CN115754277A - Monkey pox virus antigen detection kit and preparation method thereof - Google Patents
Monkey pox virus antigen detection kit and preparation method thereof Download PDFInfo
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- CN115754277A CN115754277A CN202211383428.XA CN202211383428A CN115754277A CN 115754277 A CN115754277 A CN 115754277A CN 202211383428 A CN202211383428 A CN 202211383428A CN 115754277 A CN115754277 A CN 115754277A
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Abstract
The invention belongs to the technical field of in-vitro diagnosis, and relates to a monkeypox virus antigen detection kit and a preparation method thereof. The detection card comprises an upper cover, a lower cover and a monkeypox virus antigen detection test strip, wherein the monkeypox virus antigen detection test strip comprises a sample pad, a latex combination pad, a solid-phase antibody reaction membrane, absorbent paper and a PVC base plate, and a sample adding hole, an observation window and an upper adhesive sticker window are arranged on the upper cover. The kit for quickly detecting the monkeypox antigen is developed based on the immunochromatography principle of the specific binding reaction of a latex immunochromatography method and an antigen-antibody, and is one of the methods for quickly screening monkeypox patients. The invention is characterized in that biological raw materials are pre-fixed on corresponding carriers through processes of coating, marking, drying, assembling and the like, a user only needs to mix collected samples into diluent, then adds the diluted samples onto products, and waits for time to read results.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnosis, and relates to a monkeypox virus antigen detection kit and a preparation method thereof.
Background
Monkeypox is a monkey occurring in rainforest in the middle and west parts of africa, and can also infect other animals, and occasionally can infect humans, and the clinical manifestations are similar to the patterns of the day, but the disease condition is mild. This disease is caused by monkeypox virus. It belongs to a group of viruses including variola viruses, viruses employed in variola vaccines and vaccinia viruses. It is required to be identified with smallpox and chicken pox. The virus can be transmitted from animals to human bodies through direct and close contact, and can also be transmitted from human to human, and the transmission route mainly comprises blood and body fluid.
Monkeypox epidemic in 2022 was first discovered by the uk at 2022 years 5 months 7 at local time. The world health organization confirms that an emergency meeting is held for monkeypox with the confirmation and suspected monkeypox cases of more than 100 in europe, at 5 months and 20 days of local time. At local time 2022, 5 month 29, the world health organization announced disease information announcements and evaluated monkeypox for moderate global public health risk.
According to the 24 th message of 6 months and 2022 years of Russian satellite communication society, the general affairs of the world health organization Tan Desai shows that the number of confirmed monkeypox cases all over the world is over 3200 cases. At the local time of 2022, 7, 23, afternoon, 15 o' clock, the world health organization summons the memorandum in the geneva, the general affair Tan Desai announces that the monkeypox epidemic is classified as an international public health incident of sudden concern.
Monkey pox is a viral zoonosis caused by monkey pox virus infection, monkey pox virus is an enveloped double-stranded DNA virus, belongs to orthopoxvirus of poxviridae with variola virus and vaccinia virus, and the main host is African rodent (such as African squirrel, tree squirrel, ojaya kangaroo, sleeping rat, etc.) [1-2]. In 2022, 5/7/month, UK confirmed the first cases of diagnosis of monkeypox [3], and monkeypox epidemics spread rapidly in several non-monkeypox endemic countries, such as the United states, spain, brazil, france, germany, UK, peru, the Netherlands, canada, etc. Day 23/7/2022, world Health Organization (WHO) announced that monkeypox epidemic constitutes an emergent public health event of international concern [4], and by day 1/8/2022, american centers for disease control and prevention (US CDC) statistics showed that 25047 monkeypox cases were reported in 76 non-monkeypox endemic countries, with the largest number in the united states being 6325 cases [5].
Monkeypox virus can be transmitted by direct contact with infectious ulcers, body fluids, respiratory droplets, and contaminated clothing. The vast majority of monkeypox cases are males, most of which occur in andropause, bisexual, and male actors [6]. The monkey pox has similar clinical manifestations to the smallpox, but the infectivity is inferior to the smallpox, the clinical manifestations of monkey pox cases in areas outside Africa are atypical, the rash is limited to genitals, perineum, perianal or perioral, and symptoms such as lymph node swelling, fever and pain appear.
