Heavy chain and light chain variable regions of tetracycline monoclonal antibody and applications thereof

Abstract

The present invention discloses a tetracycline monoclonal antibody and its application, belonging to the field of biotechnology. The monoclonal antibody contains a heavy chain variable region and a light chain variable region, both of which are composed of a complementary determining region and a framework region, and both of which are composed of CDR1, CDR2 and CDR3. The monoclonal antibody can be used to prepare a tetracycline detection product. The tetracycline colloidal gold detection test strip provided by the present invention has the characteristics of high sensitivity, good specificity and strong stability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a heavy chain and light chain variable region of a tetracycline monoclonal antibody and applications thereof. Background Art

[0002] Tetracycline antibiotics are a class of broad-spectrum antibiotics with a phenanthane nucleus discovered in the 1940s. Their molecular formula is C 22 H 24 N2O8. The tetracycline antibiotics mainly include chlortetracycline, oxytetracycline and tetracycline. The tetracycline antibiotics have a common chemical structure matrix, which are both acidic and alkaline compounds. Chlortetracycline and oxytetracycline are both derivatives of tetracycline, the former is chlorotetracycline and the latter is oxytetracycline. Tetracycline itself and its salts are yellow or light yellow crystals, which are extremely stable in a dry state. Except for chlortetracycline, the aqueous solutions of other tetracyclines are quite stable. Tetracyclines are soluble in dilute acids, dilute alkalis, etc., slightly soluble in water and lower alcohols, but insoluble in ether and petroleum ether. For most Gram-positive and Gram-negative bacteria, tetracycline has an inhibitory effect on them. High concentrations of tetracycline have a bactericidal effect, especially for Gram-negative bacteria.

[0003] Tetracycline antibiotics are widely used to treat infections caused by Gram-positive and Gram-negative bacteria, intracellular mycoplasmas, chlamydia, and rickettsia. The mechanism of action of tetracycline is mainly to prevent the binding of aminoacyl groups to ribonucleoproteins, prevent the growth of peptide chains and protein synthesis, and thus inhibit the growth of bacteria. In most countries around the world, tetracycline, as a broad-spectrum antibiotic, is widely used as a feed additive to feed animals and enter the food chain. Long-term intake of food containing tetracycline residues can cause allergic reactions, gastrointestinal discomfort, and even liver toxicity in humans. Therefore, it is imperative to establish a safe and efficient biological detection method for tetracycline.

[0004] The immunoassay method has the advantages of low cost, high efficiency, high sensitivity, and relatively low requirements for technicians, and is suitable for rapid detection of a large number of samples. The purpose of the present invention is to provide a monoclonal antibody with extremely high affinity and detection sensitivity for tetracycline, so as to establish a colloidal gold test strip detection method and lay a foundation for the research, development and promotion of colloidal gold test strip products. Summary of the invention

[0005] To this end, the present invention provides a heavy chain and light chain variable region of a tetracycline monoclonal antibody and applications thereof.

[0006] In order 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 tetracycline 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] The heavy chain variable region and the light chain variable region are both composed of complementarity determining regions and framework regions, and the complementarity determining regions are both 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 in SEQ ID No. 5;

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

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

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

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

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

[0017] In a second aspect, the above-mentioned tetracycline 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] In a third aspect, the present invention provides a use of a tetracycline monoclonal antibody in preparing a test strip for detecting tetracycline.

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

[0022] The present invention has the following advantages:

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

[0024] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0025] Figure 1 This is the SDS-PAGE identification result after monoclonal antibody purification;

[0026] Figure 2 This is the result of the sequence alignment of the heavy chain variable region gene of monoclonal antibody 5E1-1B7;

[0027] Figure 3 This is the result of amino acid sequence alignment of the heavy chain variable region of monoclonal antibody 5E1-1B7;

[0028] Figure 4 This is the result of sequence alignment of the light chain variable region gene of monoclonal antibody 5E1-1B7;

[0029] Figure 5 This is the result of amino acid sequence alignment of the light chain variable region of monoclonal antibody 5E1-1B7;

[0030] Figure 6 This is a schematic diagram of the interpretation of colloidal gold test strip results. DETAILED DESCRIPTION

[0031] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1 Synthesis of Tetracycline Artificial Antigen

[0033] 1. Preparation of tetracycline immune antigen

[0034] S1. Prepare 0.2M acetate buffer at pH 5.5, weigh 550 mg BSA and dissolve it in 80 mL acetate buffer solution (0.2M, pH 5.5), and place it in an ice water bath for later use;

[0035] S2. Weigh 100 mg of tetracycline hydrochloride and dissolve it in 20 mL of dimethyl sulfoxide (DMSO). Keep it away from light and place it in an ice water bath to cool it down for later use.

