rendering the virus non-invasive. The neutralizing antibody has the characteristics of strong specificity and high affinity, is greatly different from IgM, igG and IgA antibodies detected clinically in function, is one of important indexes in the research and development and clinical evaluation processes of vaccines, and the level of the neutralizing antibody in a body is a main index for the evaluation of immune protection efficacy. Therefore, it is very meaningful to establish a method for rapidly detecting the novel crown neutralizing antibody.

Patent application CN202010218566.7 discloses a novel coronavirus IgM antibody enzyme-linked immunoassay kit and detection method thereof, through peridium on the peridium board with new coronavirus antigen, form peridium board of peridium new coronavirus antigen in advance, during detection, need to wait to detect blood sample and drip into on the peridium board after diluting, then adopt enzyme linked immunosorbent assay to survey, this survey method needs to use specific instrument, and the sample is blood, belong to invasive sample, and the operation is complicated, unsuitable ordinary crowd is suitable for.

The immunoglobulins in saliva are mainly of class 2: igA (90% -98%) and IgG (1% -10%), with the balance being very small amounts of IgM, igD and IgE. IgG and IgA in serum enter the oral cavity through the ultrafiltration of gingival crevicular fluid, mucous membrane exudate and salivary gland acinus, so that the detection of the novel coronavirus antibody in saliva is realized. Patent application CN202011230093.9 discloses a new coronavirus detection test strip and a preparation method and a use method thereof, and the new coronavirus detection test strip mainly comprises a sample pad, a gold-labeled pad, a detection pad and a water absorption plate, wherein detection is carried out by identifying and combining sialic acid micromolecules and S protein on the surface of the new coronavirus.

Disclosure of Invention

In order to solve the problems, the invention provides a simple and quick detection kit for saliva new corona neutralizing antibodies, which comprises a detection plate and a drying agent.

In some preferred embodiments, the method for preparing the detection kit comprises: sealing the detection plate and desiccant in a sealed bag.

In some preferred embodiments, the assay plate comprises: reagent strips, pipette tips and plastic parts.

In some preferred embodiments, the reagent strip comprises: sample pad, reagent pad, NC membrane, absorbent paper, bottom plate.

In some preferred embodiments, the dipstick is a PBS-buffered dipstick; preferably, the treatment method is to immerse the pipette in PBS buffer until the pipette is completely soaked, and then dry overnight.

In some preferred embodiments, the amount of PBS buffer used is not particularly limited; preferably, the PBS buffer is used in an amount of 300mL per 1000 pipette tips.

In some preferred embodiments, the PBS buffer has a molarity of 30-65mM; preferably, the molar concentration of the PBS buffer is 50mM.

In some preferred embodiments, the PBS buffer contains 0.25-0.35mg/mL sodium phosphate monobasic dihydrate, 2-4mg/mL sodium phosphate dibasic dodecahydrate, 0.7-1.0% (w/v) sodium chloride; preferably, the composition of the PBS buffer comprises: 0.2968mg/mL sodium dihydrogen phosphate dihydrate, 2.98mg/mL disodium hydrogen phosphate dodecahydrate, 0.85% (w/v) sodium chloride.

In some preferred embodiments, the sample pad is selected from one of filter paper, glass fiber, non-woven fabric, polyester film; preferably, the sample pad is glass fiber; further preferably, the sample pad is glass fiber treated by the treatment solution.

In some preferred embodiments, the glass fibers have a size of 254mm by 300mm.

In some preferred embodiments, the treatment solution treatment is specifically to soak each glass fiber with 40mL of sample pad treatment solution until the surface is bubble-free, dry overnight, then cut to 20mm width, dry and seal.

In some preferred embodiments, the treatment fluid is an aqueous sodium tetraborate buffer.

The sodium tetraborate buffer solution is 0.05-0.2M; preferably, the aqueous sodium tetraborate solution is 0.1M.

In some preferred embodiments, the treatment solution further comprises: 0.2-0.7% (w/v) CaseinNa, 0.8-2.0% (w/v) EDTA, 0.1-0.3% (w/v) sodium cholate, 0.3-2.5% (w/v) BSA, 3-6% (w/v) surfactant; preferably, the treatment liquid further contains: 0.5% (w/v) CaseinNa, 1% (w/v) EDTA, 0.3% (w/v) sodium cholate, 1% (w/v) BSA, 4% (w/v) surfactant.

In some preferred embodiments, the surfactant comprises at least one of Tetronic 1307, S17 (surfactant S17 RHODASURF ON-870), CHAPS (3- [ (3-cholesterylaminopropyl) dimethylamino ] -1-propanesulfonic acid), tritonx-100; preferably, the surfactant is Tetronic 1307, S17, CHAPS, tritonx-100.

