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

Abstract

The application discloses a lincomycin 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 lincomycin detection products. The lincomycin 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 lincomycin monoclonal antibody and their applications. Background Technology

[0002] Lincomycin (LIN) is an amide antibiotic produced by Streptomyces lincolnensis, primarily used clinically to treat infections caused by Gram-positive bacteria. Given its high medicinal and economic value, lincomycin is also widely used in veterinary medicine and feed additives to prevent and treat animal diseases and promote growth.

[0003] However, the misuse and improper use of lincomycin have led to residue problems in animal-derived foods (such as meat, eggs, and dairy products). These residues can not only cause allergic reactions and gut microbiota imbalance in consumers, but also exacerbate the development and spread of bacterial resistance, posing a potential threat to human health. Therefore, regulatory agencies and food safety organizations around the world have established strict maximum residue limits (MRLs) for lincomycin. my country's national food safety standard GB 31650-2019, "Maximum Residue Limits for Veterinary Drugs in Food," specifies in detail the maximum residue limits for different target tissues in various animals (cattle, sheep, pigs, poultry, chickens, and fish).

[0004] To effectively monitor lincomycin residues in food, it is crucial to develop rapid, sensitive, high-throughput, and cost-effective detection technologies. Currently, while methods such as high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) are accurate and reliable, they suffer from drawbacks such as expensive equipment, complex operation, and long processing times, making it difficult to meet the needs of large-scale on-site screening.

[0005] Against this backdrop, immunological detection based on antigen-antibody specific reactions, particularly colloidal gold immunochromatographic test strips, has become an ideal screening tool due to its ease of use, speed, and low cost. The core of these technologies lies in high-performance monoclonal antibodies. Therefore, obtaining a monoclonal antibody that specifically recognizes lincomycin and possesses high affinity and high sensitivity is crucial for developing efficient diagnostic kits.

[0006] The purpose of this invention is to provide a monoclonal antibody with high affinity and detection sensitivity for lincomycin, laying a solid foundation for the development and promotion of colloidal gold test strips and fluorescent immunoassay test strips, as well as related products. Summary of the Invention

[0007] Therefore, the present invention provides a heavy chain and light chain variable region of a lincomycin monoclonal antibody and its application.

[0008] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions: In a first aspect, the present invention provides a lincomycin monoclonal antibody, characterized in that: the monoclonal antibody comprises 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.

[0009] Secondly, the present invention provides the lincomycin monoclonal antibody described above, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3; The nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 4.

[0010] Thirdly, the present invention provides the application of a lincomycin monoclonal antibody in the preparation of a detection product for detecting lincomycin.

[0011] Preferably, the test product consists of a lincomycin test strip and a microwell reagent; Preferably, the test strip is a colloidal gold test strip.

[0012] The present invention has the following advantages: 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 lincomycin monoclonal antibody heavy and light chain variable region and its applications, laying the foundation for the research and development and promotion of ELISA, colloidal gold test strips, and fluorescent immunoassay strips. Attached Figure Description

[0013] 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 in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0014] Figure 1 The results of SDS-PAGE identification of purified monoclonal antibodies; Figure 2 This is a schematic diagram for interpreting the results of colloidal gold test strips. Detailed Implementation

[0015] 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.

[0016] Example 1: Synthesis of Lincomycin Artificial Antigen 1. Preparation of Lincomycin Immunogen S1. Accurately weigh 100 mg of lincomycin hydrochloride (LIN-HCl) and 42 mg of succinic anhydride, place them in a 40 mL dry round-bottom flask, add 2 mL of anhydrous pyridine, and react with magnetic stirring in an oil bath at 60-70 °C for 4-6 h under nitrogen protection. S2. After the reaction is complete, the solvent (pyridine) is evaporated to dryness under reduced pressure using a rotary evaporator. The residue is dissolved in 10 mL of methanol and evaporated to dryness again. This process is repeated 2-3 times to completely remove the residual pyridine. The resulting oily or solid residue is lincomycin-hemisuccinate (hapten). S3. Weigh 15 mg EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and 8 mg NHS (N-hydroxysuccinimide) 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 containing succinimide active ester. S4. Weigh 20 mg of bovine serum albumin (BSA) and dissolve it in 0.01 M PBS (pH=7.4) buffer. Stir well and slowly add it dropwise to the succinimide activation solution. Stir at room temperature for 5 h. S5. Dialyze the obtained reaction solution with 0.01M PBS (pH=7.4) buffer, changing the dialysis solution every 4 hours, and dialyze for 48 hours to obtain lincomycin immunogenic antigen (LIN-BSA).

