Preparation method of monkey pox virus monoclonal antibody and antigen detection kit thereof

Abstract

The invention relates to the technical field of monoclonal antibody preparation and antigen detection kit preparation, in particular to a preparation method of a monkey pox virus monoclonal antibody and an antigen detection kit of the monkey pox virus monoclonal antibody. The mouse anti-human monoclonal antibody 1 and the mouse anti-human monoclonal antibody 2 are respectively used as a colloidal gold kit marker and a coating antibody, and the mouse anti-human monoclonal antibody 1 and the mouse anti-human monoclonal antibody 2 are prepared on the basis of monkey pox virus A29L protein and are prepared by a milk ball microparticle method. The kit disclosed by the invention can be used for early screening and early diagnosis of suspected people, so that monkey pox can be quickly, conveniently and accurately diagnosed.

Description

Technical Field

[0001] This invention relates to the field of reagent kit technology, specifically to a method for preparing a monkeypox virus monoclonal antibody and its antigen detection kit. Background Technology

[0002] Monkeypoxvirus (MPXV) is a member of the genus Orthopoxvirus in the family Poxviridae. It is an enveloped double-stranded DNA virus. Monkeypox is mainly prevalent in Central and West Africa, but a large number of cases have emerged in non-endemic areas caused by the West African branch, exhibiting clear characteristics of human-to-human transmission. On July 23, 2022, the World Health Organization declared the escalating global monkeypox outbreak a Public Health Emergency of International Concern (PHEIC). Given these circumstances, establishing efficient, rapid, and highly sensitive methods for detecting monkeypox virus is crucial for the prevention and control of this disease.

[0003] Antibodies are immunoglobulins produced by plasma cells differentiated from B cells in response to antigen stimulation, specifically binding to the corresponding antigen. Monoclonal cells are cells capable of producing a single, specific antibody. Culturing these cells yields a genetically identical cell population, formed by the division and proliferation of a single cell; this is called a monoclonal cell. Antibodies synthesized by monoclonal cells against a single antigenic determinant are called monoclonal antibodies. Monoclonal antibodies are highly specific, have efficient targeting, high sensitivity, and can be produced in large quantities.

[0004] Colloidal gold test strips prepared using colloidal gold immunochromatography (GICA) are one method for detecting antigens or antibodies in monkeypox. This technique is based on the specific immune reaction between antigens and antibodies, using colloidal gold particles as tracers. The labeled tracers undergo an immune reaction on the control line (C line) and test line (T line) under solvent chromatography. The detection result is obtained from the color of the T line. This method offers advantages such as ease of use, high sensitivity, low cost, and good stability. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a monoclonal antibody against monkeypox virus and its antigen detection kit, which can enable early screening and diagnosis of suspected cases, thereby quickly, conveniently and accurately diagnosing monkeypox.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A monkeypox virus monoclonal antibody antigen detection kit is prepared through the following steps:

[0008] S1. Reagent preparation:

[0009] Weigh 0.195g of morpholine ethoxylic acid solid and dissolve it in 20mL of purified water. Adjust the pH to about 6 and store it in a refrigerator at 4℃ for later use.

[0010] Weigh 0.049 g of TRIS solid and dissolve it in 20 mL of purified water. Adjust the pH to about 8.5 to obtain 20 mL of TRIS washing solution. Store the solution in a refrigerator at 4 °C for later use.

[0011] Weigh out 0.02 g of sodium dihydrogen phosphate and 0.024 g of disodium hydrogen phosphate and dissolve them in 40 mL of purified water. Adjust the pH to about 7.5 to obtain a 20 mM PB labeling reaction solution. Store the solution in a refrigerator at 4 °C for later use.

[0012] Dilute 5 mL of 20 mM PB with purified water to 10 mL of 10 mM PB, add 0.5 g of sucrose, adjust the pH to 7.5, and obtain the antibody coating solution. Store at 4°C for later use.

[0013] Weigh 1g of bovine serum albumin and dissolve it in 10mL of purified water to obtain 10% casein sodium salt blocking solution, and store it in a refrigerator at 4℃ for later use.