In some preferred embodiments, the mass ratio of Tetronic 1307, S17, CHAPS and Tritonx-100 is 1: (0.7-1.3): (0.8-1.5): (0.6-1.1); preferably, the mass ratio of the Tetronic 1307, the S17, the CHAPS and the Tritonx-100 is 1:1:1:1.

the glass fiber used as a sample pad has the advantages of compact and uniform structure, good hydrophilicity and fast permeability, but the phenomenon of sample 'backflow' is easy to occur. The applicant found that by pretreating the sample pad material, in particular, by using a pretreatment solution containing 0.2-0.7% (w/v) CaseinNa, 0.8-2.0% (w/v) EDTA, 0.1-0.3% (w/v) sodium cholate, 0.3-2.5% (w/v) BSA, 3-6% (w/v) surfactant and 0.05-0.2M sodium tetraborate, the sample pad material can be treated, so that the condition can be remarkably improved, and the release rate of the sample can be increased. Presumably, the CaseinNa contains various hydrophilic groups and hydrophobic groups, and can act with other components in a synergistic manner to further reduce the surface tension of the sample pad and promote the wetting and dispersing effects of the saliva sample in the sample pad, and the sample becomes a certain gel when entering the reagent, so that the phenomenon of sample backflow is prevented, the concentration of the sample on the surface of the sample pad is increased, and the CaseinNa also has the effect of stabilizing the sample, so that the sensitivity and the accuracy of the detection kit are improved.

In some preferred embodiments, the treatment solution is used in an amount of 30 to 50mL per glass fiber; preferably, the amount of the treatment solution is 40mL per glass fiber.

In some preferred embodiments, the reagent pad is obtained by spreading the diluted S-RBD-labeled antibody or mouse IgG-labeled antibody on glass fiber and drying overnight; preferably, the specific preparation process of the reagent pad is as follows: diluting the S-RBD labeled antibody or mouse IgG labeled antibody by a diluent of 40% (v/v) for 15 times to obtain a labeled diluent, then treating the glass fiber, drying overnight, cutting to 5mm width, drying and sealing to obtain the antibody.

In some preferred embodiments, the 40% (v/v) dilution is obtained by diluting the dilution with water.

In some preferred embodiments, the diluent is a Tris buffer containing sucrose, trehalose, triton x 100; preferably, the diluent is 65-75mM Tris buffer containing 0.8-1.3% (w/v) BSA, 8-13% (w/v) sucrose, 3-5.5% (w/v) trehalose, 0.35-0.5% (w/v) TritonX 100; preferably, the 40% (v/v) dilution is 70mM Tris buffer containing 1% (w/v) BSA, 0% (w/v) sucrose, 5% (w/v) trehalose, 0.4% (w/v) Triton X100.

In the present application, the percentage content% (w/v) of each substance is a ratio of mass to volume, and the specific unit is 1g/100mL.

In some preferred embodiments, the 70mM Tris buffer is 70mM Tris-HCl buffer.

In some preferred embodiments, the glass fibers have a size of 200mm by 300mm.

In some preferred embodiments, the marking diluent is used in an amount of 10 to 15mL per glass fiber; preferably, the marking diluent is used in an amount of 12mL per glass fiber.

In some preferred embodiments, the S-RBD labeled antibody and the murine IgG labeled antibody are a nanocarbon-labeled S-RBD protein and a nanocarbon-labeled murine IgG.

In some preferred embodiments, the method for preparing the nanocarbon-labeled S-RBD protein includes the steps of:

s1, dispersing nano carbon and then carrying out ultrasonic homogenization;

s2, preparing an EDC solution and an NHS solution respectively by using an MES coupling solution;

s3, putting 400 mu L of nano-carbon obtained in the step S1 into a 1.5mL centrifuge tube, adding the EDC solution and the NHS solution prepared in the step S2, uniformly mixing, standing and activating at room temperature for 20-40min;

s4, adding 0.1M MES coupling solution diluted labeled antibody S-RBD protein into S3, and coupling for 20-40min at room temperature;

s5, sequentially adding the confining liquid A and the confining liquid B into the S4, uniformly mixing, sealing at room temperature for 25-35min, centrifuging at 12000-16000 rpm for 10-20min, and removing a supernatant;

s6, adding the nano-carbon preservation solution into the substance obtained in the S5, performing ultrasonic homogenization, then centrifuging at 12000-16000 rpm for 10-20min, and removing the supernatant;

and S7, continuously adding the nano-carbon preservation solution into the substance obtained in the step S6, uniformly performing ultrasonic treatment, and preserving at 4 ℃.

