Application of monoclonal antibody against monkeypox virus A29L protein in preparation of monkeypox colloidal gold detection kit

Abstract

The application relates to the field of biological medicine detection, in particular to application of a monoclonal antibody against monkeypox virus A29L protein in preparation of a monkeypox colloidal gold detection kit. The application provides a detection kit (after preparation, a sample can be directly detected by adding the sample) which is rapid (10-15 minutes for result), equipment-free, rapid and efficient, low in sensitivity, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection, and in particular to the application of monoclonal antibodies against monkeypox virus A29L protein in the preparation of monkeypox colloidal gold detection kits. Background Technology

[0002] Real-time quantitative PCR (qPCR): As the gold standard, it offers the highest sensitivity and specificity. However, it relies on specialized laboratories and expensive equipment, requires professional operators, and has a long testing time.

[0003] Genome sequencing: This method yields the complete viral genome sequence, used for source tracing, mutation monitoring, and evolutionary research, and provides the richest information. However, it is the most expensive, involves complex and lengthy data analysis, and demands extremely high sample quality and bioinformatics capabilities.

[0004] ELISA is a highly sensitive and specific diagnostic tool in clinical laboratories, but the procedure is cumbersome and requires specialized equipment. It also faces the challenge of a long testing cycle.

[0005] Therefore, providing a rapid, accurate, and equipment-free screening reagent for monkeypox is of great practical significance. Summary of the Invention

[0006] In view of this, the present invention provides the application of a monoclonal antibody against monkeypox virus A29L protein in the preparation of a colloidal gold detection kit for monkeypox. This kit enables rapid, equipment-free screening and is highly effective in identifying individuals with high viral loads and strong infectivity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides the application of a monoclonal antibody against monkeypox virus A29L protein in the preparation of a monkeypox colloidal gold detection kit;

[0009] The monoclonal antibodies against monkeypox virus A29L protein include Ab-5 antibody and Ab-94 antibody;

[0010] The Ab-5 antibody includes a heavy chain variable region and a light chain variable region;

[0011] The HCDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.1;

[0012] The HCDR2 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.2;

[0013] The HCDR3 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.3;

[0014] The LCDR1 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.4;

[0015] The LCDR2 of the light chain variable region has an amino acid sequence as shown in LVS; and

[0016] The LCDR3 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.5;

[0017] The Ab-94 antibody includes a heavy chain variable region and a light chain variable region;

[0018] The HCDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 6;

[0019] The HCDR2 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7;

[0020] The HCDR3 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 8;

[0021] The LCDR1 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.9;

[0022] The LCDR2 of the light chain variable region has the amino acid sequence shown in SEQ ID NO. WAS; and

[0023] The LCDR3 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.10.

[0024] In some specific embodiments of the present invention, the heavy chain variable region of the Ab-5 antibody has an amino acid sequence as shown in SEQ ID NO. 12; and

[0025] The light chain variable region of the Ab-5 antibody has an amino acid sequence as shown in SEQ ID NO.14;

[0026] The heavy chain variable region of the Ab-94 antibody has the amino acid sequence shown in SEQ ID NO. 16; and

[0027] The light chain variable region of the Ab-94 antibody has an amino acid sequence as shown in SEQ ID NO.18.

[0028] In some specific embodiments of the present invention, the nucleic acid molecule encoding the monoclonal antibody includes:

[0029] The heavy chain variable region of the Ab-5 antibody has a nucleotide sequence as shown in SEQ ID NO. 11; and

[0030] The light chain variable region of the Ab-5 antibody has a nucleotide sequence as shown in SEQ ID NO.13;

[0031] The heavy chain variable region of the Ab-94 antibody has a nucleotide sequence as shown in SEQ ID NO. 15; and

[0032] The light chain variable region of the Ab-94 antibody has a nucleotide sequence as shown in SEQ ID NO.17.

[0033] In some specific embodiments of the present invention, the method for preparing the monoclonal antibody against monkeypox virus A29L protein includes: using monkeypox virus core protein A29L as an immunogen to immunize animals, screening out positive clones that specifically bind to monkeypox virus core protein A29L, and obtaining the monoclonal antibody against monkeypox virus A29L protein.

