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

Abstract

The application discloses a ceftriaxone 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 ceftriaxone detection product. The ceftriaxone colloidal gold detection test strip provided by the application has the characteristics of high sensitivity, good specificity and strong stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a heavy chain and light chain variable region of a cefetamet pivoxil monoclonal antibody and application thereof. BACKGROUND

[0002] Cefetamet pivoxil antibiotic is a broad-spectrum antibiotic discovered in the 1960s, and its molecular formula is C 19 H 17 N5O7S3. In 1988, cefetamet pivoxil was first approved in the United States for the treatment of respiratory bacterial diseases in cattle. Subsequently, its application range has been extended to respiratory diseases and other infectious diseases of various livestock and poultry such as pigs, horses, sheep, chickens, etc.

[0003] Cefetamet pivoxil antibiotic is a third-generation cephalosporin antibiotic specially used in veterinary clinics, which has a broad-spectrum and high-efficiency bactericidal effect on gram-positive bacteria and gram-negative bacteria including beta-lactamase-producing bacterial strains, and is the best new drug for clinical treatment of bacterial diseases. It is mainly used for the treatment of sensitive bacterial infections in livestock and poultry, such as infectious pleurisy, actinobacillus, pasteurella multocida, pre-and post-natal high fever in livestock, endometritis, mastitis, pneumonia, etc. On the other hand, non-standard use causes its residues and accumulation in animal bodies, which endangers human health. Therefore, some countries and regions have made regulations on the residue limits of cefetamet pivoxil in animal-derived food, and our country has also made regulations on the residue limits in different animal species such as muscle, fat, liver, kidney and milk in the "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Food" (GB 31650-2019), and the maximum residue limit in milk is 100 μg / kg. Therefore, it is imperative to establish a safe and efficient biological detection method for cefetamet pivoxil.

[0004] The immunoassay method has the advantages of low cost, high efficiency, high sensitivity, and relatively low requirement for technical personnel, and is suitable for rapid detection of a large number of samples. The purpose of the present application is to provide a monoclonal antibody with high affinity and detection sensitivity for cefetamet pivoxil, to lay a foundation for the development and popularization of a colloidal gold test strip detection method and a colloidal gold test strip product. SUMMARY

[0005] To this end, the present application provides a heavy chain and light chain variable region of a cefetamet pivoxil monoclonal antibody and application thereof.

[0006] In order to achieve the above-mentioned purpose, the embodiments of the present application provide the following technical solutions:

[0007] In a first aspect, the present application provides a cefetamet pivoxil 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 as SEQ ID No. 1;

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

[0010] 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;

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

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

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

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

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

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

[0017] In a second aspect, the above-mentioned cefftiofur monoclonal antibody is characterized in that:

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

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

[0020] In a third aspect, the present application provides a cefftiofur monoclonal antibody for preparing a test strip for detecting cefftiofur.

[0021] Preferably, the test strip is a colloidal gold detection test strip, or a fluorescent microsphere detection test strip, or a latex microsphere detection test strip;

[0022] The present application has the following advantages:

[0023] The monoclonal antibody has the characteristics of high specificity and high sensitivity, and can be used as a raw material for enzyme-linked immunoassay and colloidal gold immunoassay. The purpose of the application is to provide a cefetico monoclonal antibody heavy chain and light chain variable region and its detection application, and to lay a foundation for the research and development of ELISA, colloidal gold test paper and fluorescent immunoassay test paper. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required to be used in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only exemplary, and other drawings can be obtained by the provided drawings without creative labor for those skilled in the art.

[0025] Figure 1 SDS-PAGE identification results of the purified monoclonal antibody;

[0026] Figure 2 Schematic diagram for colloidal gold test paper result interpretation. DETAILED DESCRIPTION

[0027] The embodiments of the present application will be described below by specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the specification. Obviously, the described embodiments are part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0028] Example 1 Synthesis of cefetico artificial antigen

[0029] 1. Preparation of cefetico immunogen

[0030] S1, accurately weigh 500 mg of cefetico raw material, completely dissolve in 20 mL of dichloromethane; cool to 0℃, add bromoacetic acid tert-butyl ester 164 mg dropwise, stir uniformly;

[0031] S2, weigh 76 mg of sodium hydroxide and add to the reaction system, stir at room temperature 25℃ for 14 h;