Since the world wide elimination of smallpox was announced by the WHO in 1980, smallpox vaccination was stopped in each country, and in monkeypox cases occurring in this year, 74% of those without smallpox vaccination were present. At present, no specific treatment scheme aiming at the monkeypox virus exists, and smallpox can be used for local epidemic situations.
Disclosure of Invention
The invention aims to provide a monkeypox virus antigen detection kit and a preparation method thereof, which can be suitable for detecting oropharyngeal swabs, body fluids, blood and skin lesion tissue (including vesicular fluid, pustule fluid and crusty) samples, have high sensitivity, and can effectively shorten the window period of virus diagnosis so as to reduce the invisible transmission risk brought by the latent period and effectively control the large-scale transmission of epidemic situations.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the utility model provides a monkey pox virus antigen detect reagent box, includes detection card and sample diluent, and the detection card includes upper cover, lower cover and monkey pox virus antigen detect test paper strip, monkey pox virus antigen detect test paper strip includes sample pad, latex combination pad, solid-phase antibody reaction film, absorbent paper and PVC bottom plate, covers on and is equipped with application of sample hole and observation window and goes up the non-setting adhesive window.
The solid-phase antibody reaction membrane is pasted on a PVC plate, the two ends of the solid-phase antibody reaction membrane are respectively lapped with one end of a latex combination pad and one end of absorbent paper, the other end of the latex combination pad is pasted on the PVC plate, the other end of the latex combination pad is lapped with one end of a sample pad, the other end of the sample pad is pasted on the PVC plate, and the other end of the absorbent paper is pasted on the PVC plate.
The sample diluent comprises 1.2% by mass of tris (hydroxymethyl) aminomethane, 0.1% by mass of Casein-Na, 0.7% by mass of Triton X-100, 2% by mass of KCl, 1% by mass of glycine, 0.04% by mass of CMC, 1% by mass of sodium carbonate, 0.5% by mass of fatty alcohol-polyoxyethylene ether, 0.2% by mass of BND-10 and 0.05% by volume of Proclin-300.
A method for manufacturing the simipox virus antigen detection kit according to claim 1, comprising a method for preparing a latex conjugate pad, a method for preparing a sample pad and a method for preparing a solid-phase antibody reaction membrane, wherein the method for preparing the latex conjugate pad comprises the following steps:
(1) Carrying out vortex and ultrasonic treatment on latex, wherein the latex is particles with the particle size of 300 nm;
(2) Mixing latex and cleaning solution according to the volume ratio of 1:4, carrying out heavy suspension;
(3) Centrifuging at 4 deg.C and 12000rpm for 15min, removing supernatant, resuspending with the same volume of cleaning solution, and treating with ultrasound;
(4) Centrifuging at 12000rpm at 4 deg.C for 15min, removing supernatant, resuspending with the same volume of reaction buffer and treating with ultrasound;
(5) Adding EDAC into the latex reaction solution according to 20% of the volume of the original latex, and performing vortex incubation for 30min at room temperature;
(6) Adding NHS into the latex reaction solution according to 10% of the volume of the original latex, and continuing vortex incubation for 30min at room temperature;
(7) Centrifuging at 12000rpm at 4 deg.C for 15min, removing supernatant, resuspending with the same volume of conjugate diluent and sonicating;
(8) Adding a marked monkeypox virus monoclonal antibody according to the proportion of 1.0mg/mL, and performing vortex incubation for 1.5h at normal temperature;
(9) Centrifuging at 12000rpm at 4 deg.C for 15min, removing supernatant, resuspending with blocking solution of the same volume, and treating with ultrasound; sealing for 30min;
(10) Centrifuging at 4 deg.C and 12000rpm for 15min, removing supernatant, diluting the precipitate with redissolution to eight times the volume of original latex, and performing resuspension and ultrasonic treatment to obtain latex-labeled working stock solution;
(11) The latex-labeled working stock solution was sputtered onto glass fibers at 1.5ul/cm and dried overnight at 45 ℃ to produce a latex conjugate pad.
And (3) the cleaning liquid in the step (2) comprises 3.8 mass percent of borax.
The reaction buffer of step (4) comprises 50mM MES buffer at pH 5.5.
The dilution of the conjugate in the step (7) is 1.2% by mass of Na 2 HPO 4 ·12H 2 O and NaH with the mass percent of 0.148% 2 PO 4 ·2H2O。
The confining liquid in the step (9) comprises 0.09 mass percent of Na2OH with the pH value of 8.0, 1 mass percent of sodium caseinate and 0.618 mass percent of borax.