In S1, the specific operation of the nanocarbon dispersion treatment includes the following steps:

(1) Ultrasonically dispersing nano carbon by using a solvent;

(2) Washing the nanocarbon obtained in the step (1) by centrifugation, heavy suspension, centrifugation, heavy suspension and ultrasound;

(3) Diluting the nano carbon obtained in the step (2), and storing for later use.

Preferably, the specific operation of the nanocarbon dispersion treatment in S1 includes the following steps:

(1) Adding nanocarbon into a crushing cup, adding a mixed solvent consisting of pure water and 5-13% (w/v) PVP aqueous solution, and carrying out ultrasonic treatment in an ice-water bath for 12-24h;

(2) After the nano carbon obtained in the step (1) is subpackaged, purified water is respectively added, the nano carbon is centrifuged for 8-15min at 3000-7500rpm, then supernatant fluid is taken, the nano carbon is centrifuged for 0.5-1h at 8000-12000rpm and the supernatant fluid is removed, MES coupling solution with the concentration of 0.05-0.2M is added for heavy suspension, the nano carbon is centrifuged for 0.5-1h at 8000-12000rpm and the supernatant fluid is removed, MES coupling solution with the concentration of 0.05-0.15M is added for heavy suspension, and the cleaning is finished by ultrasonic;

(3) And (3) debugging the nano carbon obtained in the step (2) by using 0.05-0.2M MES coupling solution until A400 is 0.105 +/-0.005, and storing for later use.

In some preferred embodiments, the concentration of the nanocarbon in (1) is 9 to 10mg/mL; preferably, the concentration of the nano carbon in the (1) is 9.6mg/mL.

In some preferred embodiments, the volume ratio of pure water to 5-13% (w/v) PVP in the mixed solvent is (20-28): 1; preferably, the volume ratio of pure water to 5-13% (w/v) PVP in the mixed solvent is 24:1.

in some preferred embodiments, the volume ratio of the nano-carbon subpackaged in the step (2) to the purified water is 1: (4-6), the volume ratio of the split-packaged nano carbon to 0.05-0.2M MES coupling solution is 1: (4-5.5), the volume ratio of the split-packaged nano-carbon to 0.05-0.15M MES coupling solution is 1: (3-4.5); preferably, the volume ratio of the subpackaged nano-carbon to the purified water is 1:5, the volume ratio of the split-packaged nano carbon to 0.05-0.2M MES coupling solution is 1:5, the volume ratio of the split-packaged nano carbon to 0.05-0.15M MES coupling solution is 1:4.

in S2, the molar concentration of the MES coupling solution is 0.07-0.5M, the final concentration of the EDC solution is 0.5-1.5mg/mL, and the final concentration of the NHS solution is 0.5-1.7mg/mL; preferably, the MES conjugate solution has a molar concentration of 0.1M, the EDC solution has a final concentration of 0.8mg/mL, and the NHS solution has a final concentration of 0.8mg/mL.

In S3, the volume ratio of the EDC solution to the nanocarbon is 1: (3-5.5), wherein the volume ratio of the NHS solution to the nano carbon is 1: (3.5-5); preferably, the volume ratio of the EDC solution to nanocarbon is 1:4, the volume ratio of the nhs solution to the nanocarbon was 1:4.

in S4, the concentration of the diluted labeled antibody S-RBD protein is 0.3-0.5 mug/muL, and the adding volume of the 0.1M MES coupling solution is 80-120 muL; preferably, the concentration of the diluted labeled antibody S-RBD protein is 0.4. Mu.g/. Mu.L, and the addition volume of the 0.1M MES coupling solution is 100. Mu.L.

In S5, the blocking solution A is 47-55mM Tris-HCl aqueous solution, the blocking solution A also contains 1-3% BSA, and the blocking solution B is 8-15% Tween20; preferably, the blocking solution A is 50mM Tris-HCl aqueous solution, the blocking solution A also contains 2% BSA, and the blocking solution B is 10% Tween20.

The volume ratio of the sealing liquid A to the nano carbon in the S3 is (0.5-1): 1, the volume ratio of the sealing liquid B to the nano carbon in the S3 is (0.1-0.4): 1; preferably, the volume ratio of the sealing liquid A to the nano carbon in the S3 is 0.75:1, the volume ratio of the sealing liquid B to the nano carbon in the S3 is 0.25:1.

in S6, the nano-carbon preservation solution is 47-55mM Triss-HCl aqueous solution, the nano-carbon preservation solution also contains 0.25-0.6% BSA, and the volume ratio of the nano-carbon preservation solution to the nano-carbon in S3 is (0.8-1.3): 1; preferably, the nanocarbon preservation solution is 50mM Tris-HCl aqueous solution, the nanocarbon preservation solution further contains 0.5% BSA, and the volume ratio of the nanocarbon preservation solution to the nanocarbon in S3 is 1:1.

in S7, the volume ratio of the nano-carbon preservation solution to the nano-carbon in S3 is (0.3-0.6): 1; preferably, the volume ratio of the nanocarbon preservation solution to the nanocarbon in the S3 is 0.5:1.

in some preferred embodiments, the diluted mouse IgG antibody has a concentration of 0.3-0.5. Mu.g/. Mu.L, and the 0.1M MES conjugate solution is added in a volume of 80-120. Mu.L; preferably, the diluted mouse IgG antibody has a concentration of 0.4. Mu.g/. Mu.L, and the 0.1M MES conjugate solution is added in a volume of 100. Mu.L.