[0017] 2. Preparation of lincomycin detection antigen S1. Accurately weigh 500 mg of lincomycin-hemisuccinate (hapten) and completely dissolve it in DMF. First, add 12 mg of NHS, stir to dissolve, and then add 22 mg of EDC. Stir and react at room temperature in the dark for 3 hours. S2. After the reaction is complete, the lincomycin-NHS active ester reaction solution is obtained. S3. Weigh 50 mg of chicken ovalbumin OVA and dissolve it in 5 mL of 0.01 M PBS (pH=7.4) buffer. Stir gently until completely dissolved. Under magnetic stirring, slowly add the lincomycin-NHS active ester reaction solution (about 1 mL) prepared in S2 dropwise to the OVA solution. S4. After adding the S4, continue stirring the reaction at room temperature in the dark for 4-6 hours, or react overnight in a refrigerator at 4°C. S5. Dialyze the obtained reaction solution with 0.01M PBS (pH=7.4) buffer, changing the dialysis solution every 4 hours, and dialyze for 48 hours to obtain the lincomycin detection antigen (LIN-OVA).

[0018] Example 2: Preparation of Lincomycin Monoclonal Antibody 1. Mouse immunization Three female Balb / c mice aged 6-8 weeks were immunized with 20 μg of LIN-BSA artificial antigen each. For the first immunization, the LIN-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, incomplete Freund's adjuvant was used for antigen emulsification, and the dosage and method of immunization remained unchanged. One week after the three immunizations, 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.

[0019] The serum titer of mouse #2 was the highest after immunization, reaching 1:6.4×10. 4 The inhibition rate reached a maximum of 54.67%. 40 μg of LIN-BSA artificial antigen was diluted with 1×PBS to 200 μL and injected intraperitoneally to boost the immunization of mice. Cell fusion could be performed 3 days later.

[0020] Table 1 Serum titers and inhibition detection in immunized mice

[0021] 2. Culture of SP2 / 0 myeloma cells 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 50mL sterile centrifuge tube. 10mL of preheated 1640 complete culture medium was slowly added dropwise to the centrifuge tube. The tube was centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. The cell clumps were gently dispersed, and the cells were resuspended in 5mL of culture medium and transferred to a T75 cell culture flask. An additional 5mL 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.

[0022] 3. Cell fusion (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 h, the tubes were centrifuged at 4000 rpm for 10 min and the serum was collected as a positive control for subsequent screening of monoclonal antibodies. The mice were euthanized by dislocation of the neck and then disinfected by soaking in 75% alcohol.

[0023] (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.

[0024] (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 SP20 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.

[0025] (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.

[0026] (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.

[0027] 4. Screening of positive hybridoma cells Seven days after cell fusion, when the cell clusters were relatively large, the cell supernatant was analyzed using an indirect ELISA method. The LIN-OVA artificial antigen (1 μg / mL) was used as the detection antigen; the positive control (PC) was serum from fused mice, and the negative control (NC) 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.