[0036] S3, keeping the solution in step S2 in an ice-water bath, slowly add dropwise to the solution in step S1, and mix thoroughly;

[0037] S4. Use a pipette to slowly add 800uL of 25% formaldehyde aqueous solution dropwise;

[0038] S5. Place the reaction solution in a 4°C environment and slowly stir the reaction for 16 hours. After the reaction is completed, let it stand for 3 days and concentrate it by ultrafiltration to obtain tetracycline immune antigen (TC-BSA).

[0039] 2. Preparation of Tetracycline Detection Antigen

[0040] S1. Prepare 0.2 M acetate buffer solution at pH 5.5, weigh 550 mg OVA and dissolve it in 80 mL acetate buffer solution (0.2 M, pH 5.5), and place it in an ice water bath for later use;

[0041] S2. Weigh 100 mg of tetracycline hydrochloride and dissolve it in 20 mL of dimethyl sulfoxide (DMSO). Keep it away from light and place it in an ice water bath to cool it down for later use.

[0042] S3, keeping the solution in step S2 in an ice-water bath, slowly add dropwise to the solution in step S1, and mix thoroughly;

[0043] S4. Use a pipette to slowly add 800uL of 25% formaldehyde aqueous solution dropwise;

[0044] S5. Place the reaction solution in a 4°C environment and slowly stir the reaction for 16 hours. After the reaction is completed, let it stand for 3 days and concentrate it by ultrafiltration to obtain the tetracycline detection antigen (TC-OVA).

[0045] Example 2 Preparation of Tetracycline Monoclonal Antibody

[0046] 1. Mouse immunization

[0047] Three female Balb / c mice aged 6-8 weeks were taken, and each was immunized with 20ug of TC-BSA artificial antigen. During the first immunization, the TC-BSA artificial antigen was emulsified with Freund's complete adjuvant in equal volumes and injected subcutaneously at multiple points to immunize the mice. Immunization was performed once every two weeks, for a total of three immunizations. The antigen was emulsified with Freund's incomplete adjuvant for the second and third immunizations, and the immunization dose and method remained unchanged. One week after the third immunization, the tail vein of the mice was bled, and the serum was taken and its titer and inhibition were measured by indirect ELISA method. The results are shown in Table 1. After immunization, the serum titer of mouse 3# was the highest, reaching 1:3.2×10 4 The inhibition rate can reach up to 50.10%. Take 40ug of TC-BSA artificial antigen and dilute it to 200uL with 1×PBS, and inject it intraperitoneally to boost the immunity of mice. Cell fusion can be performed three days later.

[0048] Table 1. Serum titer and inhibition test of immune mice

[0049]

[0050] 2. Cultivation of SP2 / 0 Myeloma Cells

[0051] Take out a piece of SP2 / 0 myeloma cells frozen in liquid nitrogen and immediately transfer it to a 37℃ constant temperature water bath. Gently shake the cryotube from time to time and take it out when the cells melt to a semi-ice crystal state. Operate in a sterile environment, transfer the SP2 / 0 cells in the cryotube into a 50 mL sterile centrifuge tube, take 10 mL of preheated 1640 complete medium and slowly drop it into the centrifuge tube, centrifuge at 1000 r / min for 5 min, and discard the supernatant. Gently break up the cell clusters, take 5 mL of medium to resuspend the cells and transfer them to a T75 cell culture flask. Add another 5 mL of culture medium, shake the cell bottle in the "cross" direction, put it in a CO2 cell culture incubator, and culture it at 37℃. Observe the cell state under a microscope, and subculture the SP2 / 0 cells when the density is about 80%.