[0034] The monkeypox virus core protein A29L has the amino acid sequence shown in SEQ ID NO.19;

[0035] The nucleic acid molecule encoding the monkeypox virus core protein A29L has a nucleotide sequence as shown in SEQ ID NO.20.

[0036] In some specific embodiments of the present invention, the Ab-94 antibody is used as a streaking antibody, and the Ab-5 antibody is used as a labeling antibody; or

[0037] The Ab-94 antibody is used as a labeling antibody, and the Ab-5 antibody is used as a streaking antibody.

[0038] Secondly, the present invention also provides a kit for detecting monkeypox virus using colloidal gold, comprising the Ab-5 antibody and the Ab-94 antibody;

[0039] The Ab-5 antibody includes a heavy chain variable region and a light chain variable region;

[0040] The HCDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.1;

[0041] The HCDR2 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.2;

[0042] The HCDR3 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.3;

[0043] The LCDR1 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.4;

[0044] The LCDR2 of the light chain variable region has an amino acid sequence as shown in LVS; and

[0045] The LCDR3 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.5;

[0046] The Ab-94 antibody includes a heavy chain variable region and a light chain variable region;

[0047] The HCDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 6;

[0048] The HCDR2 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7;

[0049] The HCDR3 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 8;

[0050] The LCDR1 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.9;

[0051] The LCDR2 of the light chain variable region has the amino acid sequence shown in SEQ ID NO. WAS; and

[0052] The LCDR3 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.10.

[0053] In some specific embodiments of the present invention, the heavy chain variable region of the Ab-5 antibody has an amino acid sequence as shown in SEQ ID NO. 12; and

[0054] The light chain variable region of the Ab-5 antibody has an amino acid sequence as shown in SEQ ID NO.14;

[0055] The heavy chain variable region of the Ab-94 antibody has the amino acid sequence shown in SEQ ID NO. 16; and

[0056] The light chain variable region of the Ab-94 antibody has an amino acid sequence as shown in SEQ ID NO.18.

[0057] In some specific embodiments of the present invention, the nucleic acid molecule encoding the monoclonal antibody includes:

[0058] The heavy chain variable region of the Ab-5 antibody has a nucleotide sequence as shown in SEQ ID NO. 11; and

[0059] The light chain variable region of the Ab-5 antibody has a nucleotide sequence as shown in SEQ ID NO.13;

[0060] The heavy chain variable region of the Ab-94 antibody has a nucleotide sequence as shown in SEQ ID NO. 15; and

[0061] The light chain variable region of the Ab-94 antibody has a nucleotide sequence as shown in SEQ ID NO.17.

[0062] In some specific embodiments of the present invention, the method for preparing the monoclonal antibody against monkeypox virus A29L protein includes: using monkeypox virus core protein A29L as an immunogen to immunize animals, screening out positive clones that specifically bind to monkeypox virus core protein A29L, and obtaining the monoclonal antibody against monkeypox virus A29L protein.

[0063] The monkeypox virus core protein A29L has the amino acid sequence shown in SEQ ID NO.19;

[0064] The nucleic acid molecule encoding the monkeypox virus core protein A29L has a nucleotide sequence as shown in SEQ ID NO.20.

[0065] In some specific embodiments of the present invention, the Ab-94 antibody is used as a streaking antibody, and the Ab-5 antibody is used as a labeling antibody; or

[0066] The Ab-94 antibody is used as a labeling antibody, and the Ab-5 antibody is used as a streaking antibody.

[0067] This invention provides a rapid detection (results in 10-15 minutes), equipment-free colloidal gold detection kit (samples can be added directly after preparation). It is fast and efficient, with low sensitivity, and has promising application prospects. Attached Figure Description

[0068] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0069] Figure 1 A schematic diagram showing the SDS-PAGE verification of West African antigen and monkeypox-specific monoclonal antibodies (MPXV Antibody #5, MPXV Antibody #94);

[0070] Figure 2 The results of the Ab-5 underlined and Ab-94 labeled tests are shown.

[0071] Figure 3 This shows the detection results of Ab-94 underlined + Ab-5 marked.