The patent adopts a nanocarbon marking method antibody, is completely different from a colloidal gold marking method, has high detection sensitivity and good stability, is environment-friendly in carbon material, has lower cost than colloidal gold in a nanocarbon raw material, and is easier to distinguish from black and white color of an NC (numerical control) film. The applicant finds that 400 mu L of the dispersed nano carbon is taken, and then 0.3-0.5 mu g/mu L of labeled antibody S-RBD protein and mouse IgG antibody are added, so that the dispersibility of the nano carbon is good, the antibody can be fully labeled by the nano carbon, the total neutralizing antibody generated by a human body can be fully detected, the sensitivity of the detection kit is provided, and the detection limit is reduced. Particularly, when the concentration of the labeled antibody is 0.4 mug/muL, the combination of the nano carbon and the labeled antibody is good, the labeling stability is enhanced, and the storage stability of the nano carbon and the resolution of the detection kit are improved.

In some preferred embodiments, the NC membrane is coated with a detection line T line and a quality control line C line which are parallel to each other, the separation distance between the detection line T line and the quality control line C line is 5mm, the detection line T line is close to the reagent pad, and the quality control line C line is close to the absorbent paper.

In some preferred embodiments, the NC film is prepared by: adhering the NC film to a bottom plate, then respectively coating the T-line antibody and the C-line antibody on the NC film, drying overnight, adding a drying agent, and sealing; preferably, the preparation method of the NC membrane comprises the following steps: tearing off the protective paper in the middle of the PVC base plate, pasting the NC film along the lower edge of the protective paper, respectively coating the T-line antibody and the C-line antibody on the NC film by adopting a gold spraying film scratching instrument, drying overnight, adding a drying agent, and sealing.

In some preferred embodiments, the PVC base plate has dimensions of 60mm by 280mm.

In some preferred embodiments, the NC membrane is 1UN14ER100020NT.

In some preferred embodiments, the T-line antibody is a T-line coating solution obtained by diluting S-RBD protein of the coated antibody with a coating diluent.

In some preferred embodiments, the C-line antibody is a C-line coating solution obtained by diluting a goat anti-mouse IgG polyclonal antibody with a coating diluent.

In some preferred embodiments, the coating diluent is a PBS buffer solution with a pH of 7.4; preferably, the coating diluent is a PBS buffer solution containing sucrose at pH 7.4; further preferably, the coating diluent is a 10mM PBS buffer solution containing 10g/L sucrose at pH 7.4.

In some preferred embodiments, the T-line antibody has a mass concentration of 1 to 2mg/mL and the C-line antibody has a mass concentration of 1.5 to 2.5mg/mL; preferably, the mass concentration of the T line antibody is 1.5mg/mL, and the mass concentration of the C line antibody is 2.0mg/mL.

In some preferred embodiments, the gold spraying film scratching instrument has a liquid scratching amount of 0.5 to 1.7 mu L/cm and a film scratching speed of 50 to 75mm/s; preferably, the scratching liquid volume of the gold spraying scratching film instrument is 1 mu L/cm, and the scratching speed is 60mm/s.

In some preferred embodiments, the reagent strip is composed of a sample pad, a reagent pad, an NC membrane and absorbent paper which are adhered on a bottom plate; preferably, the specific preparation operation of the reagent strip is as follows: and (3) attaching 17mm of absorbent paper to the bottom plate coated with the NC film, and then sequentially attaching one reagent pad (mouse IgG labeled antibody) with the width of 5mm, one reagent pad (S-RBD labeled antibody) with the width of 5mm and one sample pad with the width of 20 mm.

In some preferred embodiments, the test plate is prepared by cutting the reagent strip to a width of 3-4mm, mounting the reagent strip on a bottom plate of a plastic member, attaching an upper cover, attaching a pipette stick, and pressing the cover.

The invention provides a simple and rapid detection kit for the saliva new crown neutralizing antibody, and an application of the detection kit in saliva new crown detection.