[0014] Add 0.25g of sucrose to 5mL of 20mM Tris, adjust the pH to about 8.5, and store the resuspended solution at 4℃.

[0015] Take 20 mL of 20 Mm PB, 0.4 g of NaCl, 120 μL of Trixon-100, 0.1 g of BSA, 20 μL of proclin300, and 1 g of trehalose. Mix them together and adjust the pH to about 7.5 to obtain the sample buffer. Store it in a 4°C refrigerator for later use.

[0016] Take 40 mL of 20 mM Tris, 40 μL of Tween-20, and 0.4 g of casein, mix them, and adjust the pH to 8.5 to obtain the gold label pad treatment buffer. Store it in a 4°C refrigerator for later use.

[0017] Weigh 0.2g of EDC and dissolve it in 20mL of purified water to obtain an EDC solution, which should be prepared and used immediately.

[0018] Weigh 0.2g of NHS and dissolve it in 20mL of purified water to obtain an NHS solution, which should be prepared and used immediately.

[0019] S2, Preparation of antibody labeling

[0020] Add 500 μL of MES buffer and 25 μL of 1 mg microsphere solution (41 mg / mL) to two 1.5 mL EP tubes. Centrifuge at 12000 rpm for 10 min, remove the supernatant, and reconstitute with 500 μL of MES buffer. Then add 15 μL of EDC and 15 μL of NHS to each of the two EP tubes. Mix the two tubes in the dark by rotating for 30 min. Centrifuge at 12000 rpm for 10 min, remove the supernatant, and reconstitute with 500 μL of MES buffer to each tube.

[0021] Add 0.1 mg of mouse anti-human monoclonal antibody 1 to one tube and label it as monoclonal antibody 1. Add 0.1 mg of chicken anti-IgY to the other tube and label it as chicken anti-IgY. Mix them separately by pipetting and incubating in the dark for 2 hours. Centrifuge at 12000 rpm for 10 minutes and discard the supernatant. Add 500 μL of blocking buffer to each tube and centrifuge for 1 hour. Centrifuge at 12000 rpm for 10 minutes and discard the supernatant. Add 500 μL of 20 mM Tris to each tube and mix by pipetting and incubating. Wash 2-3 times. Centrifuge and discard the supernatant. Add 500 μL of resuspension buffer to each tube and mix by incubation. Sonicate for 45 minutes. Due to sonication, the ultrapure water will naturally warm up to about 33°C. S3: Prepare antibody coating.

[0022] The T-line was identified as mouse anti-human monoclonal antibody 2, and its concentration was diluted to 2 mg / mL with antibody coating buffer. The T-line was drawn on the NC membrane at a concentration of 1 μL / cm. The C-line was identified as goat anti-chicken IgY antibody, and its concentration was 1 mg / mL. The C-line was drawn on the NC membrane at a concentration of 2 μL / cm.

[0023] S4, Gold Spraying

[0024] Cut the fiberglass mat into strips 4mm wide, soak them in the gold standard mat treatment solution for 2 hours, dry them in an oven at 33℃ for 16 hours, and then store them in a sealed container at room temperature for later use.

[0025] Mix the monoclonal antibody 1 and chicken anti-IgY labeled microspheres in a 1:1 ratio in a new EP tube, and mix by pipetting and shaking. Prepare a 30cm long pre-treated gold-labeled conjugate pad, select the gold spraying program on the gold spraying machine, adjust the spraying volume to 4μL / cm and the aspiration volume to 120μL, place the gold-labeled conjugate pad in a suitable position and spray gold, and place it in a 37℃ drying oven for about 20 minutes.

[0026] This invention also provides a method for preparing monkeypox virus monoclonal antibodies, namely, a method for preparing mouse anti-human monoclonal antibody 1 and mouse anti-human monoclonal antibody 2, comprising the following steps:

[0027] Monkeypox virus A29L protein was diluted with PBS to 1 μg / mL, and 100 μL was added to each well of an ELISA plate and incubated overnight at 4°C. Then, 200 μL of 1% BSA was added to each well, the plate was sealed, and incubated at 37°C for 1 hour. The serum from immunized mice was then diluted with PBS, and 50 μL of secondary antibody was added to each well. The plate was incubated at 37°C for 1 hour. TMB was used for color development, and the reaction was terminated with 1M sulfuric acid. The absorbance of each well was read at 450 nm.