[0052] 3. Cell fusion

[0053] (1) Blood was collected from the orbits of the experimental mice after booster immunization and collected in EP tubes. After standing at 37°C for 2 hours, the blood was centrifuged at 4000 rpm for 10 minutes. The serum was collected for subsequent screening of monoclonal antibodies as a positive control. The mice were killed by dislocating the neck and then immersed in 75% alcohol for disinfection.

[0054] (2) Preparation of spleen cells: Use sterilized scissors and tweezers to cut open the mouse skin in a biosafety cabinet, replace a new set of sterilized scissors and tweezers to cut open the mouse abdominal cavity, and then use a new set of sterilized scissors and tweezers to carefully remove the spleen and trim off excess fat. Prepare a sterile 15mL centrifuge tube, add 10mL DMEM culture medium, place the spleen in the centrifuge tube, and carefully discard the excess culture medium after the spleen is moistened. Pipette another 10 mL of DMEM medium into a sterile dish, grind the spleen with a frosted glass slide to make a single-cell suspension and filter it through a 200-mesh nylon mesh into a sterile centrifuge tube, add 30 mL of DMEM into 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 30 mL of DMEM medium to resuspend, and centrifuge again, then discard the supernatant, gently break up the cell clumps by hand, and add 10 mL of DMEM medium to resuspend.

[0055] (3) Cell fusion: 1000rpm, 5min centrifugation to collect well-growing SP2 / 0 cells in a 50 mL centrifuge tube, gently break up the SP20 cell clusters, add 30mL DMEM medium to resuspend, centrifuge once more, add 10mL DMEM medium to resuspend, then mix the spleen cell suspension with the SP2 / 0 cell suspension, centrifuge at 1000rpm for 5min, discard the supernatant, and gently break up the cell clusters. Place in a 37℃ water bath environment, draw 1mL PEG fusion agent and drop it into the centrifuge tube, add 1ml within 1min, at this time the cell state is red homogenous quicksand, and the tube wall feels like frosted glass when rotating.

[0056] (4) Termination of fusion: 9 mL of preheated DMEM medium was aspirated to terminate fusion, which was divided into three stages. The first stage was to add 1 mL within 1 minute, the second stage was to add 1 mL within 1 minute, and the third stage was to add the remaining 7 mL of medium within 3 minutes. Then, the cells were placed in a 37°C water bath for 5 minutes and centrifuged at 800 rpm for 5 minutes.

[0057] (5) Plating: Discard the supernatant, gently break up the cell clumps, add HAT medium (for example, if you want to plate 5 96-well plates, add 200uL / well, excluding the 100uL / well feeder layer cells that have been plated in advance, then add 50mL of HAT medium). After mixing the cells, evenly plate the fused cell suspension in the 96-well cell plate that has been added with the feeder layer cells, 100uL / well, and culture in a CO2 cell culture incubator at 37°C.

[0058] 4. Screening of positive hybridoma cells

[0059] 7 days after cell fusion, when the cell clusters are relatively large, the cell supernatant is detected by indirect ELISA. 1μg / mL TC-OVA artificial antigen is used as the antigen for detection, the positive control is the serum of fused mice, and the negative control is the serum of PBS-immunized mice. The hybridoma cell wells with the strongest color reaction are selected as positive wells. The screened positive hybridoma cells are subcloned by limiting dilution. The hybridoma cell lines that can stably secrete monoclonal antibodies identified after subcloning are expanded and transferred to T75 cell flasks. When the cell number is about 80%, they can be collected to prepare ascites.

[0060] 5. Ascites Preparation

[0061] Add 10 mL of sterile 1×PBS to the cell bottle, blow off the cell layer, resuspend and transfer it to a 15 mL centrifuge tube, and centrifuge at 1000 r / min for 10 min. Discard the supernatant, take another 1 mL of sterile 1×PBS to resuspend the precipitate and mix it well, and draw it up with a 1 mL syringe. Inject about 500 μL of cell suspension into each mouse, pay attention to the growth status of the mouse, and collect ascites when the mouse abdomen swells after one week. Collect the mouse ascites in a centrifuge tube, centrifuge at 8000 r / min for 20 min, and draw up the middle ascites layer.