Compared with the prior art, the invention has the following beneficial effects:

(1) The kit obtained by the application adopts a nanocarbon immunochromatography technology and a double-antigen sandwich method to detect the content of the new crown neutralizing antibody in saliva. The reagent strip adopts an NC membrane as a substrate, and the reagent pad adopts nano-carbon labeled S-RBD and mouse IgG, S-RBD protein and goat anti-mouse IgG as a detection line T and a quality control line C. When the saliva sample is detected, the saliva sample flows forwards under the capillary action, and the nano-carbon markers on the reagent pad are dissolved and then move forwards together with the sample. After the sample reaches the detection area, the S-RBD marked by the nano carbon, the neutralizing antibody in the sample and the S-RBD coated on the detection line form a compound, the compound is shown as that the detection line is gray black, and the content of the antibody in the sample is in positive correlation with the color development intensity of the detection line. After the reaction is finished, the color development condition is directly observed by naked eyes for testing, the content of the neutralizing antibody can be judged through the color development intensity of the nano carbon on the detection line, and the detection method is simple and convenient.

(2) The detection kit obtained by the application has high sensitivity, intuitive result, low infection risk, safety and no wound, and is a simple, accurate and economic field rapid detection method.

(3) The obtained detection kit of this application resolution ratio is high, easy judgement, and the sample is saliva moreover, and the sample collection patient collects easily by oneself and accepts, and need not to consider improper results misjudgement such as save, transportation, processing.

(4) According to the invention, a sandwich method is adopted to detect the total neutralizing antibody of the new corona, and the S-RBD of the S protein of the new corona virus with high specificity is selected, so that the total neutralizing antibody generated by a human body, including IgA, igM and IgG, can be effectively detected, and the problems of low resolution and difficulty in interpretation when the concentration of the neutralizing antibody is low in a colloidal gold competition method are solved.

Drawings

FIG. 1A plan view of the structure of a test board obtained in example 1.

FIG. 2 is a sectional view showing the structure of a test plate obtained in example 1.

FIG. 3 shows the result of detection of a negative sample in the detection kit obtained in example 1.

FIG. 4 shows the gradient detection result of quality control material of the detection kit obtained in example 1.

FIG. 5 shows the gradient detection result of the control in the detection kit obtained in example 2.

FIG. 6 shows the gradient detection result of the control in the detection kit obtained in example 2.

Description of reference numerals: 1 is a liquid absorption rod; 2 is a sample pad; 3 is a reagent pad; 4 is NC (nitrocellulose) film; 5 is absorbent paper; 6 is a bottom plate

Detailed Description

Example 1

1. A simple and rapid detection kit for saliva new corona neutralizing antibody comprises a detection plate and a drying agent.

The preparation method of the detection kit comprises the following steps: sealing the detection plate and desiccant in a sealed bag.

The detection board includes: reagent strips, pipette tips and plastic parts.

The reagent strip comprises: sample pad, reagent pad, NC membrane, absorbent paper, bottom plate.

The liquid absorbing rod is treated by PBS buffer solution; the treatment method is to immerse the pipette in PBS buffer until the pipette is completely soaked, and then dry the pipette at 37 ℃ overnight.

The amount of PBS buffer used was 300mL per 1000 pipette sticks.

The PBS buffer was 50mM PBS.

The PBS buffer contained 0.2968mg/mL of sodium dihydrogen phosphate dihydrate, 2.98mg/mL of disodium hydrogen phosphate dodecahydrate, and 0.85% (w/v) of sodium chloride.

The liquid absorbent stick is purchased from Bo-Filter specialty fibers (Ningbo) Inc.

The sample pad is glass fiber treated by the treatment solution.

The size of the glass fiber was 254mm × 300mm (glass fiber 8964, purchased from biotech ltd, korea, shanghai).

The treatment of the treatment solution is to soak each glass fiber with 40mL of sample pad treatment solution until no bubbles exist on the surface, dry the glass fiber at 37 ℃ overnight, then cut the glass fiber to be 20mm wide, dry and seal the glass fiber.

The treatment fluid is a sodium tetraborate buffer aqueous solution.

The sodium tetraborate buffer solution is 0.1M.

The treatment solution contained 0.5% (w/v) CaseinNa, 1% (w/v) EDTA, 0.3% (w/v) sodium cholate (C1254 available from sigma), 1% (w/v) BSA, and 4% (w/v) surfactant.

The surfactant is Tetronic 1307, S17 (surfactant S17 RHODASUREF ON-870), CHAPS (3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid) and Tritonx-100.

The mass ratio of Tetronic 1307, S17, CHAPS and Tritonx-100 is 1:1:1:1.

the dosage of the treatment liquid is 40mL per glass fiber.

The specific preparation process of the reagent pad comprises the following steps: diluting the S-RBD labeled antibody by 15 times with 40% (v/v) diluent to obtain labeled diluent, then treating the glass fiber, drying at 37 ℃ overnight, cutting to 5mm width, drying and sealing to obtain the final product.