[0072] Figure 4This diagram illustrates the assembly of a colloidal gold test strip. The strip mainly consists of four parts: a sample pad, a gold pad, a nitrocellulose membrane (NC membrane), and absorbent paper. These parts are connected end-to-end and adhered to a PVC base. Colloidal gold-labeled antibodies are adsorbed onto the gold pad, fixing the specific antibodies in a strip-like pattern on the membrane (T-line). When the antigen to be tested is added to the sample pad at one end of the strip, the sample moves forward through capillary action. It dissolves the colloidal gold-labeled antibodies on the conjugate pad and reacts with them. Upon reaching the area where the antibody is fixed, the conjugate of the analyte and the gold-labeled reagent binds to the specific antibody and is retained, accumulating on the detection band. The resulting colorimetric result can be observed with the naked eye. Detailed Implementation

[0073] This invention discloses the application of a monoclonal antibody against monkeypox virus A29L protein in the preparation of a colloidal gold detection kit for monkeypox. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0074] The raw materials and reagents used in the application of the monoclonal antibody against monkeypox virus A29L protein provided by this invention in the preparation of a monkeypox colloidal gold detection kit are all commercially available.

[0075] reagents and consumables

[0076] Ab-5 antibody (antibody without biotin labeling);

[0077] Ab-94 antibody (antibody without biotin labeling);

[0078] Table 1. Amino acid sequence of the CDR region of the antibody.

[0079]

[0080] MPXV Antibody #5 (Ab-5)-VH:

[0081] DNA sequence (SEQ ID NO.11):

[0082] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGTCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGAGCATTAATCCTACCTATGGTGATACTAACTTCACTGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGGATTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0083] amino acid sequence:

[0084] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.12):

[0085] QVQLQQPGAELVKPGSSVKLSCKAS GYTFTSYY MYWVKQRPGQGLEWIGS INPTYGDT NFTEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYC TRWDY WGQGTTLTVSS

[0086] MPXV Antibody #5(Ab-5)-VL:

[0087] DNA sequence (SEQ ID NO.13):

[0088] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTAACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGAAAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGTTGGCAAGGTACACATTTTCCCCAAACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0089] amino acid sequence:

[0090] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.14):

[0091] DVVMTQTPLTLSVTIGQPASISCKSSQ SLLESDGKTY LNWLLQRPGQSPKRLIY LVS KVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQT FGGGTKLEIK

[0092] MPXV Antibody #94(Ab-94)-VH:

[0093] DNA sequence (SEQ ID NO.15):

[0094] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTAGTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGAGCTATGGTTACGACGGGGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0095] amino acid sequence:

[0096] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.16):

[0097] EVQLVESGGGLVKPGGSLKLSCAAS GFTFSDYY MYWVRQTPEKRLEWVAT ISDGGSYT YYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYC ARELWLRRGWFAY WGQGTLVTVSA

[0098] MPXV Antibody #94(Ab-94)-VL:

[0099] DNA sequence (SEQ ID NO.17):

[0100] GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATTTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAGCAGCTATCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0101] amino acid sequence:

[0102] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.18)

[0103] DIVMTQSHKFMSTSVGDRVSITCKAS QDVGTA VAWYQQKPGQSPKLLIY WAS TRHTGVPDRFTGSGFGTDFTLTISNVQSEDLADYFCQ QYSSYLT FGAGTKLELK

[0104] The amino acid sequence of the purified protein of A29L antigen protein is as follows (SEQ ID NO.19):

[0105] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKCCKHNDEVLFRLENHAETLRAAMISLAKKIDVQTGRRPYEEFGGGSHHHHHH

[0106] The nucleotide sequence of A29L (codon optimized) is as follows (SEQ ID NO.20):

[0107]

[0108] West African antigen (40891-V08E, from China).

[0109] Sheep anti-rodent HRP (30003, Nanjing Huading);

[0110] PBS (SH30256.01, Shanghai Unimicron Biotechnology Co., Ltd.)

[0111] PBST (P3563-10PAK, Merck KGaA)

[0112] TMB developer (AR1104, Wuhan Bosideng);

[0113] Termination solution (AR1105, Wuhan Boside);

[0114] Quality control line coating antibody: goat anti-mouse (30004, Nanjing Huading);

[0115] Absorbent paper: Millipore;

[0116] Base plate: Millipore;

[0117] Fiberglass (Gold Label Release Pad): Millipore;

[0118] Glass fiber (sample chromatography pad): Millipore;

[0119] Nitrocellulose membrane: (Sartorius, CN140);

[0120] Colloidal gold solution: (GN0101, Changzhou Boyi Biotechnology Co., Ltd.);

[0121] Blocking solution: (3% concentration of BSA, BSA catalog number: B2064-100G, Merck KGaA).