[0028] 5. Preparation of ascites Monoclonal antibodies were prepared using an in vivo induction method. Each mouse was injected with 0.5 mL of Freund's incomplete adjuvant to purify the peritoneal cavity. After 7 days, the number of hybridoma cells in the logarithmic growth phase was adjusted to 1 × 10⁻⁶. 6Inoculate 0.5 mL of the solution into the peritoneum of each mouse. Observe the ascites production of the mice daily at intervals of approximately 7 days. When the mice's abdomens are significantly distended, their mental state deteriorates, and they become immobile and near death, collect the ascites, centrifuge at 3000 rpm for 10 min, remove the surface fat, carefully aspirate the supernatant for ammonium sulfate precipitation, and store at -20℃.

[0029] 6. Purification of monoclonal antibodies 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 .

[0030] Example 3: Sensitivity and specificity detection of lincomycin monoclonal antibody The sensitivity and specificity of lincomycin monoclonal antibodies were detected using an indirect competitive ELISA method. LIN-OVA artificial antigen was coated at a concentration of 1 μg / mL, and 1 mg / mL of monoclonal antibody 2E3-1E3 was serially diluted to verify the antibody's sensitivity. The results are shown in Table 2. The sensitivity of monoclonal antibody 2E3-1E3 reached 1:40000 dilution, with an inhibition rate as high as 73.40%. Lincomycin artificial antigen, clindamycin artificial antigen, erythromycin artificial antigen, tylosin artificial antigen, streptomycin artificial antigen, and gentamicin artificial antigen were coated at a concentration of 1 μg / mL, and 1 mg / mL of monoclonal antibody 2E3-1E3 was diluted 1:5000 to verify the antibody's specificity. The results are shown in Table 3. The purified monoclonal antibody showed no cross-reactivity with clindamycin artificial antigen, erythromycin artificial antigen, tylosin artificial antigen, streptomycin artificial antigen, and gentamicin artificial antigen, indicating that the purified monoclonal antibody had good specificity.

[0031] Table 2. Monoclonal antibody sensitivity validation

[0032] Table 3. Validation of Monoclonal Antibody Specificity

[0033] Example 4: Cloning of the variable region gene of lincomycin monoclonal antibody 1. Hybridoma cell culture and total RNA extraction Hybridoma cells 2E3-1E3 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).

[0034] 2. Synthesis of the first strand of cDNA 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.

[0035] 3. Gene amplification Design downstream primers and upstream universal primers for Lambda, Kappa, and Heavy chains.

[0036] Primer: F (SEQ ID No. 11): AAGCGTGGTATCAACGCAGA Rκ (SEQ ID No. 12):AACATTGATGTCTTTGGGGTAGAA Rλ (SEQ ID No.13):AATCGTACACACCAGTGTGTGGG R H (SEQ ID No.14):AGGGATCCAGAGTTCCAGGT 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.

[0037] 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 12 cycles; 72℃ for 30s; 98℃ for 15s, 56℃ for 30s, 72℃ for 30s, for 15 cycles; and the program ended at 72℃ for 7min.

[0038] 4. Cloning and screening of PCR amplification products 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.

[0039] 5. The variable region gene sequence and amino acid sequence of the lincomycin monoclonal antibody in this embodiment are as follows: (1) Heavy variable region gene sequence (SEQ ID No. 3): CAGGTCCAACTGCGCATGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAATTGTCCTGCAAGGCTTCTGGCTACACTTTCACCTTTTACCAGATGTACTCAACGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGGACATTCATGCAAGCAATGGTGGTACTAACTTCAATGAGAAGTTCAAGAAGAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCATACATGCAACTCAGCAGCCTGACATCCATCGACTCTGCGGTCTATTACTGTACAAGAGAAGGGGCTATGCGGGGCCAAGGGACTCTGGTCACGGTCTCTGCA。

[0040] (2) Amino acid sequence of the heavy variable region (SEQ ID No.1): QVQLRMSGAELVKPGASVKLSCKASGYTFTFYQMYSTKQRPGQGLEWIGDIHASNGGTNFNEKFKKKATLTVDKSSSTAYMQLSSLTSIDSAVYYCTREGAMRGQGTLVTVSA。