[0032] S3, monitor the reaction liquid by TLC, confirm that the reaction is complete, add 15 mL of purified water for quenching, stand and separate, retain the aqueous phase; use 3M hydrochloric acid aqueous solution to adjust the pH of the system to 4.5±0.1, add ethyl acetate 20 mL for extraction twice, combine the organic phase; wash with saturated sodium chloride 20 mL twice, spin dry the organic phase, and obtain about 520 mg of cefetico hapten;

[0033] S4, accurately take 300 mg of cefftiofur hapten, dissolve into 3 mL of DMF;

[0034] S5, take 90 mg of EDC and 50 mg of NHS, slowly add them to the reaction system, stir the reaction at room temperature 25 °C for 6 h, to obtain an activated liquid of succinimide;

[0035] S6, take 400 mg of bovine serum albumin BSA, dissolve it in 0.01 mol / L of PBS (pH = 7.4) buffer, stir it uniformly, slowly drop it into the activated liquid of succinimide, stir it at room temperature for 4 h;

[0036] S7, use 0.01 mol / L of PBS (pH = 7.4) buffer to dialyze the obtained reaction liquid, change the dialysis liquid every 4 h during the dialysis, dialyze for 48 h, to obtain cefftiofur immune antigen (SF-BSA).

[0037] 2. Preparation of cefftiofur detection antigen

[0038] S1, accurately take 500 mg of cefftiofur raw material, completely dissolve it in 20 mL of dichloromethane; cool it to 0 °C, drop 164 mg of tert-butyl bromoacetate into it, stir it uniformly;

[0039] S2, take 76 mg of sodium hydroxide, add it to the reaction system, stir the reaction at room temperature 25 °C for 14 h;

[0040] S3, monitor the reaction liquid by TLC, confirm that the reaction is completed, add 15 mL of purified water to quench it, stand and separate the liquid, retain the aqueous phase; use 3M hydrochloric acid aqueous solution to adjust the pH of the system to 4.5 ± 0.1, add 20 mL of ethyl acetate to extract twice, combine the organic phases; use 20 mL of saturated sodium chloride to wash twice, spin dry the organic phase, to obtain about 520 mg of cefftiofur hapten;

[0041] S4, accurately take 300 mg of cefftiofur hapten, dissolve it into 3 mL of DMF;

[0042] S5, take 90 mg of EDC and 50 mg of NHS, slowly add them to the reaction system, stir the reaction at room temperature 25 °C for 6 h, to obtain an activated liquid of succinimide;

[0043] S6, take 400 mg of chicken egg white albumin OVA, dissolve it in 0.01 mol / L of PBS (pH = 7.4) buffer, stir it uniformly, slowly drop it into the activated liquid of succinimide, stir it at room temperature for 4 h;

[0044] S7, the obtained reaction solution was dialyzed with 0.01 mol / L PBS (pH=7.4) buffer solution, the dialysis solution was changed every 4 hours during the dialysis, and the dialysis was performed for 48 hours to obtain the cefftbuten detection antigen (SF-OVA).

[0045] Example 2 Preparation of cefftbuten monoclonal antibody

[0046] 1. Mouse immunization

[0047] Three 6-8 week old female Balb / c mice were taken, and each mouse was immunized with 20 μg of SF-BSA artificial antigen. For the first immunization, the SF-BSA artificial antigen was emulsified with an equal volume of Freund's complete adjuvant, and the mouse was immunized by subcutaneous injection at multiple points. The mouse was immunized once every two weeks, and the mouse was immunized three times. The antigen was emulsified with Freund's incomplete adjuvant for the second and third immunizations, and the immunization dose and method were unchanged. One week after the third immunization, the mouse was subjected to tail vein blood collection, and the serum was taken and its titer and inhibition were measured by indirect ELISA. The results are shown in Table 1. The serum titer of the mouse after immunization was the highest, which was 1:6.4x10 4 , and the inhibition rate was the highest, which was 53.58%. 40 μg of SF-BSA artificial antigen was diluted to 200 μL with 1xPBS, and the mouse was subjected to intraperitoneal injection for booster immunization, and the cell fusion was performed three days later.

