Heavy and light chain variable regions of a spiramycin monoclonal antibody and uses thereof

Abstract

The application discloses a spiramycin monoclonal antibody and application thereof, and belongs to the technical field of biology. The monoclonal antibody contains a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are both composed of a complementarity determining region and a framework region, and the complementarity determining region is composed of CDR1, CDR2 and CDR3. The monoclonal antibody can be used for preparing a spiramycin detection preparation. The spiramycin colloidal gold detection test strip provided by the application has the characteristics of high sensitivity, good specificity and strong stability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the heavy and light chain variable regions of a spiramycin monoclonal antibody and their applications. Background Technology

[0002] Spiramycin (SPM) is a macrolide antibiotic with the chemical formula C60. 43 H 74 N2O 14 Spiramycin has three active components, distinguishable by the degree of acylation of the third carbon atom in its 16-ring structure: Spiramycin I (SPM I) is unacylated, Spiramycin II (SPM II) is acetylated, and Spiramycin III (SPM III) is propionylated. Spiramycin is a potent antibacterial agent that inhibits bacterial protein synthesis by binding to the 50S subunit of the bacterial ribosome, thus hindering peptide chain elongation.

[0003] Spiramycin is effective against Staphylococcus aureus, hemolytic streptococci, Campylobacter fetus, Haemophilus influenzae, Bordetella pertussis, Bacteroides spp., Clostridium perfringens, Peptostreptococcus, as well as Mycoplasma, Chlamydia, Treponema pallidum, Toxoplasma gondii, and Cryptosporidium. When used as a veterinary drug, it is mainly used to treat respiratory infections and toxoplasmosis in livestock. Spiramycin has a broad antibacterial spectrum and few side effects, thus it is widely used in actual production. However, improper use leads to its residues and accumulation in animal bodies, endangering human health. Therefore, some countries and regions have set limits on the residues of spiramycin in animal-derived foods. my country has also specified residue limits in the muscle, fat, liver, kidney, and milk of different animal species (cattle, pigs, and chickens) in the National Food Safety Standard for Maximum Residue Limits of Veterinary Drugs (GB 31650-2019), with a maximum residue limit of 200 μg / kg in milk. Therefore, establishing a safe and efficient biological detection method for spiramycin is imperative.

[0004] Immunoassay methods offer advantages such as low cost, high efficiency, high sensitivity, and relatively low skill requirements, making them suitable for rapid detection of large numbers of samples. The purpose of this invention is to provide a monoclonal antibody with high affinity and detection sensitivity for spiramycin, laying the foundation for the development and promotion of colloidal gold test strips and fluorescent immunoassay test strips. Summary of the Invention

[0005] Therefore, the present invention provides a heavy chain and light chain variable region of a spiramycin monoclonal antibody and its application.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] In a first aspect, the present invention provides a spiramycin monoclonal antibody, characterized in that: the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0008] The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1;

[0009] The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2;

[0010] Both the heavy chain variable region and the light chain variable region are composed of complementary determination regions and framework regions, and the complementary determination regions are composed of CDR1, CDR2 and CDR3.

[0011] The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5;

[0012] The amino acid sequence of CDR2 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6;

[0013] The amino acid sequence of CDR3 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7;

[0014] The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8;

[0015] The amino acid sequence of CDR2 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9;

[0016] The amino acid sequence of CDR3 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 10.

[0017] Secondly, the aforementioned spiramycin monoclonal antibody is characterized by:

[0018] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3;

[0019] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 4.

[0020] Thirdly, the present invention provides the application of a spiramycin monoclonal antibody in the preparation of a detection product for detecting spiramycin.

[0021] Preferably, the test product consists of a spiramycin test strip and a microwell reagent;

[0022] Preferably, the test strip is a colloidal gold test strip.

[0023] The present invention has the following advantages:

[0024] The monoclonal antibody described in this invention possesses high specificity and sensitivity, and can be used as a raw material for enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunoassay. The purpose of this invention is to provide a variable region of the heavy and light chains of a spiramycin monoclonal antibody and its detection applications, laying the foundation for the research and development and promotion of ELISA, colloidal gold test strips, and fluorescent immunoassay strips. Attached Figure Description

[0025] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0026] Figure 1 The results of SDS-PAGE identification of purified monoclonal antibodies;

[0027] Figure 2 This is a schematic diagram for interpreting the results of colloidal gold test strips. Detailed Implementation

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Synthesis of Spiramycin Artificial Antigen

[0030] 1. Preparation of Spiramycin Immunogen

[0031] S1. Accurately weigh 100 mg of spiramycin raw material and 15 mg of maleic anhydride, place them in a 40 mL brown glass bottle, add 15 mL of pyridine, and heat and stir in an oil bath at 75 °C for 6 h.