The redissolution of the step (10) comprises 0.125 mass percent of tris (hydroxymethyl) aminomethane, 0.25 mass percent of Casein, 20 mass percent of sucrose and 5 mass percent of trehalose.
The preparation method of the sample pad comprises the following steps: the sample pad treatment solution was soaked with K8964 glass cellulose membrane, 3.3 mL/strip, and dried overnight at 45 ℃ to prepare a sample pad.
The sample pad treatment fluid comprises 0.6 mass percent of tris (hydroxymethyl) aminomethane with a pH value of 8.0, 1 mass percent of PVP-30, 0.5 mass percent of S9, 0.5 mass percent of Casein, 0.5 mass percent of TritonX-100, 1 mass percent of S-17, 0.3 mass percent of EDTA, 1 mass percent of BSA and 0.5 mass percent of fatty alcohol-polyoxyethylene ether.
The preparation method of the solid-phase antibody reaction membrane comprises the following steps: the solid-phase antibody reaction membrane is sequentially coated with a detection T line and a quality control C line, the detection T line is coated with a monkeypox monoclonal antibody, and the quality control C line is coated with a goat anti-mouse IgG antibody and DNP-BSA.
The preparation method of the solid-phase antibody reaction membrane comprises the following steps: the concentration of the monkeypox monoclonal antibody 1 is 1.0mg/mL, the concentration of the goat anti-mouse IgG antibody is 0.5mg/mL, and the concentration of DNP-BSA is 0.5mg/mL.
By adopting the technical scheme, the invention has the following advantages:
1. the invention adopts 300nm latex label, compared with the traditional colloidal gold label, the latex label adopts covalent coupling gold particle conjugate which is more stable, and avoids the influence of gold stability and uniformity caused by sample adding mode, heating time, stirring speed, reaction temperature and container; in addition, the latex is large particles with the particle size of 300nm, the sensitivity can be improved by up to 20 times, the kit is more suitable for the detection of multiple sample types, the low-content virus in the oropharyngeal swab, body fluid and blood before the rash of a patient can be detected, the window period of virus diagnosis is shortened, the invisible transmission risk brought by the latent period is effectively reduced, and the large-scale transmission of epidemic situation is controlled.
2. At present, the monkeypox virus antigen diagnostic reagent does not exist at home and abroad, and the sample compatibility of the reagent can be improved to the maximum extent by optimizing the sample pad and the sample diluent. The components in the sample diluent and the sample pad of the monkeypox virus antigen detection kit are matched with each other to finally form a composite system with strong interference resistance and strong buffering capacity, so that the electrolyte among different samples of oropharyngeal swabs, body fluid, blood and skin lesion tissues (including vesicular fluid, pustule fluid and crusts) can be effectively buffered, and the monkey-pox virus antigen detection kit is compatible with various sample types.
3. Because the surfactant, the hydrophilic polymer, the anti-RBC monoclonal antibody and the like are added into the sample diluent and the sample pad, the non-specific combination of factors such as fat, bacteria, cell fragments, heterophilic antibodies, red blood cells and the like in the sample and the antigen antibody can be effectively prevented, the generation of false positive is reduced on the basis of protecting the antigen to be detected, and the detection accuracy is improved.
In a word, the monkey pox antigen rapid detection kit developed by the invention is based on the immunochromatography principle of the specific binding reaction of a latex immunochromatography method and an antigen antibody, and is one of the methods for rapidly screening monkey pox patients. According to the invention, biological raw materials are processed by processes of coating, marking, drying, assembling and the like and are fixed on corresponding carriers in advance, so that a user only needs to mix the collected sample into diluent, then add the diluted sample to a product, and wait for time to read a result. Compared with the conventional monkey pox PCR detection mode, the monkey pox antigen rapid detection kit has lower requirements on the detection environment, the detection process is simple and understandable, and the monkey pox antigen rapid detection kit is generally considered to be suitable for personal and family self-detection, large-scale rapid screening application and the like.
Drawings
FIG. 1 is a schematic view showing the structure of a detection card in the monkeypox virus antigen detection kit according to the present invention;
FIG. 2 is a schematic structural diagram of a simian pox virus antigen detection test strip in the simian pox virus antigen detection kit of the invention;
FIG. 3 is a schematic view of the structure of the upper cover of the detection kit for monkeypox virus antigen in accordance with the present invention;
FIG. 4 is a schematic flow chart of the preparation method of the monkeypox virus antigen detection kit of the present invention (gray steps are the key points).