[0062] 6. Purification of monoclonal antibodies

[0063] The collected ascites was purified. The purity of the purified monoclonal antibody was determined by SDS-PAGE, and the purity was about 95%. Figure 1 .

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

[0065] The sensitivity and specificity of tetracycline monoclonal antibodies were detected by indirect ELISA. TC-OVA artificial antigen was coated at 1 μg / mL, and 1 mg / mL monoclonal antibody 5E1-1B7 was diluted in gradients to verify the sensitivity of the monoclonal antibody. The results are shown in Table 2. The sensitivity of monoclonal antibody 5E1-1B7 can reach 1:40000 dilution, and the inhibition rate can reach up to 71.48%. Fluoroquinolone artificial antigen, flunixin artificial antigen and sulfonamide artificial antigen were coated at 1 μg / mL, and 1 mg / mL monoclonal antibody 5E1-1B7 was diluted 1:5000 to verify the specificity of the monoclonal antibody. The results are shown in Table 3. The purified monoclonal antibody had no cross reaction with quinolone artificial antigen, flunixin artificial antigen and sulfonamide artificial antigen, indicating that the purified monoclonal antibody has good specificity.

[0066] Table 2 Monoclonal antibody sensitivity verification

[0067]

[0068] Table 3 Monoclonal antibody specificity verification

[0069]

[0070] Example 4. Cloning of variable region genes of tetracycline monoclonal antibody

[0071] 1. Hybridoma cell culture and total RNA extraction

[0072] Hybridoma cells 5E1-1B7 were cultured with RPMI 1640 complete medium at 37°C and 5% carbon dioxide. When the cell number reached 1×107, total RNA was extracted from the cells using a total RNA extraction kit (purchased from Tiangen).

[0073] 2. Synthesis of the first strand of cDNA

[0074] The total RNA extracted in step 1 was used as a template for amplification to synthesize the first-strand cDNA using a transcription kit (purchased from TAKARA).

[0075] 3. Gene amplification

[0076] Design downstream primers and upstream universal primers for Lambda chain, Kappa chain, Heavy chain.

[0077] Primer: F (SEQ ID No. 11): AAGCAGTGGTATCAACGCAGA

[0078] Rκ (SEQ ID No. 12):AACATTGATGTCTTTGGGGTAGAA

[0079] Rλ (SEQ ID No.13):AATCGTACACACCAGTGTGTGGG

[0080] RH (SEQ ID No.14):AGGGATCCAGAGTTCCAGGT

[0081] PCR amplification was performed using the first strand of cDNA as a template, and the reaction system was 50uL, including 3uL of template, 2.5uL of upstream primer (10uM), 2.5uL of downstream primer (10uM), 25uL of 2×Taq enzyme, and 17uL of sterile water.

[0082] The touchdown PCR reaction conditions were as follows: 98°C for 30 s; 98°C for 15 s, 64°C-58°C for 30 s, decreasing by 0.5°C each time until 58°C, for 10 cycles; 72°C for 30 s; 98°C for 15 s, 56°C for 30 s, 72°C for 30 s, for 15 cycles; the program was terminated at 72°C for 7 min.

[0083] 4. Cloning and screening of PCR amplification products

[0084] The PCR products were subjected to 1% agarose gel electrophoresis, and the Kappa chain, Lambda chain and Heavy chain amplified 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), transformed into DH5α competent cells (ampicillin resistance), and the recombinant positive clones were screened and sequenced.

[0085] 5. The variable region gene sequence and amino acid sequence of the tetracycline monoclonal antibody of this example are as follows:

[0086] (1) Heavy chain variable region gene sequence (SEQ ID No. 3):

[0087] ATGTACCGCATGCAGCTGCTGTCTTGCATCGCCCTGTCTCTGGCTCTGGTGACCAATAGCGAGGTGCAGCTGGTGGAAAGCGGAGGAGATCTGGTGAAGCCAGGCGGATCTCTGAAGCTGTCTTGCCGCCAGCGGCTTTACATTCAGCAACTACGCCATGTCTTGGATCCGGCAGACCCCAGAGAAGAGACTCGAGTGGGTGGCCACAAT CAGCAGCGGCGGCAGCTACACCTACTACCCAGGCAGCGTGAAGGGCAGATTCACCATCAGCAGGGACAACGCCCAGAACACCCTGTACCTGCAGATGAGCAGCCTGAGAAGCGAGGACACCGCCATGTACTATTGCGCCAGGCACGGAAAGAGCCTGAGCACAAGCGAGGGCACACCATTCGCCTATTGGGGACAGGGAACCCTCGTGACA.