[0032] S2. Dry the organic phase by rotary evaporation, and dissolve the residue in 10 mL of DMF;

[0033] S3. Weigh 40 mg EDC and 20 mg NHS and slowly add them to the S2 reaction system. Stir the reaction at room temperature (25°C) for 6 hours to obtain an activated solution that forms succinimide.

[0034] S4. Weigh 200 mg of bovine serum albumin (BSA) and dissolve it in 0.01 mol / L PBS (pH=7.4) buffer. Stir well and slowly add it dropwise to the succinimide activation solution. Stir at room temperature for 5 hours.

[0035] S5. Dialyze the obtained reaction solution with 0.01 mol / L PBS (pH=7.4) buffer, changing the dialysis solution every 4 hours, and dialyze for 48 hours to obtain spiramycin immunogenic antigen (SPM-BSA).

[0036] 2. Preparation of Spiramycin Detection Antigen

[0037] S1. Accurately weigh 100 mg of spiramycin raw material and 15 mg of maleic anhydride, place them in a 40 mL brown glass bottle, add 15 mL of pyridine, and heat and stir in an oil bath at 75 °C for 6 h.

[0038] S2. Dry the organic phase by rotary evaporation, and dissolve the residue in 10 mL of DMF;

[0039] S3. Weigh 40 mg EDC and 20 mg NHS and slowly add them to the S2 reaction system. Stir the reaction at room temperature (25°C) for 6 hours to obtain an activated solution that forms succinimide.

[0040] S4. Weigh 200 mg of chicken ovalbumin OVA and dissolve it in 0.01 mol / L PBS (pH=7.4) buffer. Stir well and slowly add it dropwise to the succinimide activation solution. Stir at room temperature for 5 h.

[0041] S5. Dialyze the obtained reaction solution with 0.01 mol / L PBS (pH=7.4) buffer, changing the dialysis solution every 4 hours, and dialyze for 48 hours to obtain the spiramycin detection antigen (SPM-OVA).

[0042] Example 2: Preparation of Spiramycin Monoclonal Antibody

[0043] 1. Mouse immunization

[0044] Three female Balb / c mice aged 6-8 weeks were immunized with 20 μg of SPM-BSA artificial antigen each. For the first immunization, the SPM-BSA artificial antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. Immunizations were repeated every two weeks for a total of three immunizations. For the second and third immunizations, Freund's incomplete adjuvant was used for antigen emulsification, and the dosage and method of immunization remained unchanged. One week after the third immunization, blood was collected from the tail vein of the mice, and serum was analyzed using an indirect ELISA method to determine its titer and inhibition. The results are shown in Table 1. Mice #2 showed the highest serum titer after immunization, reaching 1:3.2 × 10⁻⁶. 4The inhibition rate can reach up to 52.88%. 40 μg of SPM-BSA artificial antigen was diluted with 1×PBS to 200 μL and injected intraperitoneally to boost the immunization of mice. Cell fusion can be performed three days later.

[0045] Table 1 Serum titers and inhibition detection in immunized mice

[0046]

[0047] 2. Culture of SP2 / 0 myeloma cells

[0048] One vial of SP2 / 0 myeloma cells, frozen in liquid nitrogen, was immediately transferred to a 37°C water bath. The cryovial was gently agitated periodically until the cells reached a semi-ice crystal state. Under sterile conditions, the SP2 / 0 cells were transferred to a 50 mL sterile centrifuge tube. 10 mL of preheated 1640 complete culture medium was slowly added dropwise to the centrifuge tube. The tube was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cell clumps were gently dispersed, and the cells were resuspended in 5 mL of culture medium and transferred to a T75 cell culture flask. An additional 5 mL of culture medium was added, and the flask was agitated in a "cross" motion before being placed in a CO2 cell culture incubator at 37°C. Cell status was observed under a microscope. When the cell density reached approximately 80%, the SP2 / 0 cells were passaged.