The 40% (v/v) diluent is obtained by diluting the diluent with water.

The dilution contained 1% (w/v) BSA, 10% (w/v) sucrose, 5% (w/v) trehalose, 0.4% (w/v) TritonX100 in 70mM Tris buffer.

The 70mM Tris buffer is 70mM Tris-HCl buffer.

The size of the glass fiber was 200mm × 300mm (glass fiber KB50 available from biotechnology limited, korea, shanghai).

The amount of the marking diluent was 12mL per glass fiber.

The S-RBD labeled antibody and the mouse IgG labeled antibody are the S-RBD labeled antibody labeled by nano carbon and the mouse IgG labeled by nano carbon.

The preparation method of the nanocarbon-labeled S-RBD-labeled antibody comprises the following steps:

s1, dispersing nano carbon and then carrying out ultrasonic homogenization;

s3, putting 400 mu L of nano-carbon obtained in the step S1 into a 1.5mL centrifuge tube, adding the EDC solution and the NHS solution prepared in the step S2, uniformly mixing, standing and activating at room temperature for 30min;

s4, adding 0.1M MES coupling solution diluted labeled antibody S-RBD protein into S3, and coupling for 30min at room temperature;

s5, sequentially adding the confining liquid A and the confining liquid B into the S4, uniformly mixing, sealing for 30min at room temperature, centrifuging for 15min at 13000rpm, and removing supernatant;

s6, adding the nano-carbon preservation solution into the substance obtained in the S5, performing ultrasonic homogenization, then centrifuging at 13000rpm for 15min, and removing the supernatant;

(1) Adding nanocarbon into a crushing cup, adding a mixed solvent consisting of pure water and 10% (w/v) PVP aqueous solution, and carrying out ultrasonic treatment for 16h in an ice-water bath;

(2) After subpackaging the nanocarbon obtained in the step (1), respectively adding purified water, centrifuging at 600rpm for 10min, then taking supernatant, centrifuging at 9800rpm for 1h and removing the supernatant, adding 0.1M MES coupling solution for resuspension, and carrying out ultrasonic cleaning;

(3) And (3) debugging the nano carbon obtained in the step (2) to A400 of 0.105 +/-0.005 by using 0.1M MES coupling liquid, and storing for later use.

The nanocarbon was purchased from the island technologies ltd, dekko, beijing, with a model of CNT302.

The concentration of the nano carbon in the (1) is 9.6mg/mL.

The volume ratio of pure water to 10% (w/v) PVP in the mixed solvent is 24:1.

the volume ratio of the split packaged nano carbon to the purified water is 1:5, the volume ratio of the split-packaged nano carbon to 0.05-0.2M MES coupling solution is 1:5, the volume ratio of the split-packaged nano carbon to 0.05-0.15M MES coupling solution is 1:4.

in S2, the molar concentration of the MES coupling solution is 0.1M, the final concentration of the EDC solution is 0.8mg/mL, and the final concentration of the NHS solution is 0.8mg/mL.

In S3, the volume ratio of the EDC solution to the nano-carbon is 1:4, the volume ratio of the NHS solution to the nano carbon is 1:4.

in S4, the concentration of the diluted labeled antibody S-RBD protein is 0.4. Mu.g/. Mu.L, and the addition volume of the 0.1M MES coupling solution is 100. Mu.L.

In S5, the blocking solution A is 50mM Tris-HCl aqueous solution, the blocking solution A also contains 2% BSA, and the blocking solution B is 10% Tween20 (purchased from VWR under the trade name 0777).

The volume ratio of the sealing liquid A to the nano carbon in the S3 is 0.75:1, the volume ratio of the sealing liquid B to the nano carbon in the S3 is 0.25:1.

in S6, the nanocarbon preservation solution is 50mM Tris-HCl aqueous solution, 0.5% BSA is also contained in the nanocarbon preservation solution, and the volume ratio of the nanocarbon preservation solution to the nanocarbon in S3 is 1:1.

in S7, the volume ratio of the nano-carbon preservation solution to the nano-carbon in S3 is 0.5:1.

the specific implementation mode of the preparation method of the nanocarbon-labeled mouse IgG-labeled antibody is the same as that of the nanocarbon-labeled S-RBD-labeled antibody.

The concentration of the diluted mouse IgG labeled antibody is 0.4 mu g/mu L, and the addition volume of the 0.1M MES coupling solution is 100 mu L.

The NC membrane is coated with a detection line T line and a quality control line C line which are parallel to each other, the interval distance between the detection line T line and the quality control line C line is 5mm, the detection line T line is close to the reagent pad, and the quality control line C line is close to the absorbent paper.