[0088] (2) Heavy chain variable region amino acid sequence (SEQ ID No. 1):

[0089] MQLLSCIALSLALVTNSEVQLVESGGDLVKPGGSLKLSCAASGFTFSNYAMSWIRQTPEKRLEWVATISSGGSYTYYPGSVKGRFTISRDNAQNTLYLQMSSLRSEDTAMYYCARHGKSLSTSEGTPFAYWGQGTLVTVSA.

[0090] (3) Kappa chain variable region gene sequence (SEQ ID No. 4):

[0091] ATGTACCGCATGCAGCTGCTGTCTTGCATCGCTCTGTCTCTGGCCCTGGTGACCAATAGCGAGACCACAGTGACCCAGTCTCCAGCCTTCTGAGCATGGCTATCGGCGAGAAGGTCACCATCCGCTGCATCACCAGCACCGACATCAACGACGAGATGAATTGGTTCCAGCAGAAGCCCGGCGAGCCTCC TAAAGTGCTGATCAGCGAGGGCAATATCGTGAGACCCGGAGTGCCTAGCAGATTCAGCTCTAGCGGCTACGGCACCGACTTCGTCTTCACCATCGAGAACATGCTGAGCGAGGACGTGGCCGACTACTATTGCCTGCAGAGCGACAACCTGCCTCTGACATTTGGCGCCGGCACCAAGCTGGAGCTGAAA.

[0092] (4) Kappa chain variable region amino acid sequence (SEQ ID No. 2)

[0093] MYRMQLLSCIALSLALVTNSETTVTQSPASLSMAIGEKVTIRCITSTDINDEMNWFQQKPGEPPKVLISEGNIVRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGAGTKLELK.

[0094] 6. Variable region amino acid sequence and homology analysis

[0095] The heavy chain and light chain gene sequences were compared and analyzed in the NCBI database. The analysis results showed that the heavy chain variable region gene sequence of monoclonal antibody 5E1-1B7 had the highest homology with the mouse immunoglobulin heavy chain variable region (Sequence ID: AF144898.1), with a homology of 168 / 206 and a homology percentage of 82%. Figure 2 The amino acid sequence of the heavy chain variable region of monoclonal antibody 5E1-1B7 has the highest homology with the amino acid sequence of the heavy chain variable region of mouse immunoglobulin (Sequence ID: QKW93014.1), with a homology of 108 / 124 and a homology percentage of 87%. The results are shown in Figure 3The gene sequence of the light chain variable region of monoclonal antibody 5E1-1B7 has the highest homology with the mouse immunoglobulin light chain variable region (Sequence ID: M55318.1), with a homology of 236 / 321 and a homology percentage of 74%. The results are shown in Figure 4 The amino acid sequence of the light chain variable region of monoclonal antibody 5E1-1B7 has the highest homology with the amino acid sequence of the light chain variable region of mouse immunoglobulin (Sequence ID: pir|PT0357|), with a homology of 101 / 110 and a homology percentage of 92%. The results are shown in Figure 5 .

[0096] 7. CDR region analysis

[0097] The amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody 5E1-1B7 were analyzed at https: / / www.novopro.cn / tools / cdr.html to obtain its CDR region.