[0049] 3. Cell fusion

[0050] (1) Blood was collected from the orbital cavity of mice after booster immunization and placed in EP tubes. After standing at 37°C for 2 hours, the tubes were centrifuged at 4000 rpm for 10 minutes. The serum was collected as a positive control for subsequent screening of monoclonal antibodies. The mice were euthanized by cervical dislocation and then disinfected by soaking in 75% alcohol.

[0051] (2) Preparation of spleen cells: In a biosafety cabinet, use sterilized scissors and forceps to cut open the mouse skin. Replace with a new set of sterilized scissors and forceps to cut open the mouse abdominal cavity. Then, carefully remove the spleen using a set of sterilized scissors and forceps, and trim away excess fat. Prepare a sterile 15mL centrifuge tube, add 10mL of DMEM culture medium, place the spleen into the centrifuge tube, moisten the spleen, and carefully discard the excess culture medium. Take another 10 mL of DMEM culture medium and place it in a sterile Petri dish. Grind the spleen with a ground glass slide to prepare a single-cell suspension. Filter the suspension through a 200-mesh nylon mesh into a sterile centrifuge tube. Add 30 mL of DMEM to a 50 mL sterile centrifuge tube. Rinse the nylon mesh with a pipette. Centrifuge the centrifuge tube containing the spleen cell suspension at 1500 rpm for 5 min. Discard the supernatant. Gently break up the cell clumps by hand. Add another 30 mL of DMEM culture medium to resuspend the cells and centrifuge again. Discard the supernatant, gently break up the cell clumps by hand, and add another 10 mL of DMEM culture medium to resuspend the cells.

[0052] (3) Cell fusion: Collect well-grown SP2 / 0 cells by centrifugation at 1000 rpm for 5 min into a 50 mL centrifuge tube. Gently break up the SP2 / 0 cell clusters, add 30 mL of DMEM medium to resuspend, centrifuge again, add 10 mL of DMEM medium to resuspend, then mix the spleen cell suspension with the SP2 / 0 cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, and gently break up the cell clusters. Place in a 37℃ water bath, and add 1 mL of PEG fusion agent to the centrifuge tube within 1 min. At this time, the cells are red, homogeneous, and quicksand-like, and rotating the tube wall feels like frosted glass.

[0053] (4) Termination of fusion: Take 9 mL of preheated DMEM medium to terminate the fusion, which is divided into three stages. The first stage is to add 1 mL in the first 1 min, the second stage is to add 1 mL in the first 1 min, and the third stage is to add the remaining 7 mL of medium in the first 3 min. Then, let it stand in a 37℃ water bath for 5 min to stabilize, and then centrifuge at 800 rpm for 5 min.

[0054] (5) Plating: Discard the supernatant, gently break up the cell clumps, add HAT medium (for example, to plate 5 96-well plates, 200 μL / well, remove the feeder layer cells that have been pre-plated at 100 μL / well, then add 50 mL of HAT medium), mix the cells, and then evenly spread the fused cell suspension into the 96-well cell plate with the feeder layer cells added, 100 μL / well, and incubate in a CO2 cell incubator at 37°C.

[0055] 4. Screening of positive hybridoma cells

[0056] Seven days after cell fusion, when the cell clusters were relatively large, the cell supernatant was analyzed using an indirect ELISA method. A 1 μg / mL SPM-OVA artificial antigen was used as the detection antigen; the positive control was serum from fused mice, and the negative control was serum from mice immunized with PBS. Wells with the strongest chromogenic reaction were selected as positive wells. The selected positive hybridoma cells were subcloned using a limiting dilution method. The hybridoma cell lines that stably secreted monoclonal antibodies, identified after subcloning, were expanded and cultured in T75 cell flasks. When the cell count reached approximately 80%, the cells were collected for ascites preparation.

[0057] 5. Preparation of ascites

[0058] Add 10 mL of sterile 1×PBS to the cell culture flask, blow off the cell layer, resuspend it, and transfer it to a 15 mL centrifuge tube. Centrifuge at 1000 r / min for 10 min. Discard the supernatant, resuspend the precipitate in 1 mL of sterile 1×PBS, mix well, and aspirate using a 1 mL syringe. Inject approximately 500 μL of the cell suspension into each mouse, observing the mouse's growth. Collect ascites fluid one week later when the mouse's abdomen is swollen. Collect the mouse ascites fluid into a centrifuge tube, centrifuge at 8000 r / min for 20 min, and aspirate the middle ascites layer.