[0048] Table 1. Serum titer and inhibition detection of immunized mice

[0049]

[0050] 2. Culture of SP2 / 0 myeloma cells

[0051] One vial of SP2 / 0 myeloma cells stored in liquid nitrogen was taken out and immediately transferred to a 37°C constant temperature water bath, and the cryopreservation tube was gently shaken from time to time. When the cells were thawed to a semi-ice crystal state, they were taken out. In a sterile environment, the SP2 / 0 cells in the cryopreservation tube were transferred to a 50 mL sterile centrifuge tube, and 10 mL of preheated 1640 complete culture medium was slowly added dropwise to the centrifuge tube, and centrifuged at 1000 r / min for 5 min, and the supernatant was discarded. The cell mass was gently dispersed, 5 mL of culture medium was added to resuspend the cells and transferred to a T75 cell culture bottle. Another 5 mL of culture medium was added, and the cell bottle was shaken in a "cross" direction, and placed in a CO2 cell culture box and cultured at 37°C. The cell state was observed under a microscope, and when the density was about 80%, the SP2 / 0 cells were subcultured.

[0052] 3. Cell fusion

[0053] (1) Take the experimental mouse after booster immunization to take the blood in the orbit, collect in the EP tube, 37℃ rest for 2h, 4000rpm centrifugal 10 min, collect the serum for the follow-up screening of monoclonal antibody as positive control. The mouse is executed by decapitation and is immersed in 75% alcohol for disinfection.

[0054] (2) Preparation of spleen cells: in the biological safety cabinet, use sterilized scissors and forceps to cut the mouse skin, replace a new set of sterilized scissors and forceps to cut the mouse abdominal cavity, and then use a set of sterilized scissors and forceps to carefully take out the spleen, and cut off the excess fat. Prepare a sterile 15mL centrifuge tube, add 10mL DMEM medium, put the spleen into the centrifuge tube, and carefully discard the excess medium after the spleen is wet. Then 10mL of DMEM medium is taken and placed in a sterile dish, and the spleen is ground with a glass sheet to prepare a single cell suspension and filtered through a 200 mesh nylon mesh into a sterile centrifuge tube. Add 30mL of DMEM to a 50mL sterile centrifuge tube, rinse the nylon mesh with a pipette, centrifuge the centrifuge tube containing the spleen cell suspension at 1500rpm for 5min, discard the supernatant, and gently disperse the cell clumps with your hand, then add 30mL of DMEM medium to resuspend and centrifuge again, then discard the supernatant, gently disperse the cell clumps with your hand, and then add 10mL of DMEM medium to resuspend.

[0055] (3) Cell fusion: centrifuge the well-grown SP2 / 0 cells in a 50mL centrifuge tube at 1000rpm for 5min, gently disperse the SP20 cell clumps, add 30mL of DMEM medium to resuspend, centrifuge again, add 10mL of DMEM medium to resuspend, and then mix the spleen cell suspension with the SP2 / 0 cell suspension, centrifuge at 1000rpm for 5min, discard the supernatant, and gently disperse the cell clumps. Place in a 37℃ water bath environment, take 1mL of PEG fusion agent and add it to the centrifuge tube, add 1mL in 1min, at this time the cell state is red homogeneous sand, and the rotation of the tube wall feels like frosted glass.

[0056] (4) Stop fusion: take 9mL of preheated DMEM medium to stop fusion, which is divided into three stages. The first stage is to add 1mL in the first minute, the second stage is to add 1mL in 1min, and the third stage is to add the remaining 7mL of medium in 3min. Then stand stable in a 37℃ water bath for 5min, and centrifuge at 800rpm for 5min.

[0057] (5) Plating: Discard the supernatant, gently disperse the cell mass, add HAT medium (for example, to plate 5 96-well plates, 200 μL / well, remove the feeder layer cells that have been pre-plated 100 μL / well, then add 50 mL of HAT medium), mix the cells, and then evenly plate the fused cell suspension into the 96-well cell plates with the feeder layer cells, 100 μL / well, and place in a CO2 cell incubator at 37°C.

[0058] 4. Screening of positive hybridoma cells

[0059] Seven days after cell fusion, the supernatant of the cells was detected by indirect ELISA when the cell mass was relatively large. The SF-OVA artificial antigen was used as the detection antigen at a concentration of 1 μg / mL, the positive control was the serum of the fused mouse, and the negative control was the serum of the PBS immunized mouse. The well with the strongest color reaction was selected as the positive well. The screened positive hybridoma cells were subcloned by limiting dilution. The hybridoma cell strain that was identified to stably secrete monoclonal antibodies after subcloning was expanded and transferred to a T75 cell bottle. When the cell number was about 80%, the cells were collected for the preparation of ascites.