[0028] After fusing spleen cells and myeloma cells from immunized BABL / c mice in a certain ratio, the medium was changed every two and a half days. After screening and culturing for one week, the medium was changed to HT medium and cultured for 3 days in a biosafety cabinet. Finally, it was changed to RPMI-1640 complete medium and cultured for 1-1.5 months.

[0029] The titer of antibodies against hybridoma cells after screening and culture was determined by ELISA, and positive wells were selected.

[0030] Seed the positive cells in a new 96-well plate at densities of 20 cells / well, 10 cells / well, 5 cells / well, 2.5 cells / well, 1.25 cells / well, and 0.625 cells / well, with 100 μL per well and two columns for each density. After culturing for 7-10 days, collect the supernatant to detect antibodies. Repeat 2-5 times until the antibody positivity rate of a single clone well is 100%. Finally, transfer the cells to a 24-well plate for further expansion culture.

[0031] The supernatant of cloned cells was collected by ELISA to detect antibody titer and antibody subtype. HRP-labeled antibodies were purified by affinity column chromatography, and antibodies with high pairing were screened by orthogonal checkerboard ELISA experiment as mouse anti-human monoclonal antibody 1 and coating antibody as mouse anti-human monoclonal antibody 2.

[0032] Furthermore, spleen cells from immunized BABL / c mice were fused with myeloma cells at a ratio of 5:1.

[0033] The kit of this invention can be used for early screening and diagnosis of suspected cases, thereby enabling rapid, convenient and accurate diagnosis of monkeypox. Attached Figure Description

[0034] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0035] Figure 1 This is a comparison of the OD values ​​of polyclonal antibodies in serum after four immunizations.

[0036] In the figure: *: P < 0.05 indicates a significant difference; **: P < 0.01 indicates a highly significant difference; ***: P < 0.001 indicates a highly significant difference.

[0037] Figure 2 These are hybridoma cells observed under a microscope.

[0038] Figure 3 The image shows a clone cluster observed under a microscope.

[0039] Figure 4 The prepared labeled antibodies (left: chicken anti-IgY, right: antibody 1).

[0040] Figure 5 For well-coated antibodies.

[0041] Figure 6 For spraying gold.

[0042] Figure 7 The C-line was developed after adding the sample diluent.

[0043] Figure 8 When a sample diluent containing antigen is added, both the C and T lines of the test result will show color.

[0044] Figure 9 This is a test card for displaying the C line.

[0045] Figure 10 Test card for displaying C and T lines. Detailed Implementation

[0046] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0047] Example 1

[0048] 1.1 Experimental Materials:

[0049] Laboratory animals: Four SPF-grade BABL / c female mice, with an average weight of approximately 20g (License No.: 2019-0008).

[0050] Main reagents and chemicals: Monkeypox virus A29L protein (Nanjing Baikang Biotechnology Co., Ltd.), 0.01% chloroauric acid, 1% trisodium citrate, goat anti-mouse IgG (BH20210206, Hangzhou Bohang Ruida), chicken anti-IgY (BH2021117, Hangzhou Bohang Ruida), goat anti-chicken IgY (BH2021119, Hangzhou Bohang Ruida), fetal bovine serum (Gemini), double antibiotics (Hyclone), RPMI-1640 basal medium (Hyclone), glutamine (Biotopped), HRP rapid labeling kit (Luoyang Baiaotong), Freund's complete adjuvant and Freund's incomplete adjuvant, Hybridoma Feeder additives, HAT selection medium, HT selection medium, PEG, and mouse myeloma cells SP2 / 0 were all provided by Beijing Bio-Long Immunotherapy Co., Ltd. Sample pads, gold labeling pads, absorbent pads, and nitrocellulose membranes (NC membranes) were all provided by Hangzhou Fenghang Technology Co., Ltd. Plastic boxes were provided by Guangzhou Wondfo Biotech Service Co., Ltd.