[0122] Experimental instruments and materials

[0123] Electric thermostatic incubator (Tianjin Tester);

[0124] ELISA reader (Beijing Pulang Medical);

[0125] Plate washer (Beijing Putian Xinqiao);

[0126] Centrifuge: Mini-14k model, Hangzhou Aosheng Instrument Co., Ltd.;

[0127] Spraying system (film scribing machine): HM3030, Shanghai Jinbiao;

[0128] Chopping machine: ZQ2002 model, Shanghai Jinbiao.

[0129] The present invention will be further illustrated below with reference to the embodiments:

[0130] Example 1: SDS-PAGE Detection

[0131] (1) Mixing adhesives (top layer adhesive, bottom layer adhesive);

[0132] (2) Antigen-antibody denaturation (after adding sample buffer, heat in a metal bath at 95-100℃ for 5 minutes).

[0133] (3) SDS-electrophoresis (start with 120V for electrophoresis, then adjust to 200V after the proteins run to the stacking gel until electrophoresis is complete).

[0134] (4) Coomassie brilliant blue staining and destaining;

[0135] (5) Imaging analysis results.

[0136] The results are as follows Figure 1 As shown. West African antigen (40891-V08E, Sinopharm) and monkeypox-specific monoclonal antibody were analyzed by SDS-PAGE electrophoresis (e.g., Figure 1 The sample size is correct and there are no extraneous bands. This indicates that the monkeypox West African antigen and the specific monoclonal antibody are of high purity.

[0137] (1) West African antigen: There is a clear band at 11KD in lane 2 of the first gel, which is the correct size and has no impurities as expected;

[0138] (2) The light and heavy chains of the MPXV Antibody #5 showed obvious bands at 25 KD and 55 KD in lane 3 of the first gel, respectively. The bands were of the correct size and there were no extraneous bands, which met expectations.

[0139] (3) The light and heavy chains of the MPXV Antibody #94 showed obvious bands at 25 KD and 55 KD in lane 4 of the first gel, respectively. The bands were of the correct size and there were no extraneous bands, which met expectations.

[0140] Example 2: Indirect ELISA titer detection

[0141] (1) Design the coating plate according to the experimental requirements and mark it on the strip.

[0142] (2) Dilute the West African antigen to the required concentration with PBS, mix well and add 100 μL to each well of the enzyme label strip, and incubate overnight at 4°C.

[0143] Coating antigen: West African antigen

[0144] Coating concentration: 1 μg / mL, 100 μL / well

[0145] Coating buffer: Phosphate-buffered saline (PBS, pH 7.4)

[0146] (3) After coating, discard the coating solution, wash the plate 3 times, add 200 μL of blocking solution to each well, and incubate at 37℃ for 1 h. Remove the microplate, discard the inner solution, and wash the plate 3 times.

[0147] (4) Start with 1 μg / mL antibody, dilute 2 times, 100 μL per well, and incubate at 37℃ for 1 h.

[0148] (5) Remove the ELISA plate, discard the internal liquid, wash the plate 3 times, and add 100 μL of diluted ELISA-labeled secondary antibody to each well. ELISA-labeled secondary antibody: goat anti-mouse-HRP, 1:20,000. Incubate at 37℃ for 1 h.

[0149] (6) Remove the microplate, discard the inner liquid, wash the plate 4 times, add 100 μL of TMB colorimetric solution to each well, and determine the color development time according to the color intensity, generally 37 ℃, 15 min.

[0150] (7) Add 100 μL of 1 M HCl solution to each well to stop the reaction. Immediately take a reading at 450 nm on the microplate reader. The dilution corresponding to the well with an OD value that is 2.1 times greater than the set negative control OD value is defined as the potency of the sample.

[0151] The specific results are shown in Table 2:

[0152] Table 2 Results of antibody indirect titer detection

[0153]

[0154] Conclusion: Both Ab-5 and Ab-94 antibodies showed good titers against West African antigens.