[0060] 5. Preparation of ascites

[0061] 10 mL of sterile 1xPBS was added to the cell bottle, the cell layer was blown down, and then resuspended and transferred to a 15 mL centrifuge tube. Centrifugation was performed at 1000 r / min for 10 min. The supernatant was discarded, and the precipitate was resuspended with 1 mL of sterile 1xPBS and mixed well. 1 mL of a syringe was used to aspirate. Each mouse was injected with about 500 μL of cell suspension, and the growth state of the mouse was observed. The ascites was collected when the mouse's abdomen was swollen after one week. The mouse ascites was collected in a centrifuge tube and centrifuged at 8000 r / min for 20 min. The middle ascites layer was aspirated.

[0062] 6. Purification of monoclonal antibodies

[0063] The collected ascites was purified. SDS-PAGE was used to identify the purity of the purified monoclonal antibodies, and the purity was about 95%. The results are shown in Figure 1 .

[0064] Example 3: Specificity and sensitivity detection of cefuroxime monoclonal antibodies

[0065] The sensitivity and specificity of the cefuroxime monoclonal antibodies were detected by indirect ELISA. The SF-OVA artificial antigen was coated at a concentration of 1 μg / mL, and the monoclonal antibody 4D2-1C10 was diluted at a concentration of 1 mg / mL to verify the sensitivity of the monoclonal antibody. The results are shown in Table 2. The sensitivity of the monoclonal antibody 4D2-1C10 can reach 1:40000 dilution, and the highest inhibition rate can reach 72.34%.

[0066] Table 2 Sensitivity verification of monoclonal antibody

[0067]

[0068] Ceftriaxone artificial antigen, β-lactam artificial antigen (cephalosporin C drug coupling), tetracycline artificial antigen, fluoroquinolone artificial antigen and sulfonamide artificial antigen were coated at 1 μg / mL, 1 mg / mL of monoclonal antibody 4D2-1C10 was diluted at 1:5000 to verify the specificity of the monoclonal antibody, and the results are shown in Table 3. The purified monoclonal antibody had no cross reaction with β-lactam artificial antigen (cephalosporin C drug coupling), tetracycline artificial antigen, fluoroquinolone artificial antigen and sulfonamide artificial antigen, indicating that the purified monoclonal antibody had good specificity.

[0069] Table 3 Specificity verification of monoclonal antibody

[0070]

[0071] Example 4, Cloning of Ceftriaxone Monoclonal Antibody Variable Region Gene

[0072] 1. Hybridoma cell culture and total RNA extraction

[0073] The hybridoma cell 4D2-1C10 was cultured in RPMI 1640 complete medium at 37°C, 5% carbon dioxide, and the cell number reached 1×10 7 The total RNA in the cells was extracted by total RNA extraction kit (purchased from Tiangen).

[0074] 2. Synthesis of cDNA first strand

[0075] The total RNA extracted in step 1 was used as the amplification template to synthesize the cDNA first strand using the reverse transcription kit (purchased from TAKARA).

[0076] 3. Gene amplification

[0077] The downstream primer and upstream universal primer of Lambda chain, Kappa chain and Heavy chain were designed.

[0078] Primer: F (SEQ ID No. 11): AAGCAGTGGTATCAACGCAGA

[0079] Rκ (SEQ ID No. 12): AACATTGATGTCTTTGGGGTAGAA

[0080] Rλ (SEQ ID No. 13): AATCGTACACACCAGTGTGTGGG

[0081] RH (SEQ ID No. 14): AGGGATCCAGAGTTCCAGGT

[0082] PCR reaction system was 50 μL. Template 3 μL, upstream primer (10 uM) 2.5 μL, downstream primer (10 uM) 2.5 μL, 2 × Taq enzyme 25 μL, sterile water 17 μL.

[0083] The touchdown PCR reaction conditions were as follows: 98℃ 30s; 98℃ 15s, 64℃-58℃ 30s, each time down 0.5℃, until 58℃, cycle 10 times; 72℃ 30s; 98℃ 15s, 56℃ 30s, 72℃ 30s, cycle 15 times; 72℃ 7min end program.

[0084] 4. Cloning and screening of PCR amplification products

[0085] The PCR products were electrophoresed on 1% agarose gel, and the Kappa chain, Lambda chain and Heavy chain amplification fragments were recovered by PCR product recovery kit (purchased from Tiangen). The recovered and purified target fragments were inserted into pLB vector by pLB zero background rapid cloning kit (purchased from Tiangen), and transformed into DH5α competent cells (ampicillin resistance). The recombinant positive clones were screened and sequenced.