[0051] Major instruments and equipment: Microplate reader (Gene Company Limited), ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.), high-speed centrifuge (Xiangyi H1650-W), magnetic stirrer (HZ85-2 type), HGS510 benchtop streak plate sprayer, HGS201 programmable strip cutter (Hangzhou Fenghang Technology Co., Ltd.), clean bench (Shaoxing Jingmai Instrument Equipment Co., Ltd.), biochemical incubator (SPX-70BⅢ), biosafety cabinet (The Baker Company), flow cytometer (BDAccuri C6).

[0052] Experimental methods

[0053] 1.2 Preparation of main reagents

[0054] Preparation of RPMI-1640 complete culture medium: Mix RPMI-1640 basal culture medium, fetal bovine serum, and antibiotics together at a mass ratio of 89:10:1.

[0055] Preparation of HAT medium: Take 100 mL of RPMI-1640 complete medium, add 2 mL of 50× HAT concentrate and 10 mL of 10% Hybridoma Feeder additive, mix thoroughly, and set aside at 4℃ for later use.

[0056] Preparation of HT medium: Take 100 mL of RPMI-1640 complete medium, add 2 mL of 50× HT concentrate and 5 mL of 5% Hybridoma Feeder additive, mix thoroughly, and set aside at 4℃ for later use.

[0057] 1.3 Experimental Procedure

[0058] Immunization program: Four female BABL / c mice were randomly divided into two groups of three each: two experimental groups and one control group. Each mouse was injected with 0.4 mL of antigen (French adjuvant / French incomplete adjuvant + A29L protein). The control group received no injection. The experimental groups were immunized a total of four times, with injections every three weeks.

[0059] Serum collection: Blood was obtained 3 days after the last injection using the tail-cutting blood collection method. After centrifugation at 3000 rpm for 5 min, the supernatant was collected and stored at -20℃ for later use.

[0060] Enzyme-linked immunosorbent assay (ELISA): Monkeypox virus A29L protein was diluted with PBS to 1 μg / mL, and 100 mL was added to each well of the ELISA plate and incubated overnight at 4°C. Then, 200 μL of 1% BSA was added to each well, the plate was sealed, and incubated at 37°C for 1 h. The serum of each group was then diluted with PBS by 1000, 5000, 10000, 30000, 50000, 100000, 200000, 300000, and 500000 times, respectively. Then, 50 μL of secondary antibody was added to each well and incubated at 37°C for 1 h. The plate was developed with TMB, and the reaction was terminated with 1M sulfuric acid. The absorbance of each well was read at 450 nm.

[0061] Cell fusion: Spleen cells and myeloma cells from BABL / c mice (immunized mice) were fused at a ratio of 5:1. The medium was changed every two and a half days. After screening and culturing for one week, the medium was changed to HT medium and cultured for 3 days in a biosafety cabinet. Finally, the medium was changed to RPMI-1640 complete medium and cultured for 1-1.5 months.

[0062] Selection of hybridoma cells (ELISA screening): The antibody titer of the cultured hybridoma cells was identified by ELISA, and positive wells were selected.

[0063] Clonal culture (limiting dilution method): Seed positive well cells into new 96-well plates at densities of 20 cells / well, 10 cells / well, 5 cells / well, 2.5 cells / well, 1.25 cells / well, and 0.625 cells / well, with 100 μL per well and two columns for each density. After culturing for 7-10 days, collect the supernatant to detect antibodies. Repeat 2-5 times until the antibody positivity rate of a single clonal well reaches 100%. Finally, transfer the cells to 24-well plates for further expansion culture.

[0064] Antibody titer identification: The supernatant of cloned cells was collected by ELISA to detect antibody titer.

[0065] Antibody subtype detection: Refer to the instructions for the purchased kit for the method.

[0066] HRP-labeled antibody: Refer to the instructions for the purchased kit for the method.

[0067] Affinity column chromatography: The antibody was purified using the rProtein A Beads method, following the manufacturer's instructions.

[0068] Screening for paired antibodies: An orthogonal checkerboard ELISA assay was used to screen for antibody pairs with excellent affinity that could bind to monkeypox antigen.