[0041] (3) Gene sequence of the Kappa variable region (SEQ ID No.4): CAAATTGTTCTCACTTCATCTCCAGCAATCATGTCTGCATCTCCTGGGGAGAAGGTCACCATGACCTGCAGTGCCAGATCAAGTGTAAGTTCCACCTACTTACGGTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATGGCAGGATCAACCTGGCTTCTCGAGTCCCTGCTCGCTTCAGTGGTTCCGGGTCTGGGACCTCTTATTCTCTGTGTATCAGCAGCATGGAGACTGAAGATGCTGCCACTTCCGTCTGCCAACAATACAGTGGTTACCCATCCACGTCTCCCTCGGGGAACCAGCCAGAAATCCAA。

[0042] (4) Amino acid sequence of the Kappa variable region (SEQ ID No.2) QIVLTSSPAIMSASPGEKVTMTCSARSSVSSTYLRWYQQKPGSSPKLWIYGRINLASRVPARFSGSGSGTSYSLCISSMETEDAATSVCQQYSGYPSTSPSGNQPEIQ.

[0043] 6. Variable region amino acid sequence and homology analysis 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 monoclonal antibody 2E3-1E3 had the highest homology with the mouse 1F4 anti-CVA6 antibody mRNA sequence (Sequence ID: LC900916.1), with a homology of 310 / 346 (90%). The amino acid sequence of the heavy chain variable region of monoclonal antibody 2E3-1E3 had the highest homology with the mouse immunoglobulin heavy chain variable region amino acid sequence (Sequence ID: AAG39147.1), with a homology of 86 / 97 (89%). The light chain variable region gene sequence of monoclonal antibody 2E3-1E3 had the highest homology with the mouse immunoglobulin IgVk ad4 gene sequence (Sequence ID: AJ231212.1), with a homology of 267 / 295 (91%). The amino acid sequence of the light chain variable region of the monoclonal antibody 2E3-1E showed the highest homology with the amino acid sequence of the variable region of mouse V-type κ chain H1 anti-benzoxazole immunoglobulin (Sequence ID: prf||1202258G), with a homology of 85 / 96 and a homology percentage of 89%.

[0044] 7. CDR Area Analysis The amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody 2E3-1E3 were analyzed at https: / / www.novopro.cn / tools / cdr.html to obtain its CDR region.

[0045] Antibody heavy chain CDR region: CDR-H1 (SEQ ID No.5): FYQMY CDR-H2 (SEQ ID No.6):DIHASNGGTNFNEKFKK CDR-H3 (SEQ ID No.7): EGAM Antibody light chain CDR region: CDR-L1 (SEQ ID No.8): SARSSVSSTYLR CDR-L2 (SEQ ID No.9): GRINLAS CDR-L3 (SEQ ID No.10): QQYSGY

[0046] Example 5: Preparation of Lincomycin Colloidal Gold Test Strip 1. Preparation of chloroauric acid 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 stirrer with a stirring speed of 1. After the water boils, increase the stirring speed to 6. Once the liquid is stirring, quickly add 1 mL of 0.9% reducing agent and adjust the stirring speed to 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, clear, and free of precipitates and floating matter.

[0047] 2. Preparation of Lincomycin Monoclonal Antibody-Colloidal Gold Label (1) Add bare gold: Mix the colloidal bare gold well and take 1L of colloidal gold solution; (2) Add K2CO3: Add 0.2M K2CO3 at a ratio of 3μL / mL colloidal gold and vortex to mix; (3) Adding antibody: Mix 1 mL of 1 mg / mL lincomycin 2E3-1E3 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; (4) Leveling: Reduce the rotation speed to stabilize the vortex at a drop of about 1 cm, and then rebalance for 1 hour; (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. (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; (7) Centrifugation: Use a benchtop high-speed centrifuge at 11,000 rpm and 4°C for 6 min; (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; (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; (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. (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. (12) Lyophilization of microwell reagent: The microwell reagent solution was prepared by mixing lincomycin monoclonal antibody-colloidal gold label and lyophilization buffer, stirring and mixing for 20 min, and dispensed into 96-well microplates at a ratio of 60 μL / well; wherein the lyophilization buffer is composed of 0.05 M phosphate buffer, 1% BSA, 2% sucrose, and 1% Triton X-100; the ratio of lincomycin colloidal gold to the lyophilization buffer in the microwell reagent solution is 1:5; the coated 96-well microplate was placed in a vacuum freeze dryer with a preset freeze-drying program for processing. After the freeze-drying process was completed, the lyophilized gold was removed, the rubber cap was closed, and it was placed in an aluminum foil bag with desiccant for storage.