[0086] 5. The variable region gene sequence and amino acid sequence of the cefuroxime monoclonal antibody of the embodiment are as follows:

[0087] (1) Heavy chain variable region gene sequence (SEQ ID No. 3):

[0088] ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGATGCTGGTGGAGTCTGTGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGAATTCACTCTCAGTACCTATGTCATGTCTTGGTTTCGTCAGACTACAGAGAAGAGGCTGGAGTGGGTCGCAACCACTCTTAGTGGTGGTTACTATCGCTACTATCAAGACAGTGTGAAGCGGCGCTTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAGGTGTGAGGACACGGCCATGTATTACTGTACAAGAGGGATTTACTACGGTAGTAGGACCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0089] (2) Heavy chain variable region amino acid sequence (SEQ ID No. 1):

[0090] MNFGLSLIFLVLVLKGVQCEVMLVESVGGLVKPGGSLKLSCAASEFTLSTYVMSWFRQTTEKRLEWVATTLSGGYYRYYQDSVKRRFTISRDNAKNTLYLQMSSLRCEDTAMYYCTRGIYYGSRTWYFDVWGAGTTVTVSS.

[0091] (3) Kappa chain variable region gene sequence (SEQ ID No. 4):

[0092] ATGCGGTGCTCTCTTCAGTTCCTGGGGGTGCTTATGTTCTGGATCTCTGGAGTCAGTGGGGATATTGTGATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTCGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACTAACTTGAATTGGTTTCTTCAGAGACCAAGACAATCTCCTCAGCTCCTGATATCTATGATGTCCACCCGTGCATCAGGAGACTCAGTCCGGTTTAGGGGCAGCGGGTCAGGAATAGATTTCACACTGGAAATCAGTGGAGTGAAGGCTGAGAATGTGGGTGTGTATTACTGTCAACAACTTGTAGAGTATCCATTCACGTTCGGCTCGGGGACAAAGCCGCAAATAATC.

[0093] (4) Kappa chain variable region amino acid sequence (SEQ ID No. 2)

[0094] MRCSLQFLGVLMFWISGVSGDIVITQDELSNPVTSRESVSISCRSSKSLLYKDGKTNLNWFLQRPRQSPQLLISMMSTRASGDSVRFRGSGSGIDFTLEISGVKAENVGVYYCQQLVEYPFTFGSGTKPQII.

[0095] 6. Variable region amino acid sequence and homology analysis

[0096] The heavy chain and light chain gene sequences were analyzed by alignment analysis in the NCBI database, and the analysis results showed that the monoclonal antibody 4D2-1C10 heavy chain variable region gene sequence had the highest homology with the mouse immunoglobulin TF2-76 variable region gene sequence (Sequence ID: M13280.1), the homology was 330 / 352, and the homology percentage was 93.75%. The monoclonal antibody 4D2-1C10 heavy chain variable region amino acid sequence had the highest homology with the mouse immunoglobulin heavy chain variable region amino acid sequence (Sequence ID: AAK71611.1), the homology was 91 / 106, and the homology percentage was 85.85%. The monoclonal antibody 4D2-1C10 light chain variable region gene sequence had the highest homology with the mouse chromosome 6 clone gene RP23-321G20 (Sequence ID: AC122322.4), the homology was 295 / 311, and the homology percentage was 94.86%. The monoclonal antibody 4D2-1C10 light chain variable region amino acid sequence had the highest homology with the mouse immunoglobulin v-kappa167 light chain variable region amino acid sequence (Sequence ID: AAA39032.1), the homology was 108 / 119, and the homology percentage was 90.76%.

[0097] 7. CDR region analysis

[0098] The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4D2-1C10 were analyzed at https: / / www.novopro.cn / tools / cdr.html, and the CDR regions were obtained.

[0099] Antibody heavy chain CDR region:

[0100] CDR-H1 (SEQ ID No. 5): TYVMS

[0101] CDR-H2 (SEQ ID No. 6): TTLSGGYYRYYQDSVKR

[0102] CDR-H3 (SEQ ID No. 7): GIYYGSRTWYFDV

[0103] Antibody light chain CDR region:

[0104] CDR-L1 (SEQ ID No. 8): RSSKSLLYKDGKTNLN

[0105] CDR-L2 (SEQ ID No. 9): MMSTRAS

[0106] CDR-L3 (SEQ ID No. 10): QQLVEYPFT.