Wherein the reference numbers: 3-monkeypox virus antigen detection test paper strip, 4-PVC board, 5-sample pad, 6-latex combined pad, 7-solid phase antibody reaction membrane, 8-absorbent paper, 9-detection T line, 10-quality control C line, 11-sample adding hole, 12-observation window, 13-upper cover, 14-lower cover and 15-upper non-setting adhesive window.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, in which specific conditions are not specified, and which are performed according to conventional conditions or conditions recommended by manufacturers. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.
The embodiments described are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without any inventive work belong to the protection scope of the present invention.
Example 1:
as shown in fig. 1-4, a monkeypox virus antigen detection kit, including detection card and sample diluent, the detection card includes upper cover 13, lower cover 14 and monkeypox virus antigen detection test paper strip 3, upper cover 13 and lower cover 14 buckle each other and connect, form between upper cover 13 and the lower cover 14 and hold the chamber, monkeypox virus antigen detection test paper strip 3 is located and holds the intracavity, monkeypox virus antigen detection test paper strip 3 includes sample pad 5, latex bond pad 6, solid phase antibody reaction membrane 7, absorbent paper 8 and PVC board 4, solid phase antibody reaction membrane 7 pastes on PVC board 4, the both ends of solid phase antibody reaction membrane 7 overlap joint respectively have one end of latex bond pad 6 and one end of absorbent paper 8, the other end of latex bond pad 6 adheres on PVC board 4, the other end overlap joint of latex bond pad 6 has one end of sample pad 5, the other end of sample pad 5 adheres on PVC board 4, the other end of absorbent paper 8 adheres on PVC board 4, the width of monkeypox virus antigen detection test paper strip 3.5mm, be equipped with 13 sample hole and sample pad 11 sample hole and corresponding to the sample observation window, the sample hole 11 can be measured on the corresponding to the sample window 11.
A method for preparing a monkey pox virus antigen detection kit comprises a method for preparing a latex combined pad, a method for preparing a sample pad and a method for preparing a solid-phase antibody reaction membrane.
The preparation method of the latex combined pad comprises the following steps:
(1) Performing vortex and ultrasonic treatment on latex (particles with the particle size of 300 nm);
(2) Mixing the latex and a cleaning solution according to the proportion of 1:4 (volume ratio 1:4, sample 200ul latex: 800ul wash) for resuspension;
(3) Centrifuging at 4 deg.C and 12000rpm for 15min, removing supernatant, resuspending with the same volume of cleaning solution, and treating with ultrasound;
(4) Centrifuging at 12000rpm at 4 deg.C for 15min, removing supernatant, resuspending with the same volume of reaction buffer and treating with ultrasound;
(5) Adding EDAC into the latex reaction solution according to 20% of the volume of the original latex, and performing vortex incubation for 30min at room temperature;
(6) Adding NHS into the latex reaction solution according to 10% of the volume of the original latex, and continuing vortex incubation for 30min at room temperature;
(7) Centrifuging at 12000rpm at 4 deg.C for 15min, removing supernatant, resuspending with the same volume of conjugate diluent and sonicating;
(8) Adding a marked monkeypox virus monoclonal antibody according to the proportion of 1.0mg/mL, and performing vortex incubation for 1.5h at normal temperature;
(9) Centrifuging at 12000rpm at 4 deg.C for 15min, removing supernatant, resuspending with blocking solution of the same volume, and treating with ultrasound; sealing for 30min;
(10) Centrifuging at 4 deg.C and 12000rpm for 15min, removing supernatant, diluting the precipitate with redissolution to eight times the volume of original latex, and performing resuspension and ultrasonic treatment to obtain latex-labeled working stock solution;
(11) The latex-labeled working stock solution was sputtered onto glass fibers at 1.5ul/cm and dried overnight at 45 ℃ to produce a latex conjugate pad.
In the step (1), the latex is particles with the particle size of 300 nm.
The cleaning liquid in the step (2) comprises 3.8 mass percent of borax.
The buffer in step (4) should comprise 50mM MES buffer at pH 5.5.