[0098] Antibody heavy chain CDR region:

[0099] CDR-H1 (SEQ ID No.5): NYAMS

[0100] CDR-H2 (SEQ ID No.6): TISSGGSYTYYPGSVKG

[0101] CDR-H3 (SEQ ID No. 7): HGKSLSTSSEGTPFAY

[0102] Antibody light chain CDR region:

[0103] CDR-L1 (SEQ ID No.8): ITSTDINDEMN

[0104] CDR-L2 (SEQ ID No.9):EGNIVRP

[0105] CDR-L3 (SEQ ID No.10): LQSDNLPLT

[0106] Example 5. Preparation of Tetracycline Colloidal Gold Test Strips

[0107] 1. Preparation of chloroauric acid

[0108] Weigh 800mL of ultrapure water into a 1000mL conical flask, add 8mL of 1% chloroauric acid to the ultrapure water, place on a magnetic heating rod stirrer to mix and heat, temperature 500℃, speed 1. After the water boils, increase the speed to 6, quickly add 1mL of 1.2% reducing agent after the liquid is stirred, and adjust the speed to 5. Stop heating after heating for 6 minutes and 30 seconds, and let it cool to room temperature. The prepared colloidal gold has a pure, transparent appearance, without precipitation and floating objects.

[0109] 2. Preparation of tetracycline monoclonal antibody-colloidal gold label

[0110] (1) Add bare gold: Mix colloidal gold and bare gold and take 1L of colloidal gold solution;

[0111] (2) Add K2CO3: Add 0.2M K2CO3 at a ratio of 5uL / mL colloidal gold and vortex to mix;

[0112] (3) Adding antibodies: Mix 1 mL of 1 mg / mL tetracycline 5E1-1B7 antibody with 4 mL of 10% BSA (IgG), then add the mixed antibody dropwise to the gold using a 1 mL pipette and equilibrate for 5 min.

[0113] (4) Smooth mixing: Reduce the speed until the vortex drops by about 1 cm, and then balance for 1 hour;

[0114] (5) Add BSA: Increase the speed until the vortex drops by about 3 cm, pour in 10 mL of 10% BSA (Xibao), balance for 3 min, reduce the speed to stabilize until the vortex drops by about 1 cm, and balance for another 30 min;

[0115] (6) Add PEG: Increase the rotation speed until the vortex drops by about 3 cm, pour in 10 mL of 10% PEG 20000, and equilibrate for 5 min;

[0116] (7) Centrifugation: Use a desktop high-speed centrifuge, place two centrifuge tubes at opposite corners of the rotor, 12000 rpm / min, 8 min, 4°C;

[0117] (8) Discard the supernatant: After centrifugation, gently remove the centrifuge bucket and place it on the laboratory table of the negative pressure aspiration device. Do not shake or bump it to avoid disturbing the gold precipitate in the centrifuge bucket; stop aspirating when it is close to the bottom of the centrifuge bucket (about 5-6 mL remaining), and use a 1 mL pipette to carefully remove the remaining supernatant to avoid aspirating the gold precipitate;

[0118] (9) Resuspension: Stop aspirating when the centrifuge bucket is close to the bottom (about 2-3 mL remaining), gently shake the centrifuge bucket to resuspend, and transfer the resuspended gold precipitate to a blue-capped bottle;

[0119] (10) Rinse the bucket: Add about 20 mL of rehydration solution to the centrifuge bucket, shake gently to wash away the remaining gold precipitate, transfer the liquid to the other three centrifuge buckets in turn, and finally transfer it to the blue-capped bottle, and repeat once;

[0120] (11) Make up the volume: Finally, use a blue-capped bottle to make up the volume to 1 / 10 of the gold solution, i.e. 100 mL. Mix well and set aside.

[0121] 3. Preparation of sample absorption pad

[0122] The sample absorption pad was immersed in phosphate buffer containing 0.5% BSA, pH=7.2, 0.1 mol / L for 2 h, and dried at 37°C for 2 h to obtain the sample absorption pad.

[0123] 4. Preparation of nitrocellulose membrane

[0124] Tetracycline artificial antigen was diluted to 10 mg / mL with phosphate buffer and coated on the detection line T on the nitrocellulose membrane with a biodot film spotter, with a coating amount of 1.0 μg / cm. Sheep anti-mouse IgG antibody was diluted to 200 μg / mL with 0.01 mol, pH 7.2 PBS buffer and coated on the quality control line C on the nitrocellulose membrane with a biodot film spotter. The coated reaction membrane was dried at 37°C for 2 h to obtain a nitrocellulose membrane.