[0107] Example 5, Preparation of Cefuroxime Colloidal Gold Test Strips

[0108] 1. Preparation of chloroauric acid

[0109] Take 800 mL of ultrapure water into a 1000 mL conical flask, add 8 mL of 1% chloroauric acid in ultrapure water, mix and heat on a magnetic heating rod stirrer, temperature 500℃, speed 1st gear. After the water boils, increase the speed to 6th gear, and after the liquid is stirred, quickly add 1 mL of 0.9% reducing agent, and adjust the speed to 5th gear. After heating for 6 minutes and 30 seconds, stop heating, and let it cool to room temperature. The prepared colloidal gold is pure in appearance, transparent, and free of precipitate and floaters.

[0110] 2. Preparation of Cefuroxime Monoclonal Antibody-Colloidal Gold Label

[0111] (1) Add bare gold: mix the colloidal gold bare gold, take 1 L of colloidal gold solution;

[0112] (2) Add K2CO3: add 0.2M K2CO3 at a ratio of 3 μL / mL of colloidal gold, vortex mix;

[0113] (3) Add antibody: mix 1 mL of 1 mg / mL cefuroxime 4D2-1C10 antibody with 5 mL of 10% BSA (IgG), then add the mixed antibody drop by drop into the gold using a pipette, and balance for 5 minutes;

[0114] (4) Mix evenly: reduce the speed and stabilize at about 1 cm below the vortex, and balance for 1 hour;

[0115] (5) Add BSA: increase the speed to about 3 cm below the vortex, pour in 10 mL of 10% BSA (Ribo), balance for 3 minutes, reduce the speed and stabilize at about 1 cm below the vortex, and balance for 30 minutes;

[0116] (6) Add PEG: increase the speed to about 3 cm below the vortex, pour in 10 mL of 10% PEG 20000, balance for 5 minutes;

[0117] (7) Centrifuge: use a desktop high-speed centrifuge, 10500 rpm, 4℃, centrifuge for 6 minutes;

[0118] (8) Discard supernatant: after centrifugation, gently remove the centrifuge bucket and place it on the experimental bench of the negative pressure liquid suction device. Do not shake or knock to avoid disturbing the gold precipitate in the centrifuge bucket; stop suction when close to the bottom of the centrifuge bucket (about 5-6 mL remaining), carefully take the remaining supernatant with a 1 mL pipette, avoiding suction of the gold precipitate;

[0119] (9) Resuspend: stop pipetting when close to the bottom of the centrifuge bucket (about 2-3 mL left), resuspend by gently shaking the centrifuge bucket, and transfer the resuspended gold precipitate to a blue cap bottle;

[0120] (10) Rinse the bucket: add about 20 mL of the reconstitution solution to the centrifuge bucket, shake gently to wash away the remaining gold precipitate, and transfer the liquid to the other three centrifuge buckets in turn, and finally to the blue cap bottle. Repeat once.

[0121] (11) Volumetric: finally, volumetrically adjust the blue cap bottle to 1 / 10 of the gold solution, i.e. 100 mL, and mix well.

[0122] (12) Micro-pore reagent freeze-drying: use cefquinome colloidal gold and freeze-drying buffer to prepare a micro-pore reagent solution, stir and mix well for 20 min, and then aliquot 60 μL / well of the micro-pore reagent solution into a 96 micro-pore plate. The freeze-drying buffer is composed of 0.05 M phosphate buffer, 1% BSA, 3% trehalose, and 1% Triton X-100. The ratio of cefquinome colloidal gold to the freeze-drying buffer in the micro-pore reagent solution is 1:5. Place the coated 96 micro-pore plate into a vacuum freeze-drying machine with a pre-set freeze-drying program for processing. After the freeze-drying process is completed, remove the freeze-dried gold, put on a rubber cap, and then place it into an aluminum foil bag with a drying agent for storage and standby use.

[0123] 3. Preparation of sample absorption pad

[0124] Soak the sample absorption pad in a 0.5% BSA, pH=7.2, 0.1 mol / L phosphate buffer for 2 h, and then dry it at 37°C for 2 h to obtain the sample absorption pad.