In the step (7), the mass percent of the conjugate diluent is 1.2 percent of Na 2 HPO 4 ·12H 2 O and NaH with the mass percent of 0.148% 2 PO 4 ·2H 2 O。
In the step (9), the sealing liquid comprises 0.09% by mass of Na with the pH value of 8.0 2 OH, 1% by mass of sodium caseinate and 0.618% by mass of borax.
The redissolution in the step (10) comprises 0.125 mass percent of tris (hydroxymethyl) aminomethane, 0.25 mass percent of Casein, 20 mass percent of sucrose and 5 mass percent of trehalose.
The preparation method of the sample pad is as follows:
the sample pad treatment solution was soaked with K8964 glass cellulose membrane, 3.3 mL/strip, and dried overnight at 45 ℃ to prepare a sample pad.
The sample pad treatment solution included 0.6 mass% tris (hydroxymethyl) aminomethane, 1 mass% PVP-30, 0.5 mass% S9, 0.5 mass% Casein, 0.5 mass% triton x-100, 1 mass% S-17, 0.3 mass% EDTA, 1 mass% BSA, and 0.5 mass% fatty alcohol polyoxyethylene ether at a pH of 8.0.
The preparation method of the solid-phase antibody reaction membrane comprises the following steps:
a detection T line 9 and a quality control C line 10 are sequentially coated on the solid-phase antibody reaction membrane, a monkeypox monoclonal antibody is coated on the detection T line 9, and a goat anti-mouse IgG antibody and DNP-BSA are coated on the quality control C line 10.
The concentration of the monkeypox monoclonal antibody is 1.0mg/mL, the concentration of the goat anti-mouse IgG antibody is 0.5mg/mL, and the concentration of DNP-BSA is 0.5mg/mL.
The position of the observation window 12 corresponds to the positions of the detection T line 9 and the quality control C line 10.
The sample diluent comprises 1.2% by mass of tris (hydroxymethyl) aminomethane, 0.1% by mass of Casein-Na, 0.7% by mass of Triton X-100, 2% by mass of KCl, 1% by mass of glycine, 0.04% by mass of CMC, 1% by mass of sodium carbonate, 0.5% by mass of fatty alcohol-polyoxyethylene ether, 0.2% by mass of BND-10, and 0.05% by volume of Proclin-300.
And (3) evaluating the result:
the detection method of the monkeypox virus antigen detection kit comprises the following steps:
(1) And (3) detection:
a. the detection samples comprise oropharyngeal swabs, blood and skin lesion tissues;
b. adding 0.4mL of sample diluent into an attached reagent tube;
c. wiping tonsil at root and two sides of the tongue with cotton swab for at least three times, collecting Eleopus scaber with disinfecting forceps, soaking the collected sample in sample diluent in a reagent tube, and stirring to obtain sample solution to be detected;
d. dripping 80 mu L of whole blood or the sample liquid to be detected prepared in the step c into the sample adding hole of the detection card, allowing the sample liquid to be detected to reach the test strip through the sample adding hole, observing the result in 15 minutes, and making the result invalid after 20 minutes;
(2) And (5) judging a result:
a. negative: only one red band appears on the quality control C line 10, and no red band appears in the detection T line 9.
b. Positive: two red bands appear. One red strip is located in the detection T line 9, and the other red strip is located in the quality control C line 10.
c. And (4) invalidation: the absence of a red band on the quality control C-line 10 indicates an incorrect procedure or a damaged test card.
The sensitivity and specificity of the kit are verified as follows:
(1) Sensitivity of the composition
The sensitivity studies were carried out with the recombinant antigens of the monkey pox virus purchased externally, diluted in a gradient to 1000, 500, 200, 20, 2, 0.2pg/mL, respectively, and the results are given in table 1 below (sensitivity studies):
TABLE 1 sensitivity study
Remarking: "+ + + +" represents strong positive, "+" represents weak positive, "-" represents negative.
The lowest detection limit for detecting the simian pox virus recombinant antigen is 2pg/mL.
(2) Analysis of specificity
The following table demonstrates that cross-reactive pathogenic microorganisms, which are susceptible to the same or similar symptoms, have good specificity for cross-reactive species at the following concentrations:
TABLE 2 study of assay specificity
In order to better prove that the kit of the invention has better sensitivity and specificity, the kit of the invention in the embodiment 1 is taken as a reference, and the kit of the invention in the embodiment 1 and the kit of the comparative examples 1-2 are used for detecting negative samples at the same time through 2 comparative examples, and the specific detection results are shown in table 3; the recombinant proteins with different concentrations are detected, and the specific detection results are shown in Table 4.