[0059] 6. Purification of monoclonal antibodies

[0060] The collected ascites fluid was purified. SDS-PAGE was used to assess the purity of the purified monoclonal antibody, which was approximately 95%. The results are shown below. Figure 1 .

[0061] Example 3: Specificity and sensitivity detection of spiramycin monoclonal antibody

[0062] The sensitivity and specificity of spiramycin monoclonal antibody were detected using an indirect competitive ELISA method. SPM-OVA artificial antigen was used for coating at a concentration of 1 μg / mL. 1 mg / mL of monoclonal antibody 5D7-1C1 was serially diluted to verify the antibody's sensitivity. The results are shown in Table 2. The sensitivity of monoclonal antibody 5D7-1C1 reached a 1:40000 dilution, with an inhibition rate as high as 74.30%.

[0063] Table 2. Monoclonal antibody sensitivity validation

[0064]

[0065] Spiramycin artificial antigen, erythromycin artificial antigen, lincomycin artificial antigen, spectinomycin artificial antigen, tylosin artificial antigen, and tilmicosin artificial antigen were respectively coated at 1 μg / mL. The monoclonal antibody 5D7-1C1 at 1 mg / mL was diluted 1:5000 to verify the specificity of the monoclonal antibody. The results are shown in Table 3. The purified monoclonal antibody showed no cross-reactivity with erythromycin artificial antigen, lincomycin artificial antigen, spectinomycin artificial antigen, tylosin artificial antigen, and tilmicosin artificial antigen, indicating that the purified monoclonal antibody had good specificity.

[0066] Table 3. Validation of Monoclonal Antibody Specificity

[0067]

[0068] Example 4: Cloning of the variable region gene of spiramycin monoclonal antibody

[0069] 1. Hybridoma cell culture and total RNA extraction

[0070] Hybridoma cells 5D7-1C1 were cultured in RPMI 1640 complete medium at 37°C and 5% CO2 until the cell number reached 1×10⁻⁶. 7 Total RNA was extracted from cells using a total RNA extraction kit (purchased from Tiangen).

[0071] 2. Synthesis of the first strand of cDNA

[0072] The first strand of cDNA was synthesized using a reverse transcription kit (purchased from TAKARA) with the total RNA extracted in step 1 as the amplification template.

[0073] 3. Gene amplification

[0074] Design downstream primers and upstream universal primers for the Lambda, Kappa, and Heavy chains.

[0075] Primer: F (SEQ ID No. 11): AAGCGTGGTATCAACGCAGA

[0076] Rκ (SEQ ID No. 12):AACATTGATGTCTTTGGGGTAGAA

[0077] Rλ (SEQ ID No.13):AATCGTACACACCAGTGTGTGGG

[0078] R H (SEQ ID No.14):AGGGATCCAGAGTTCCAGGT

[0079] PCR amplification was performed using the first strand of cDNA as a template in a 50 μL reaction volume. The reaction volume consisted of 3 μL template, 2.5 μL upstream primer (10 μM), 2.5 μL downstream primer (10 μM), 25 μL 2×Taq enzyme, and 17 μL sterile water.

[0080] The landing PCR reaction conditions were as follows: 98℃ for 30s; 98℃ for 15s, 64℃-58℃ for 30s, decreasing by 0.5℃ each time until reaching 58℃, for 10 cycles; 72℃ for 30s; 98℃ for 15s, 56℃ for 30s, 72℃ for 30s, for 15 cycles; and the program ended at 72℃ for 7min.

[0081] 4. Cloning and screening of PCR amplification products

[0082] The PCR products were subjected to 1% agarose gel electrophoresis. The Kappa, Lambda and Heavy chain amplification fragments were recovered using a PCR product recovery kit (purchased from Tiangen). The recovered and purified target fragments were inserted into the pLB vector using a pLB zero-background rapid cloning kit (purchased from Tiangen). The vector was then transformed into DH5α competent cells (ampicillin resistant). Recombinant positive clones were screened and sequenced.