[0125] 4. Preparation of nitrocellulose membrane

[0126] Dilute the cefquinome artificial antigen to 1 mg / mL with a phosphate buffer, and coat it on the test line T of the nitrocellulose membrane with a biodot dot membrane instrument at a coating amount of 1.0 μg / cm. Dilute the goat anti-mouse IgG antibody to a concentration of 200 μg / mL with a 0.01 M, pH 7.2 PBS buffer, and coat it on the control line C of the nitrocellulose membrane with a biodot dot membrane instrument. Dry the coated reaction membrane at 37°C for 3 h to obtain the nitrocellulose membrane.

[0127] 5. Assembly of cefquinome colloidal gold test strip

[0128] Assemble the sample absorption pad, nitrocellulose membrane, and water absorption pad in order on the base plate, with the beginning of the sample absorption pad connected to the end of the nitrocellulose membrane, the beginning of the nitrocellulose membrane connected to the end of the water absorption pad, the end of the sample absorption pad aligned with the end of the base plate, and the beginning of the water absorption pad aligned with the beginning of the base plate, to form the colloidal gold test strip.

[0129] Example 6, Application of Cefuroxime Colloidal Gold Test Strip

[0130] 1. Test strip detection

[0131] The required test strip and sample diluent were restored to room temperature (20-25℃). 200µL of the sample solution to be tested was taken with a micropipette into a microwell, slowly sucked and mixed well with the reagent in the microwell. After incubation at 40℃ for 3 min, the labeled test strip was inserted into the microwell and immersed in the solution. After incubation at 40℃ for 5 min, the test strip was taken out and the results were determined according to the schematic diagram (Figure 1), and other times were determined to be invalid. Figure 2

[0132] 2. Interpretation of test results

[0133] Negative (-): C line and T line developed color and T line developed color was stronger than C line, indicating that the concentration of cefuroxime in the sample was lower than the detection limit.

[0134] Positive (+): C line developed color, T line developed color was the same as C line, T line developed color was weaker than C line or T line did not develop color, indicating that the concentration of cefuroxime in the sample was equal to or higher than the detection limit.

[0135] Invalid: No C line appeared, indicating incorrect operation process or test strip had deteriorated and was invalid.

[0136] In addition to naked eye interpretation, NBReader can be used for result interpretation.

[0137] 3. Sensitivity detection of cefuroxime colloidal gold test strip

[0138] The concentration of cefuroxime standard was diluted to 0.75ppb and 1.5ppb, and the milk sample was detected by adding standard according to the test strip detection method, and the minimum detection limit of the product was verified, and the results were shown in Table 4. The sensitivity of the test strip provided by the present application for the detection of cefuroxime standard in milk was 1.5ppb.

[0139] Table 4 Sensitivity of cefuroxime colloidal gold test strip

[0140]

[0141] 4. Specificity detection of cefuroxime colloidal gold test strip

[0142] ​According to the detection method, 2000 ppb of ceftriaxone, tetracycline, fluoroquinolone and sulfonamide were detected, and the data are shown in Table 5, and the results were all negative. According to the detection method, 2000 ppb of β-lactam drugs (total of 23 kinds) were detected, and the data are shown in Table 6, and the results were all negative, and the cross-reactivity with cefquinome was less than 0.075%. In summary, it is indicated that the test strip provided by the application does not have cross-reaction with other antibiotic drugs, and has good specificity.

[0143] Table 5 Specificity 1 of ceftriaxone colloidal gold detection test strip

[0144]

[0145] Table 6 Specificity 2 of ceftriaxone colloidal gold detection test strip

[0146]

[0147] 5. Stability detection of ceftriaxone colloidal gold detection test strip

[0148] The prepared test strip was placed in 4℃ and 37℃ environment for accelerated test, and 1.5 ppb of ceftriaxone standard was detected by the test strip at 0 d, 7 d, 14 d and 28 d, respectively, error analysis of the detected concentration and the actual sample concentration value was carried out, and the results are shown in Table 7. The CV is less than 10%, indicating that the test strip provided by the application has good stability.

[0149] Table 7 Stability of ceftriaxone colloidal gold detection test strip

[0150]

[0151] Although the application has been fully described in the foregoing by general description and specific embodiments, some modifications or improvements can be made on the basis of the application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the application, all belong to the scope of protection claimed by the application.

Claims

1. A ceftiofur 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 ceftiofur 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 ceftiofur monoclonal antibody according to claim 1 in the preparation of ceftiofur detection products.

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

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