Comparative example 1:
different from the embodiment 1, the gold bonding pad is prepared by the following steps, namely, the latex bonding pad is adopted and labeled by latex in the embodiment 1, the colloidal gold bonding pad is adopted and labeled by the colloidal gold in the conventional process in the comparative example 1, and the rest operations are the same as follows: the preparation method of the colloidal gold bonding pad comprises the following steps: and (3) regulating the pH value of the 40nm colloidal gold solution to 9.0 by 0.2Mol/L K2CO3, adding 10 mu g/mL monkeypox virus monoclonal antibody 2, fully reacting, centrifuging at 4 ℃ and 10000rpm for 30min, removing supernatant, and concentrating by 25 times to obtain the colloidal gold labeled antibody. Sputtering on a glass cellulose membrane by a gold spraying instrument according to the concentration of 1.5 mu L/cm, and drying at 45 ℃ overnight to prepare the colloidal gold combined pad.
Comparative example 2:
different from example 1, the sample pad of comparative example 2 was prepared by adding RBC instead of TritonX-100 and S-17, and the following operations were performed:
the preparation method of the sample pad is as follows:
soaking K8964 glass cellulose membrane in a sample pad treatment solution, drying at 45 ℃ overnight to prepare a sample pad, wherein the sample pad treatment solution comprises 0.6 mass percent of tris (hydroxymethyl) aminomethane with the pH value of 8.0, 1 mass percent of PVP-30, 0.5 mass percent of S9, 0.5 mass percent of Casein, 0.3 mass percent of EDTA, 1 mass percent of BSA, 0.5 mass percent of fatty alcohol-polyoxyethylene ether and 1 mass percent of RBC.
TABLE 3
| Examples | Oropharynx swab 100 cases | 100 examples of body fluid | Blood sample 100 | Crust bark 100 cases |
| Example 1 | 100％ | 100％ | 100％ | 100％ |
| Comparative example 1 | 86.2％ | 87.7％ | 95.2％ | 93.3％ |
| Comparative example 2 | 100％ | 100％ | 100％ | 100％ |
TABLE 4
The studies in tables 3 and 4 show that, compared with comparative examples 1 and 2, the kit in example 1 of the present invention has higher sensitivity and stronger specificity in practical detection applications.
Finally, it should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and the embodiments are examples, wherein the details that are not described are all the common general knowledge of those skilled in the art, and the technical solutions described in the foregoing embodiments can be modified or some technical features can be equivalently replaced by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A monkey pox virus antigen detect reagent box, its characterized in that: including detection card and sample diluent, the detection card includes upper cover, lower cover and monkey pox virus antigen test paper strip, monkey pox virus antigen test paper strip includes sample pad, latex combination pad, solid-phase antibody reaction membrane, absorbent paper and PVC bottom plate, covers on and is equipped with application of sample hole and observation window and goes up the non-setting adhesive window.
2. The monkeypox virus antigen detection kit of claim 1, characterized in that: the solid-phase antibody reaction membrane is adhered to a PVC plate, two ends of the solid-phase antibody reaction membrane are respectively lapped with one end of a latex combination pad and one end of absorbent paper, the other end of the latex combination pad is adhered to the PVC plate, the other end of the latex combination pad is lapped with one end of a sample pad, the other end of the sample pad is adhered to the PVC plate, and the other end of the absorbent paper is adhered to the PVC plate; the sample diluent comprises 1.2% by mass of tris (hydroxymethyl) aminomethane, 0.1% by mass of Casein-Na, 0.7% by mass of Triton X-100, 2% by mass of KCl, 1% by mass of glycine, 0.04% by mass of CMC, 1% by mass of sodium carbonate, 0.5% by mass of fatty alcohol-polyoxyethylene ether, 0.2% by mass of BND-10 and 0.05% by volume of Proclin-300.