[0155] Example 3 Chromatography Experiment

[0156] like Figure 4 As shown, the labeled antibody is completed in step 3.1, laid on a gold pad and dried for later use (e.g., Ab94 labeling). The streaking antibody is completed in step 3.2 and applied to the membrane (e.g., Ab5 streaking), forming a double-antibody sandwich method. When the target antigen is detected, the target band will appear at the T line. In addition, a goat anti-mouse antibody is placed at the C line for quality control of the entire colloidal gold test strip.

[0157] 3.1 Colloidal gold labeling

[0158] Take one 1.5 mL centrifuge tube and rinse it twice with ultrapure water. Add 1 mL of colloidal gold to each tube, add an appropriate amount of 0.2 M potassium carbonate to adjust the pH, shake the tube to mix thoroughly, add 10 μg of antibody, mix well, and react for 30 min. Then add 50 μL of 10% BSA to each tube to block the reaction for 30 min. Centrifuge at 11000 r / min for 20 min. Discard the supernatant, redissolve the precipitate with 0.5 mL of gold redissolving solution, and spread it on a 7 mm × 150 mm gold pad to dry.

[0159] 3.2 point film

[0160] The streaking antibody (if the labeled antibody is Ab-94, then it is Ab-5 here; if the labeled antibody is Ab-5, then it is Ab-94 here) was diluted to 1 mg / mL with diluent and coated onto the NC membrane as the test line. Goat anti-mouse antibody was used as the quality control line, diluted to 1 mg / mL with diluent and coated onto the NC membrane. The membrane was then dried at room temperature and low humidity.

[0161] 3.3 Assembly Inspection

[0162] Adhere absorbent paper, NC membrane, gold pad, and sample chromatography pad to the base plate from top to bottom, and cut them into 3 mm wide test strips for testing.

[0163] 3.4 Experimental Results

[0164] 3.4.1 Colloidal gold method: Ab-5 streak + Ab-94 marking (marking step is 3.1, streak step is 3.2)

[0165] After diluting the West African antigen, take 50 μL of the sample and spot it, then develop the color for 15 min.

[0166] The results showed that the C line was normal, with a weak band at 62.5 ng / mL and a very weak band at 12.5 ng / mL, indicating that the detection sensitivity was around 62.5 ng / mL (see Table 3). Figure 2 .

[0167] Table 3. Detection results of Ab-5 underlining + Ab-94 marking

[0168]

[0169] 3.4.2 Colloidal gold method: Ab-94 streaking + Ab-5 marking

[0170] After diluting the West African antigen, take 50 μL of the sample and spot it, then develop the color for 15 min.

[0171] The results showed that the C line was normal, with a weak band at 62.5 ng / mL and a very weak band at 12.5 ng / mL, indicating that the detection sensitivity was around 62.5 ng / mL (see Table 4). Figure 2 .

[0172] Table 4. Detection results of Ab-94 puncturing + Ab-5 marking

[0173]

[0174] 3.6 Results:

[0175] The experimental results show that the pairing sensitivity of Ab-5 and Ab-94 is around 62.5 ng / mL, and the performance of Ab-94 streaking with Ab-5 labeling is slightly better than that of Ab-5 streaking with Ab-94 labeling.

[0176] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of monoclonal antibodies against monkeypox virus A29L protein in the preparation of monkeypox colloidal gold detection kit; The monoclonal antibodies against monkeypox virus A29L protein include Ab-5 antibody and Ab-94 antibody; The Ab-5 antibody includes a heavy chain variable region and a light chain variable region; The HCDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.1; The HCDR2 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.2; The HCDR3 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.3; The LCDR1 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.4; The LCDR2 of the light chain variable region has an amino acid sequence as shown in LVS; and The LCDR3 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.5; The Ab-94 antibody includes a heavy chain variable region and a light chain variable region; The HCDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 6; The HCDR2 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7; The HCDR3 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 8; The LCDR1 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.9; The LCDR2 of the light chain variable region has the amino acid sequence shown in SEQ ID NO. WAS; and The LCDR3 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.

10.

2. The application as described in claim 1, characterized in that, The heavy chain variable region of the Ab-5 antibody has the amino acid sequence shown in SEQ ID NO. 12; and The light chain variable region of the Ab-5 antibody has an amino acid sequence as shown in SEQ ID NO.14; The heavy chain variable region of the Ab-94 antibody has the amino acid sequence shown in SEQ ID NO. 16; and The light chain variable region of the Ab-94 antibody has an amino acid sequence as shown in SEQ ID NO.