[0083] 5. The variable region gene sequence and amino acid sequence of the spiramycin monoclonal antibody in this embodiment are as follows:

[0084] (1) Gene sequence of the variable region of the heavy chain (SEQ ID No. 3):

[0085] GAAGTGCAGCTGCAGCAGAGCGGCCCGGAACTGGTGAAAACCGGCGCGAGCGTGAAAATTAGCTGCAAAGCGAGCGGCTATAGCTTTACCGGCTATTATATGCATTGGGTGAAACAGCGCCCGGGCAAAAGCCTGGAATGGATTGGCTATCTTAACAACTATAACGGCGCGACCCGCTATA ACCAGAAATTTAAAGGCAAAGCGACCTTTACCGTGGATACCAGCAGCAGCACCGCGTATATGCAGTTTAACAGCCTGACCATCGAAGATACCGCGTTGTATTACTGCGGCAGCGAAGCGTATGGCTACCTTATGAAGCGATGGATTATTGCATCCAGGGCACCAGCGTTCCCGTGCGCAGC.

[0086] (2) Amino acid sequence of the variable region of the heavy chain (SEQ ID No. 1):

[0087] EVQLQQSGPELVKTGASVKISCKASGYSFTGYYMHWVKQRPGKSLEWIGYLNNYNGATRYNQKFKGKATFTVDTSSSTAYMQFNSLTIEDTALYYCGSEAYGYLMKRWIIASRAPAFPCA.

[0088] (3) Kappa chain variable region gene sequence (SEQ ID No. 4):

[0089] GAAATTGTGCTGACCCAGAGCCCGACCACCATGGCGGCGAGCCCGGGCGAAACAATTACCATTACCTGCAGCGCGAGCAGCTGCCTGACCAGAAACTATCTGCATTGGTATCAGCAGAAACCGGGCTCTAGCCCGAAACTGCTGATTTATCGCACCAGCAAC CTGGCGAGCGGCGTGCCGGCGCGCTTTAGCGGCGGCGGCAGCGGCACCAGCTATAGCCTGCCCATTGGCATCATGGAAGCGGAAGATGTGGCGACCTATTATTGCCAGCAGGGCCGCACCCTGCCGTTTACCTTTGGCGGCGGCACCAAACTGGAAATTAAA.

[0090] (4) The amino acid sequence of the variable region of the Kappa chain (SEQ ID No. 2)

[0091] EIVLTQSPTTMAASPGETITITCSASSCLTRNYLHWYQQKPGSSPKLLIYRTSNLASGVPARFSGGGSGTSYSLPIGIMEAEDVATYYCQQGRTLPFTFGGGTKLEIK.

[0092] 6. Variable region amino acid sequence and homology analysis

[0093] The heavy and light chain gene sequences were compared and analyzed in the NCBI database. The results showed that the heavy chain variable region gene sequence of the monoclonal antibody 5D7-1C1 had the highest homology with the mouse transgenic HEL23 antibody mRNA sequence (Sequence ID: KX431574.1), with a homology of 245 / 292, or 83.90%. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5D7-1C1 had the highest homology with the amino acid sequence of the mouse immunoglobulin heavy chain variable region (Sequence ID: CAA26304.1), with a homology of 77 / 86, or 89.53%. The light chain variable region gene sequence of the monoclonal antibody 5D7-1C1 showed the highest homology with the light chain variable region gene of the synthetic construct clone FNA1 anti-influenza virus N1 neuraminidase monoclonal antibody (Sequence ID: PP483116.1), with a homology of 272 / 323 (84.21%). The amino acid sequence of the light chain variable region of the monoclonal antibody 5D7-1C1 showed the highest homology with the amino acid sequence of the light chain variable region of mouse immunoglobulin (Sequence ID: AHX81765.1), with a homology of 87 / 100 (87%).

[0094] 7. CDR Area Analysis

[0095] The amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody 5D7-1C1 were analyzed at https: / / www.novopro.cn / tools / cdr.html to obtain its CDR region.

[0096] Antibody heavy chain CDR region:

[0097] CDR-H1 (SEQ ID No.5): GYYMH

[0098] CDR-H2 (SEQ ID No.6): YLNNYNGATRYNQKFKG

[0099] CDR-H3 (SEQ ID No.7): EAYGYLMK

[0100] Antibody light chain CDR region:

[0101] CDR-L1 (SEQ ID No.8): SASSCLTRNYLH

[0102] CDR-L2 (SEQ ID No.9):RTSNLAS

[0103] CDR-L3 (SEQ ID No. 10): QQGRTLPFT.