The preparation method of the NC film comprises the following steps: tearing off the protective paper in the middle of the PVC base plate, pasting the NC film along the lower edge of the protective paper, respectively coating the T-line antibody and the C-line antibody on the NC film by adopting a gold spraying film scratching instrument, drying overnight at 37 ℃, adding a drying agent, and sealing.

The PVC base plate has a size of 60mm × 280mm (MT 101200507 from New Material Co., mike, quzhou).

The NC membrane was 1UN14ER100020NT (Sadoris).

The T line antibody is a T line coating solution obtained by diluting S-RBD protein of the coating antibody by using a coating diluent.

The C-line antibody is C-line coating solution obtained by diluting goat anti-mouse IgG by using coating diluent.

The coating dilution was 10mM PBS buffer containing 10g/L sucrose at pH 7.4.

The mass concentration of the T line antibody is 1.5mg/mL, and the mass concentration of the C line antibody is 2.0mg/mL.

The scratching liquid quantity of the gold spraying scratching film instrument is 1 mu L/cm, and the scratching speed is 60mm/s.

The absorbent paper is purchased from 21050901 of pharmaceutical medical devices (shanghai) limited.

The reagent strip is formed by sticking a sample pad, a reagent pad, an NC membrane and absorbent paper on a bottom plate; the specific preparation operation of the reagent strip is as follows: and (3) pasting 17mm of water-absorbing paper on the bottom plate coated with the NC film, and then sequentially pasting a reagent pad and a sample pad with the width of 20 mm.

The reagent pad is a mouse IgG labeled antibody reagent pad with the width of 5mm and an S-RBD labeled antibody reagent pad with the width of 5mm, the two reagent pads are transversely arranged side by side, the reagent pad close to the sample pad is the S-RBD reagent pad, and the reagent pad close to the NC membrane is the mouse IgG reagent pad.

The lengths of the sample pad and the reagent pad are the same as the length of the PVC base plate.

The detection plate is prepared by cutting reagent strip into 3-4mm width, mounting on the bottom plate of plastic part, covering, mounting liquid absorbing rod, and pressing the cover (shown in fig. 1 and 2).

2. A simple and rapid detection kit for a saliva new crown neutralizing antibody is applied to saliva new crown detection.

Example 2

1. A simple and rapid detection kit for saliva neocorona neutralizing antibodies is different from that in example 1 in that:

the treatment liquid contains: 1.0% (w/v) CaseinNa, 1% (w/v) EDTA, 0.5% (w/v) sodium cholate, 1% (w/v) BSA, 4% (w/v) surfactant.

Example 3

in the preparation method of the S-RBD labeled antibody labeled by nano carbon,

and S3, putting 200 mu L of nanocarbon obtained in the S1 into a 1.5mL centrifuge tube, adding the EDC solution and the NHS solution prepared in the S2, uniformly mixing, standing, and activating at room temperature for 30min.

Performance testing

The detection kits obtained in the examples and the comparative examples are used for detecting real samples.

(1) Negative samples: selecting unvaccinated human saliva as a negative sample;

according to the kit instruction operation: the liquid absorbing rod is respectively placed on the tongue, the upper jaw and the lower jaw of the oral cavity and the inner wall of the oral cavity for scraping for 5 times, then placed on the tongue for buccal, the sampling can be finished when the liquid appears in the observation window and reaches the T position, the sampling is taken out, the cover is covered, the desktop is horizontally placed, and the timing is started. And (5) judging the result in 15 min. The test results are shown in fig. 3.

The test results are negative, which indicates that the test quality control line of the negative sample is normal.

(2) Quality control product gradient detection

The saliva of the non-vaccinated persons was collected as a dilution with a disposable urine cup, and the quality control of the neutralizing antibody was diluted to different concentrations of 2.5. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.1. Mu.g/mL, and 0.05. Mu.g/mL.

And (3) sucking 450 mu L of saliva diluted sample by using a pipette, directly adding the saliva diluted sample to the pipette stick of the detection plate obtained in the example 1-3, reaching the T position when liquid appears in an observation window, and timing for 15min to judge the result. The following values represent our test results, (as judged by the color card): c3.5 represents weak yang, C5 represents medium yang, and C7 represents strong yang.

The detection result of the detection kit obtained in example 1 is shown in fig. 4: the detection result is C3.5 (weak positive) at 0.1 mu g/mL, and the detection result is negative at C3 and below which are 0.05 mu g/mL, which indicates that the sensitivity of the test is 0.1 mu g/mL, the repeated detection is carried out on the quality control product of 0.5 mu g/mL, the results are consistent in color development, and the experimental result is stable.

The detection result of the detection kit obtained in example 2 is shown in FIG. 5: the sensitivity can only detect 0.5 mug/mL, weak positive C3.5.