18.

3. The application as described in claim 1 or 2, characterized in that, The nucleic acid molecule encoding the monoclonal antibody includes: The heavy chain variable region of the Ab-5 antibody has a nucleotide sequence as shown in SEQ ID NO. 11; and The light chain variable region of the Ab-5 antibody has a nucleotide sequence as shown in SEQ ID NO.13; The heavy chain variable region of the Ab-94 antibody has a nucleotide sequence as shown in SEQ ID NO. 15; and The light chain variable region of the Ab-94 antibody has a nucleotide sequence as shown in SEQ ID NO.

17.

4. The application as described in any one of claims 1 to 3, characterized in that, The method for preparing the monoclonal antibody against monkeypox virus A29L protein includes: using monkeypox virus core protein A29L as an immunogen to immunize animals, screening out positive clones that specifically bind to monkeypox virus core protein A29L, and obtaining the monoclonal antibody against monkeypox virus A29L protein. The monkeypox virus core protein A29L has the amino acid sequence shown in SEQ ID NO.19; The nucleic acid molecule encoding the monkeypox virus core protein A29L has a nucleotide sequence as shown in SEQ ID NO.

20.

5. The application as described in any one of claims 1 to 3, characterized in that, The Ab-94 antibody is used as a streaking antibody, and the Ab-5 antibody is used as a labeling antibody; or The Ab-94 antibody is used as a labeling antibody, and the Ab-5 antibody is used as a streaking antibody.

6. A reagent kit for detecting monkeypox virus using colloidal gold, characterized in that, Including the Ab-5 antibody and the Ab-94 antibody; The Ab-5 antibody includes a heavy chain variable region and a light chain variable region; The HCDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.1; The HCDR2 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.2; The HCDR3 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.3; The LCDR1 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.4; The LCDR2 of the light chain variable region has an amino acid sequence as shown in LVS; and The LCDR3 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.5; The Ab-94 antibody includes a heavy chain variable region and a light chain variable region; The HCDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 6; The HCDR2 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7; The HCDR3 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 8; The LCDR1 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.9; The LCDR2 of the light chain variable region has the amino acid sequence shown in SEQ ID NO. WAS; and The LCDR3 of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.

10.

7. The kit according to claim 6, characterized in that, The heavy chain variable region of the Ab-5 antibody has the amino acid sequence shown in SEQ ID NO. 12; and The light chain variable region of the Ab-5 antibody has an amino acid sequence as shown in SEQ ID NO.14; The heavy chain variable region of the Ab-94 antibody has the amino acid sequence shown in SEQ ID NO. 16; and The light chain variable region of the Ab-94 antibody has an amino acid sequence as shown in SEQ ID NO.

18.

8. The kit as described in claim 6 or 7, characterized in that, The nucleic acid molecule encoding the monoclonal antibody includes: The heavy chain variable region of the Ab-5 antibody has a nucleotide sequence as shown in SEQ ID NO. 11; and The light chain variable region of the Ab-5 antibody has a nucleotide sequence as shown in SEQ ID NO.13; The heavy chain variable region of the Ab-94 antibody has a nucleotide sequence as shown in SEQ ID NO. 15; and The light chain variable region of the Ab-94 antibody has a nucleotide sequence as shown in SEQ ID NO.

17.

9. The kit according to any one of claims 6 to 8, characterized in that, The method for preparing the monoclonal antibody against monkeypox virus A29L protein includes: using monkeypox virus core protein A29L as an immunogen to immunize animals, screening out positive clones that specifically bind to monkeypox virus core protein A29L, and obtaining the monoclonal antibody against monkeypox virus A29L protein. The monkeypox virus core protein A29L has the amino acid sequence shown in SEQ ID NO.19; The nucleic acid molecule encoding the monkeypox virus core protein A29L has a nucleotide sequence as shown in SEQ ID NO.

20.

10. The kit according to any one of claims 6 to 9, characterized in that, The Ab-94 antibody is used as a streaking antibody, and the Ab-5 antibody is used as a labeling antibody; or The Ab-94 antibody is used as a labeling antibody, and the Ab-5 antibody is used as a streaking antibody.

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