[0069] Experimental materials and instruments: 75% alcohol, 0.01M PBS, skim milk powder, TMB chromogenic reagent, horseradish peroxidase (HRP) labeled antibody, monkeypox virus antigen, 96-well ELISA plate, ELISA reader, pipette, 37℃ incubator, 4℃ refrigerator, vortex mixer.

[0070] Experimental procedure: Unlabeled antibodies, used as capture antibodies, were coated separately, and an ELISA assay using the orthogonal checkerboard method was performed.

[0071] (1) The purified unlabeled antibody of each cell line was coated into a 96-well microplate at a concentration of 2 μg / mL, 100 μL per well, and incubated overnight at 4°C.

[0072] (2) Use 5% skim milk powder and seal in a 37℃ constant temperature box for 2 hours;

[0073] (3) Add monkeypox antigen to each well, 0.2 μg, and incubate at 37°C for 1 h.

[0074] (4) Use HRP-labeled antibody (detection antibody) diluted at 1:2000, and apply 100 μL per well using the positive cross method. Incubate at 37°C for 40 min.

[0075] (5) Use TMB colorimetry and detect the entire plate at a wavelength of 450nm and read the values.

[0076] (6) Select samples with high OD values ​​to obtain antibody pairs with excellent affinity.

[0077] Preparation and Identification of Lactobacillus Microparticle Antigen Detection Kit

[0078] 2.1 Reagent Preparation:

[0079] Weigh 0.195g of morpholine ethoxylic acid solid and dissolve it in 20mL of purified water. Adjust the pH to about 6 and store it in a refrigerator at 4℃ for later use.

[0080] Weigh 0.049 g of TRIS solid and dissolve it in 20 mL of purified water. Adjust the pH to about 8.5 to obtain 20 mL of TRIS washing solution. Store the solution in a refrigerator at 4 °C for later use.

[0081] Weigh out 0.02 g of sodium dihydrogen phosphate and 0.024 g of disodium hydrogen phosphate and dissolve them in 40 mL of purified water. Adjust the pH to about 7.5 to obtain a 20 mM PB labeling reaction solution. Store the solution in a refrigerator at 4 °C for later use.

[0082] Dilute 5 mL of 20 mM PB with purified water to 10 mL of 10 mM PB, add 0.5 g of sucrose, adjust the pH to 7.5, and obtain the antibody coating solution. Store at 4°C for later use.

[0083] Weigh 1g of bovine serum albumin and dissolve it in 10mL of purified water and 10% casein sodium salt blocking solution. Store the solution in a refrigerator at 4℃ for later use.

[0084] Add 0.25g of sucrose to 5mL of 20mM Tris, adjust the pH to about 8.5 to obtain a resuspension, and store it in a refrigerator at 4℃ (do not store for too long).

[0085] Mix 20 mL of 20 Mm PB, 0.4 g of NaCl, 120 μ L of Trixon-100, 0.1 g of BSA, 20 μ L of proclin300, and 1 g of trehalose. Adjust the pH to approximately 7.5 to obtain the sample buffer solution. Store the buffer solution at 4 °C for later use.

[0086] Take 40 mL of 20 mM Tris, 40 μL of Tween-20, and 0.4 g of casein, mix them, and adjust the pH to 8.5 to obtain the gold label pad treatment buffer. Store it in a 4°C refrigerator for later use.

[0087] Weigh 0.2g of EDC and dissolve it in 20mL of purified water to obtain an EDC solution, which should be prepared and used immediately.

[0088] Weigh 0.2g of NHS and dissolve it in 20mL of purified water to obtain an NHS solution, which should be prepared and used immediately.

[0089] 2.2 Preparation of antibody labeling

[0090] Add 500 μL of MES buffer and 25 μL of 1 mg microsphere solution (41 mg / mL) to two 1.5 mL EP tubes. Centrifuge at 12000 rpm for 10 min, remove the supernatant, and reconstitute with 500 μL of MES buffer. Then add 15 μL of EDC and 15 μL of NHS to each of the two EP tubes. Mix the two tubes in the dark by rotating for 30 min. Centrifuge at 12000 rpm for 10 min, remove the supernatant, and reconstitute with 500 μL of MES buffer to each tube.