The detection result of the detection kit obtained in example 3 is shown in FIG. 6: the sensitivity can only detect 0.5 mug/mL, weak positive C4.

(3) And (3) real sample detection: the measurement was carried out using the detection kit obtained in example 1.

According to the kit instruction: by testing 54 vaccinees clinical specimens: 31 vaccinations of the pfeiffe vaccine, 18 vaccinations of the danio-manicure vaccine, and 5 vaccinations of the boost vaccine.

The detection result shows that 45 samples show positive; saliva samples of 64 uninoculated corona vaccinees were tested and all negative results were shown. The sensitivity of the kit is preliminarily determined to be >83%, which indicates that the detection mode is feasible.

The data of the kit detection card obtained in the embodiment 1-3 show that the detection method and the kit for detecting the novel coronavirus neutralizing antibody in saliva by the sandwich method have the advantages of simple operation, high sensitivity, visual result, low infection risk, safety and no wound, and are a simple, accurate and economic field rapid detection method.

Claims

1. A simple and rapid detection kit for saliva new corona neutralizing antibody is characterized by comprising a detection plate and a drying agent;

the detection board includes: reagent strips, pipette tips and plastic parts;

the reagent strip comprises: the kit comprises a sample pad, a reagent pad, an NC membrane, absorbent paper and a bottom plate;

the liquid absorbing rod is treated by PBS buffer solution.

2. The kit for detecting the simple and rapid saliva corona neutralizing antibody according to claim 1, wherein the sample pad is selected from one of filter paper, glass fiber, non-woven fabric and polyester film.

3. The kit for detecting the simple and rapid saliva corona neutralizing antibody according to claim 1 or 2, wherein the sample pad is glass fiber treated by a treatment solution.

4. The simple and rapid detection kit for saliva new crown neutralizing antibodies according to claim 3, wherein the treatment solution treatment is specifically to immerse glass fibers in a pretreatment solution for treatment for 0.5-1.5h, dry overnight, then cut to 20mm width, dry and seal;

the treatment fluid is a sodium tetraborate buffer aqueous solution; the sodium tetraborate buffer solution is 0.05-0.2M.

The treatment liquid also contains: 0.2-0.7% (w/v) CaseinNa, 0.8-2.0% (w/v) EDTA, 0.1-0.3% (w/v) sodium cholate, 0.3-2.5% (w/v) BSA, 3-6% (w/v) surfactant.

5. The kit for detecting the simple and rapid saliva corona neutralization antibody according to claim 1, wherein the reagent pad is prepared by spreading an S-RBD labeled antibody or a mouse IgG labeled antibody diluted by a diluent on glass fibers, and drying the glass fibers overnight.

6. The simple and rapid detection kit for saliva corona new neutralizing antibody according to claim 5, wherein the S-RBD labeled antibody and the mouse IgG labeled antibody are nano carbon labeled S-RBD labeled antibody and nano carbon labeled mouse IgG labeled antibody.

7. The simple and rapid detection kit for saliva neocorona neutralizing antibody according to claim 6, wherein the preparation method of the nanocarbon-labeled S-RBD-labeled antibody comprises the following steps:

s1, dispersing nano carbon and then carrying out ultrasonic homogenization;

8. The simple and rapid detection kit for saliva new corona neutralizing antibody according to claim 7, wherein the specific operation of dispersing the nanocarbon in the S1 solution comprises the following steps:

(1) Adding nano carbon into a crushing cup, adding a mixed solvent consisting of pure water and 5-13% (w/v) PVP (polyvinyl pyrrolidone) aqueous solution, and carrying out ultrasonic treatment in an ice-water bath for 12-24h;

(2) After the nano carbon obtained in the step (1) is subpackaged, purified water is respectively added, the nano carbon is centrifuged for 8-15min at 3000-7500rpm, then supernatant fluid is taken, the nano carbon is centrifuged for 0.5-1h at 8000-12000rpm and the supernatant fluid is removed, MES coupling fluid with 0.05-0.2M is added for resuspension, the nano carbon is centrifuged for 0.5-1h at 8000-12000rpm and the supernatant fluid is removed, MES coupling fluid with 0.05-0.15M is added for resuspension, and the cleaning is finished by ultrasonic;

9. The simple and rapid detection kit for the salivary neocoronal neutralizing antibody according to claim 1, wherein the NC membrane is prepared by a method comprising the following steps: adhering the NC film to the bottom plate, then respectively coating the T-line antibody and the C-line antibody on the NC film, drying overnight, adding a drying agent, and sealing.

10. Use of the simple and rapid detection kit for salivary corona neutralization antibody according to any one of claims 1 to 9 in salivary corona detection.