[0104] Example 5: Preparation of Spiramycin Colloidal Gold Test Strip

[0105] 1. Preparation of chloroauric acid

[0106] Weigh 800 mL of ultrapure water into a 1000 mL Erlenmeyer flask, add 8 mL of 1% chloroauric acid to the ultrapure water, and place the flask on a magnetic heating rod stirrer to mix and heat at 500℃ and speed setting 1. After the water boils, increase the speed to speed setting 6. Once the liquid is stirring, quickly add 1 mL of 0.9% reducing agent and adjust the speed to speed setting 5. After heating for 6 minutes and 30 seconds, stop heating and allow it to cool to room temperature. The prepared colloidal gold is pure, transparent, and free of precipitates and floating matter.

[0107] 2. Preparation of Spiramycin Monoclonal Antibody-Colloidal Gold Label

[0108] (1) Add bare gold: Mix the colloidal bare gold well and take 1L of colloidal gold solution;

[0109] (2) Add K2CO3: Add 0.2M K2CO3 at a ratio of 3μL / mL colloidal gold and vortex to mix;

[0110] (3) Add antibody: Mix 1 mL of 1 mg / mL spiramycin 5D7-1C1 antibody with 3 mL of 10% BSA (IgG), and then use a pipette to add the well mixed antibody dropwise to the gold, and equilibrate for 5 min;

[0111] (4) Leveling: Reduce the rotation speed to stabilize the vortex at a drop of about 1 cm, and then rebalance for 1 hour;

[0112] (5) Add BSA: Increase the rotation speed until the vortex drops by about 3cm, pour in 10mL of 10% BSA (Ruibao), balance for 3min, decrease the rotation speed to stabilize the vortex drop by about 1cm, and then balance for 30min.

[0113] (6) Add PEG: Increase the rotation speed until the vortex drops by about 3cm, pour in 10mL of 10%PEG 20000, and balance for 5min;

[0114] (7) Centrifugation: Use a benchtop high-speed centrifuge at 11,000 rpm and 4°C for 6 min;

[0115] (8) Discard the supernatant: After centrifugation, gently remove the centrifuge bucket and place it on the experimental table of the negative pressure aspiration device. Do not shake or bump it to avoid disturbing the gold precipitate in the centrifuge bucket; stop aspiration when it is close to the bottom of the centrifuge bucket (about 5-6 mL remaining), and carefully remove the remaining supernatant with a 1 mL pipette to avoid aspirating the gold precipitate;

[0116] (9) Resuspension: Stop aspirating when the liquid is close to the bottom of the centrifuge bucket (about 2-3 mL remaining), gently shake the centrifuge bucket to resuspend the gold precipitate, and transfer the resuspended gold precipitate to a blue cap bottle;

[0117] (10) Rinsing the container: Add about 20 mL of reconstitution solution to the centrifuge container, shake gently to wash away the residual gold precipitate, transfer the liquid to the other three centrifuge containers in turn, and finally transfer it to the blue cap bottle. Repeat once.

[0118] (11) Volume adjustment: Finally, adjust the volume of the gold solution to 1 / 10, i.e. 100 mL, using the blue-capped bottle, and mix well;

[0119] (12) Lyophilization of microwell reagent: Prepare microwell reagent solution by mixing spiramycin colloidal gold and lyophilization buffer, stir and mix for 20 min, and dispense 60 μL / well microwell reagent solution into 96-well plates; wherein, the lyophilization buffer is composed of 0.05M phosphate buffer, 1% BSA, 2% sucrose and 1% Triton X-100; the ratio of spiramycin colloidal gold to lyophilization buffer in the microwell reagent solution is 1:5; place the coated 96-well plates into a vacuum freeze dryer with a preset freeze-drying program for processing. After the freeze-drying process is completed, take out the lyophilized gold, put on the rubber cap, put it into an aluminum foil bag with desiccant, and store it for later use.

[0120] 3. Preparation of sample absorption pads

[0121] The sample absorption pad was immersed in 0.1 mol / L phosphate buffer containing 0.5% BSA, pH=7.2 for 2 hours and then dried at 37℃ for 2 hours to obtain the sample absorption pad.