[0091] Add 0.1 mg of mouse anti-human monoclonal antibody 1 to one tube and label it as monoclonal antibody 1. Add 0.1 mg of chicken anti-IgY to the other tube and label it as chicken anti-IgY. Mix them separately by pipetting and stirring. Place them in the dark and rotate to mix for 2 hours. Centrifuge at 12000 rpm for 10 minutes and remove the supernatant. Add 500 μL of blocking buffer to each tube and rotate for 1 hour (do not mix the monoclonal antibody 1 and the chicken anti-labeled microspheres). Centrifuge at 12000 rpm for 10 minutes and remove the supernatant. Add 500 μL of 20 mM Tris to each tube and mix by pipetting and shaking. Wash 2-3 times. After centrifugation and removal of supernatant, add 500 μL of resuspension to each tube and mix by shaking. Sonicate for 45 minutes. Due to the ultrasonic oscillation, the ultrapure water will naturally warm up to about 33°C.

[0092] 2.3 Preparation of antibody coating

[0093] The T-line was identified as mouse anti-human monoclonal antibody 2, and its concentration was diluted to 2 mg / mL with antibody coating buffer. The T-line was drawn on the NC membrane at a concentration of 1 μL / cm. The C-line was identified as goat anti-chicken IgY antibody, and its concentration was 1 mg / mL. The C-line was drawn on the NC membrane at a concentration of 2 μL / cm.

[0094] 2.4 Gold spraying

[0095] Cut the fiberglass mat into strips 1.2cm wide, soak them in the gold standard mat treatment solution for 2 hours, dry them in an oven at 33℃ for 16 hours, and then store them in a sealed container at room temperature for later use.

[0096] Mix the monoclonal antibody 2 and chicken anti-IgY labeled microspheres in a 1:1 ratio in a new EP tube, and mix by pipetting and shaking. Prepare a 30cm long pre-treated gold-labeled conjugate pad, select the gold spraying program on the gold spraying machine, adjust the spraying volume to 4μL / cm and the aspiration volume to 120μL, place the gold-labeled conjugate pad in a suitable position and spray gold, and place it in a 37℃ drying oven for about 20 minutes.

[0097] 1.4 Experimental Results

[0098] Immunization procedure results: During the experiment, the mice became less active, and hard lumps were found on the mice during the last injection.

[0099] Enzyme-linked immunosorbent assay (ELISA) results: After four immunizations, the OD values ​​of the blank control group and the experimental group were compared. At a serum dilution of 500,000 times, mouse 4 showed a highly significant difference compared to control group 1 (P < 0.001), while mouse 3 showed no significant difference compared to control group 1. Figure 1 This indicates that even when the serum was diluted 500,000 times, the experimental mice still had a large number of polyclonal antibodies, at which point cell fusion could be performed.

[0100] Cell screening and subcloning: Hybridoma cells screened on HAT medium ( Figure 2 ) and a single-celled clonal cluster derived from the division of a single cell ( Figure 3 )

[0101] Affinity chromatography separation and purification results: A total of 10 mL of eluent was collected after elution with elution buffer, which was the target protein.

[0102] Orthogonal checkerboard method for screening paired antibodies:

[0103] Table 1. Statistical table of results for the orthogonal chessboard method.

[0104]

[0105]

[0106] The results of the orthogonal checkerboard method show that a total of 9 antibody pairs were matched. Among them, 2 pairs had high affinity (in bold), 4 pairs had medium to high affinity (in bold and underlined), and 3 pairs had low affinity (in underlined). The antibody with the higher affinity was selected as the marker (i.e., mouse anti-human monoclonal antibody 1) and coating antibody (i.e., mouse anti-human monoclonal antibody 2) for the colloidal gold reagent kit prepared in this study.