[0125] 5. Assembly of Tetracycline Colloidal Gold Test Strips

[0126] The sample absorption pad, colloidal gold binding pad, nitrocellulose membrane, and absorption pad are sequentially pasted on the bottom plate, the end of the sample absorption pad is connected to the beginning of the colloidal gold binding pad, the end of the colloidal gold binding pad is connected to the beginning of the nitrocellulose membrane, the end of the nitrocellulose membrane is connected to the beginning of the absorption pad, the beginning of the sample absorption pad is aligned with the beginning of the bottom plate, and the end of the absorption pad is aligned with the end of the bottom plate to assemble a colloidal gold test strip.

[0127] Example 6: Application of Tetracycline Colloidal Gold Test Strips

[0128] 1. Test strip detection

[0129] The required test strips and sample diluents are returned to room temperature (20-25°C). Use a micropipette to draw 200µL of the sample solution to be tested into the microwell, slowly aspirate and fully mix with the reagent in the microwell. Insert the marked test strip into the microwell so that it is fully immersed in the solution. After incubating at room temperature for 5 minutes, remove the test strip and follow the schematic diagram ( Figure 6 ) judgment result, and the judgment at other times is invalid.

[0130] 2. Interpretation of test results

[0131] Negative (-): Both the C line and the T line are colored and the color of the T line is stronger than that of the C line, indicating that the tetracycline concentration in the sample is lower than the detection limit.

[0132] Positive (+): The C line is colored, and the T line is the same color as the C line, the T line is weaker than the C line, or the T line is not colored, indicating that the tetracycline concentration in the sample is equal to or higher than the detection limit.

[0133] Invalid: No C line appears, indicating incorrect operation or the test strip has deteriorated and become invalid. In this case, you should read the instructions carefully again and retest with a new test strip.

[0134] If the test strip needs to be archived, the sponge pad at the bottom must be cut off immediately after reading, and the strip must be dried and archived.

[0135] In addition to naked eye interpretation, NBReader can be used for result interpretation.

[0136] 3. Sensitivity test of tetracycline colloidal gold test strips

[0137] The concentration of the tetracycline standard was diluted to 2 ppb and 4 ppb, and the milk sample was spiked and tested according to the test strip detection method to verify the minimum detection limit of the product. The results are shown in Table 4. The test strip provided by the present invention has a detection sensitivity of 4 ppb for the tetracycline standard in milk.

[0138] Table 4 Sensitivity of tetracycline colloidal gold test strips

[0139]

[0140] 4. Specificity detection of tetracycline colloidal gold test strips

[0141] According to the detection method, 400 ppb of fluoroquinolone, flunixin and sulfonamide were tested, and the results are shown in Table 5. The results were all negative, indicating that the test strip provided by the present invention did not cross-react with other antibiotic drugs and had good specificity.

[0142] Table 5 Specificity of tetracycline colloidal gold test strips

[0143]

[0144] 5. Stability test of tetracycline colloidal gold test strips

[0145] The prepared test strips were placed in 4°C and 37°C environments for accelerated testing, and the tetracycline standard (4 ppb) was detected at 0 d, 7 d, 14 d, and 28 d, respectively. The error analysis of the measured concentration and the actual sample concentration was performed, and the results are shown in Table 6. The CVs were all <10%, indicating that the test strips provided by the present invention have good stability.

[0146] Table 6 Stability of tetracycline colloidal gold test strips

[0147]

[0148] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A tetracycline 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; The heavy chain variable region and the light chain variable region are both composed of complementarity determining regions and framework regions, and the complementarity determining regions are both composed of CDR1, CDR2 and CDR3; The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5; The amino acid sequence of CDR2 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.6; The amino acid sequence of CDR3 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.7; The amino acid sequence of CDR1 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.8; The amino acid sequence of CDR2 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.9; The amino acid sequence of CDR3 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

10.

2. The tetracycline 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. Use of the tetracycline monoclonal antibody according to claim 1 in the preparation of tetracycline detection products.

4. A tetracycline test strip, characterized in that: The test strip is a colloidal gold test strip, and the tetracycline monoclonal antibody according to claim 1 or 2 is coated on the conjugate pad.

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