3. A method for manufacturing the simian pox virus antigen detection kit according to claim 1, which comprises a method for preparing a latex conjugate pad, a method for preparing a sample pad and a method for preparing a solid phase antibody reaction membrane, wherein the method for preparing the latex conjugate pad comprises the following steps:
(1) Carrying out vortex and ultrasonic treatment on latex, wherein the latex is particles with the particle size of 300 nm;
(2) Mixing the latex and the cleaning solution according to the volume ratio of 1:4, carrying out heavy suspension;
(3) Centrifuging, removing supernatant, resuspending again with the same volume of washing solution and ultrasonically treating;
(4) Centrifuging, removing supernatant, resuspending with the same volume of reaction buffer and sonicating;
(5) Adding EDAC into the latex reaction solution according to 20% of the volume of the original latex, and performing vortex incubation at room temperature;
(6) Adding NHS into the latex reaction solution according to 10% of the volume of the original latex, and continuing vortex incubation at room temperature;
(7) Centrifuging, removing the supernatant, resuspending with the same volume of conjugate diluent and sonicating;
(8) Adding a marked monkeypox virus monoclonal antibody according to the proportion of 1.0mg/mL, and performing vortex incubation at normal temperature;
(9) Centrifuging, removing supernatant, resuspending with the same volume of confining liquid and sonicating; sealing for 30min;
(10) Centrifuging, removing supernatant, fixing the volume of the precipitate to eight times of the volume of the original latex by using a redissolution, and then carrying out heavy suspension and ultrasonic treatment to obtain a latex-labeled working stock solution;
(11) And (3) sputtering the latex marking working stock solution on glass fiber, and drying at 45 ℃ overnight to prepare the latex bonding pad.
4. The method for producing a monkeypox virus antigen detection kit according to claim 3, characterized in that: and (3) the cleaning liquid in the step (2) comprises 3.8 mass percent of borax.
5. The method for producing a monkeypox virus antigen detection kit according to claim 3, characterized in that: the reaction buffer of step (4) comprises 50mM MES buffer at pH 5.5.
6. The method for producing a monkey pox virus antigen detection kit according to claim 3, which comprisesIs characterized in that: the dilution of the conjugate in the step (7) is 1.2% by mass of Na 2 HPO 4 ·12H 2 O and NaH with the mass percent of 0.148% 2 PO 4 ·2H 2 O。
7. The method for producing a monkeypox virus antigen detection kit according to claim 3, characterized in that: the confining liquid in the step (9) comprises 0.09 mass percent of Na2OH with the pH value of 8.0, 1 mass percent of sodium caseinate and 0.618 mass percent of borax.
8. The method for producing a monkeypox virus antigen detection kit according to claim 3, characterized in that: the redissolution of the step (10) comprises 0.125 mass percent of tris (hydroxymethyl) aminomethane, 0.25 mass percent of Casein, 20 mass percent of sucrose and 5 mass percent of trehalose.
9. The method for producing a monkeypox virus antigen detection kit according to claim 3, wherein the method for preparing the sample pad is as follows: soaking K8964 glass cellulose membrane in the sample pad treatment solution, drying at 45 ℃ overnight and obtaining a sample pad, wherein each piece of the K8964 glass cellulose membrane is 3.3 mL; the sample pad treatment fluid comprises 0.6 mass percent of tris (hydroxymethyl) aminomethane with a pH value of 8.0, 1 mass percent of PVP-30, 0.5 mass percent of S9, 0.5 mass percent of Casein, 0.5 mass percent of TritonX-100, 1 mass percent of S-17, 0.3 mass percent of EDTA, 1 mass percent of BSA and 0.5 mass percent of fatty alcohol-polyoxyethylene ether.
10. The method for producing a monkeypox virus antigen detection kit according to claim 3, wherein the solid-phase antibody reaction membrane is prepared by the following method: a detection T line and a quality control C line are sequentially coated on the solid-phase antibody reaction membrane, the detection T line is coated with a monkeypox monoclonal antibody, and the quality control C line is coated with a goat anti-mouse IgG antibody and DNP-BSA; the concentration of the monkeypox monoclonal antibody is 1.0mg/mL, the concentration of the goat anti-mouse IgG antibody is 0.5mg/mL, and the concentration of DNP-BSA is 0.5mg/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116425865A (en) * | 2023-03-24 | 2023-07-14 | 北京科跃中楷生物技术有限公司 | Antibodies and their use in monkey poxvirus detection |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116425865A (en) * | 2023-03-24 | 2023-07-14 | 北京科跃中楷生物技术有限公司 | Antibodies and their use in monkey poxvirus detection |
| CN116425865B (en) * | 2023-03-24 | 2023-08-25 | 北京科跃中楷生物技术有限公司 | Antibodies and their use in monkey poxvirus detection |
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