[0107] Lactobacillus microparticle antigen detection kit:

[0108] Antibody labeling results: The final labeled antibody was purple-red. Figure 4 )

[0109] Antibody coating preparation results: After multiple experiments, when the stripping concentration was 1.0 μL / cm, the lines drawn on the NC membrane were thicker and stronger; therefore, 1.0 μL / cm was selected as the stripping concentration for this experiment. Figure 5 )

[0110] Gold spraying: After removing the supernatant, the precipitate was resuspended in gold-labeled working solution. The labeled chicken anti-IgY and antibody 1 immunogold were mixed 1:1; the spraying volume was 4 μL / cm; after gold spraying, the gold-labeled conjugate pad appeared purple-red. Figure 6 )

[0111] Assembly, slicing, and detection results: The NC membrane was attached to a PVC plate and fixed to a scribing machine. After scribing, the gold label pad was attached to the PVC plate, and the sample pad was placed on top of the gold label pad. An absorbent pad was attached to the other end. The assembled PVC plate was then cut into 4mm wide strips using a cutting machine, completing the initial gold label test strip. This experiment used sample diluent and antigen-containing sample diluent for testing. The result detected by the sample diluent was the C line color (…). Figure 7 When a sample diluent containing the antigen is used, both the C and T lines show color development. Figure 8 ).

[0112] Kit assembly: Assemble the test strips and plastic box into a test card. Figure 9 , Figure 10 )

[0113] 1.5 Conclusion

[0114] In this experiment, mouse immunization was successful, and the monkeypox virus monoclonal antibody was successfully prepared. The prepared labeled antibody was purple-red. Test strips were used to detect the sample diluent and the antigen-containing sample diluent, respectively. The test result after adding the sample diluent showed color development at the C line, while the test result after adding the antigen-containing sample diluent showed color development at both the C and T lines, proving the successful preparation of the antibody and colloidal gold chromatography test strip.

[0115] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A monkeypox virus monoclonal antibody antigen detection kit, characterized in that: The following steps were performed to obtain the following: S1. Reagent preparation: Weigh 0.195g of morpholine ethoxylic acid solid and dissolve it in 20mL of purified water. Adjust the pH to about 6 and store it in a refrigerator at 4℃ for later use. Weigh 0.049 g of TRIS solid and dissolve it in 20 mL of purified water. Adjust the pH to about 8.5 to obtain 20 mL of TRIS washing solution. Store the solution in a refrigerator at 4 °C for later use. Weigh out 0.02 g of sodium dihydrogen phosphate and 0.024 g of disodium hydrogen phosphate and dissolve them in 40 mL of purified water. Adjust the pH to about 7.5 to obtain a 20 mM PB labeling reaction solution. Store the solution in a refrigerator at 4 °C for later use. Dilute 5 mL of 20 mM PB with purified water to 10 mL of 10 mM PB, add 0.5 g of sucrose, adjust the pH to 7.5, and obtain the antibody coating solution. Store at 4°C for later use. Weigh 1g of bovine serum albumin and dissolve it in 10mL of purified water to obtain 10% casein sodium salt blocking solution, and store it in a refrigerator at 4℃ for later use. Add 0.25g of sucrose to 5mL of 20mM Tris, adjust the pH to about 8.5, and store the resuspended solution at 4℃. Mix 20 mL of 20 Mm PB, 0.4 g of NaCl, 120 μ L of Trixon-100, 0.1 g of BSA, 20 μ L of proclin300, and 1 g of trehalose. Adjust the pH to approximately 7.5 to obtain the sample buffer solution. Store the buffer solution at 4 °C for later use. Take 40 mL of 20 mM Tris, 40 μL of Tween-20, and 0.4 g of casein, mix them, and adjust the pH to 8.5 to obtain the gold label pad treatment buffer. Store it in a 4°C refrigerator for later use. Weigh 0.2g of EDC and dissolve it in 20mL of purified water to obtain an EDC solution, which should be prepared and used immediately. Weigh 0.2g of NHS and dissolve it in 20mL of purified water to obtain an NHS solution, which should be prepared and used immediately. S2, Preparation of antibody labeling Add 500 μL of MES buffer and 25 μL of 1 mg microsphere solution (41 mg / mL) to two 1.5 mL EP tubes. Centrifuge at 12000 rpm for 10 min, remove the supernatant, and reconstitute with 500 μL of MES buffer. Then add 15 μL of EDC and 15 μL of NHS to each of the two EP tubes. Mix the two tubes in the dark by rotating for 30 min. Centrifuge at 12000 rpm for 10 min, remove the supernatant, and reconstitute with 500 μL of MES buffer to each tube. Add 0.1 mg of mouse anti-human monoclonal antibody 1 to one tube and label it as monoclonal antibody 1. Add 0.1 mg of chicken anti-IgY to the other tube and label it as chicken anti-IgY. Mix them separately by pipetting and stirring, and place them in the dark for 2 hours. Centrifuge at 12000 rpm for 10 minutes and remove the supernatant. Add 500 μL of blocking buffer to each tube and centrifuge for 1 hour. Centrifuge at 12000 rpm for 10 minutes and remove the supernatant. Add 500 μL of 20 mM Tris to each tube and mix by pipetting and stirring. Wash 2-3 times. After centrifugation and removal of the supernatant, add 500 μL of resuspension to each tube and mix by shaking. Sonicate for 45 minutes. Due to the ultrasonic oscillation, the ultrapure water will naturally warm up to 33°C. S3. Preparation of antibody coating The T-line was identified as mouse anti-human monoclonal antibody 2, and its concentration was diluted to 2 mg / mL with antibody coating buffer. The T-line was drawn on the NC membrane at a concentration of 1 μL / cm. The C-line was identified as goat anti-chicken IgY antibody, and its concentration was 1 mg / mL. The C-line was drawn on the NC membrane at a concentration of 2 μL / cm. S4, Gold Spraying Cut the fiberglass mat into strips 1.2cm wide, soak them in the gold standard mat treatment solution for 2 hours, dry them in an oven at 33℃ for 16 hours, and then store them in a sealed container at room temperature for later use. Mix the monoclonal antibody 1 and chicken anti-IgY labeled microspheres in a 1:1 ratio in a new EP tube, and mix by pipetting and shaking. Prepare a 30cm long pre-treated gold-labeled conjugate pad, select the gold spraying program on the gold spraying machine, adjust the spraying volume to 4μL / cm and the aspiration volume to 120μL, place the gold-labeled conjugate pad in a suitable position and spray gold, and place it in a 37℃ drying oven for 20min.