[0122] 4. Preparation of nitrocellulose membranes

[0123] Spiramycin artificial antigen was diluted to 1 mg / mL with phosphate buffer and coated onto the detection line T of a nitrocellulose membrane using a Biodot coating instrument, with a coating amount of 1.0 μg / cm. Goat anti-mouse IgG antibody was diluted to 300 μg / mL with 0.01 M, pH 7.2 PBS buffer and coated onto the control line C of a nitrocellulose membrane using a Biodot coating instrument. The coated reaction membrane was dried at 37°C for 4 h to obtain the nitrocellulose membrane.

[0124] 5. Assembly of Spiramycin Colloidal Gold Test Strips

[0125] The sample absorption pad, nitrocellulose membrane, and absorbent pad are sequentially attached to the base plate. The beginning of the sample absorption pad is connected to the end of the nitrocellulose membrane, and the beginning of the nitrocellulose membrane is connected to the end of the absorbent pad. The end of the sample absorption pad is aligned with the end of the base plate, and the beginning of the absorbent pad is aligned with the beginning of the base plate. This assembles the colloidal gold test strip. Together with the microporous reagent, it forms the spiramycin colloidal gold test strip.

[0126] Example 6: Application of Spiramycin Colloidal Gold Test Strip

[0127] 1. Test strip detection

[0128] The required test strips and test samples should be brought to room temperature (20~25℃). Using a micropipette, pipette 200µL of the test sample into each well, slowly aspirating and thoroughly mixing it with the reagent in the well. After incubating at 40℃ for 3 min, insert the labeled test strip into the well, ensuring it is fully immersed in the solution. After incubating at 40℃ for 5 min, remove the test strip and follow the diagram (…). Figure 2 The judgment result is valid at other times; judgments made at other times are invalid.

[0129] 2. Interpretation of test results

[0130] Negative (﹣): Both C and T lines show color, with the T line showing stronger color than the C line, indicating that the concentration of spiramycin in the sample is below the detection limit.

[0131] Positive (+): C line shows color, T line shows color the same as C line, T line shows color weaker than C line, or T line shows no color, indicating that the concentration of spiramycin in the sample is equal to or higher than the detection limit.

[0132] Invalid: No C line appears, indicating incorrect operation or that the test strip has deteriorated and become ineffective.

[0133] In addition to naked-eye interpretation, NBReader can be used for result interpretation.

[0134] 3. Sensitivity detection of spiramycin colloidal gold test strips

[0135] Spiramycin standard was diluted to 1.5 ppb and 3 ppb, and milk samples were spiked and tested according to the test strip detection method to verify the product's limit of detection. The results are shown in Table 4. The test strip provided by this invention has a detection sensitivity of 3 ppb for spiramycin standard in milk.

[0136] Table 4. Sensitivity of Spiramycin Colloidal Gold Test Strips

[0137]

[0138] 4. Specificity detection of spiramycin colloidal gold test strips

[0139] The test strips were tested at 500 ppb for spiramycin, erythromycin, lincomycin, spectinomycin, tylosin, and tilmicosin according to the detection method. The data are shown in Table 5, and all results were negative. The results indicate that the test strips provided by this invention do not exhibit cross-reactivity with other antibiotics and have good specificity.

[0140] Table 5. Specificity of Spiramycin Colloidal Gold Test Strips

[0141]

[0142] 5. Stability testing of spiramycin colloidal gold test strips

[0143] The prepared test strips were subjected to accelerated testing at 4℃ and 37℃. They were used to detect spiramycin standard (3ppb) at 0, 7, 14, and 28 days. Error analysis was performed on the measured concentration and the actual sample concentration, and the results are shown in Table 6. The CV values ​​were all <10%, indicating that the test strips provided by this invention have good stability.

[0144] Table 6. Stability of Spiramycin Colloidal Gold Test Strips

[0145]

[0146] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A spiramycin monoclonal antibody, characterized in that: The monoclonal antibody includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1; The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2; Both the heavy chain variable region and the light chain variable region are composed of complementary determination regions and framework regions, and the complementary determination regions are composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5; The amino acid sequence of CDR2 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6; The amino acid sequence of CDR3 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7; The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8; The amino acid sequence of CDR2 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9; The amino acid sequence of CDR3 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

10.

2. The application of the spiramycin monoclonal antibody according to claim 1 in the preparation of spiramycin detection products.

3. A spiramycin detection product, characterized in that: The test product consists of a spiramycin test strip and a microwell reagent, wherein the test strip is a colloidal gold test strip and the microwell reagent contains the spiramycin monoclonal antibody as described in claim 1.

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