[0048] 3. Preparation of sample absorption pads The sample absorption pad was immersed in 0.1M 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.

[0049] 4. Preparation of nitrocellulose membranes Lincomycin artificial antigen was diluted to 1 mg / mL with phosphate buffer and coated onto a nitrocellulose membrane using a coating apparatus to form the detection line T, with a coating amount of 1.0 μg / cm. Goat anti-mouse IgG antibody was diluted to 300 μg / mL with PBS buffer (0.01 M, pH 7.2) and coated onto a nitrocellulose membrane as the control line C. The coated reaction membrane was then dried at 37°C for 4 hours to obtain the coated nitrocellulose membrane.

[0050] 5. Assembly of Lincomycin Colloidal Gold Test Strips 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 assembly forms the colloidal gold test strip. Together with the microporous reagent, it forms the lincomycin colloidal gold test strip.

[0051] Example 6: Application of Lincomycin Colloidal Gold Test Strip 1. Test strip detection 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 minutes, insert the labeled test strip into the well, ensuring it is fully immersed in the solution. After incubating at 40℃ for 5 minutes, 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.

[0052] 2. Interpretation of test results Negative (﹣): Both C and T lines show color, with the T line showing stronger color than the C line, indicating that the concentration of lincomycin in the sample is below the detection limit.

[0053] 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 lincomycin in the sample is equal to or higher than the detection limit.

[0054] Invalid: No C line appears, indicating incorrect operation or that the test strip has deteriorated and become ineffective.

[0055] In addition to naked-eye interpretation, a colloidal gold reader can be used to interpret the results; a T / C ≤ 1 is considered positive.

[0056] 3. Sensitivity detection of lincomycin colloidal gold test strips The concentrations of lincomycin standard were diluted to 1 ppb and 2 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 2 ppb for lincomycin standard in milk.

[0057] Table 4. Sensitivity of Lincomycin Colloidal Gold Test Strips

[0058] 4. Specificity detection of lincomycin colloidal gold test strips The test strips were tested at 500 ppb for lincomycin, clindamycin, erythromycin, tylosin, streptomycin, and gentamicin 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.

[0059] Table 5. Specificity of Lincomycin Colloidal Gold Test Strips

[0060] 5. Stability testing of lincomycin colloidal gold test strips The prepared test strips were subjected to accelerated testing at 4℃ and 37℃. They were used to detect lincomycin standard (2 ppb) 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 Tables 6-1 and 6-2. All test results were positive, and the CV values ​​were all <10%, indicating that the test strips provided by this invention have good stability.

[0061] Table 6-1 Stability index determination of lincomycin colloidal gold test strips

[0062] Table 6-2 Stability evaluation of lincomycin colloidal gold test strips

[0063] 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 lincomycin 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 lincomycin monoclonal antibody according to claim 1, characterized in that: The gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3; The gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

4.

3. The application of the lincomycin monoclonal antibody according to claim 1 in the preparation of lincomycin detection products.

4. A lincomycin detection product, characterized in that: The test product consists of a lincomycin test strip and a microwell reagent; the test strip is a colloidal gold test strip, and the microwell reagent contains the lincomycin monoclonal antibody as described in claim 1 or 2.

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