2. The monkeypox virus monoclonal antibody antigen detection kit as described in claim 1, characterized in that: The mouse anti-human monoclonal antibody 1 and mouse anti-human monoclonal antibody 2 were prepared by the following steps: Monkeypox virus A29L protein was diluted with PBS to 1 μg / mL, and 100 mL was added to each well of an ELISA plate and incubated overnight at 4°C. Then, 200 μL of 1% BSA was added to each well, the plate was sealed, and incubated at 37°C for 1 hour. The serum from immunized mice was then diluted with PBS, and 50 μL of secondary antibody was added to each well. The plate was incubated at 37°C for 1 hour. TMB was used for color development, and the reaction was terminated with 1M sulfuric acid. The absorbance of each well was read at 450 nm. After fusing spleen cells and myeloma cells from immunized BABL / c mice in a certain ratio, the medium was changed every two and a half days. After screening and culturing for one week, the medium was changed to HT medium and cultured for 3 days in a biosafety cabinet. Finally, it was changed to RPMI-1640 complete medium and cultured for 1-1.5 months. The titer of antibodies against hybridoma cells after screening and culture was determined by ELISA, and positive wells were selected. Seed the positive cells in a new 96-well plate at densities of 20 cells / well, 10 cells / well, 5 cells / well, 2.5 cells / well, 1.25 cells / well, and 0.625 cells / well, with 100 μL per well and two columns for each density. After culturing for 7-10 days, collect the supernatant to detect antibodies. Repeat 2-5 times until the antibody positivity rate of a single clone well is 100%. Finally, transfer the cells to a 24-well plate for further expansion culture. The supernatant of cloned cells was collected by ELISA to detect antibody titer and antibody subtype. HRP-labeled antibodies were purified by affinity column chromatography, and antibodies with high pairing were screened by orthogonal checkerboard ELISA experiment as mouse anti-human monoclonal antibody 1 and coating antibody as mouse anti-human monoclonal antibody 2.

3. The monkeypox virus monoclonal antibody antigen detection kit as described in claim 2, characterized in that: Spleen cells from immunized BABL / c mice were fused with myeloma cells at a ratio of 5:1.

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