Heavy and light chain variable regions of a t-2 toxin monoclonal antibody and uses thereof

Abstract

This invention discloses a T-2 toxin monoclonal antibody and its applications, 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 complementarity-determining region (CDR) and a framework region. The CDR is composed of CDR1, CDR2, and CDR3. The monoclonal antibody can be used to prepare T-2 toxin detection products. The T-2 toxin colloidal gold test strip provided by this invention features 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 T-2 toxin monoclonal antibody and their applications. Background Technology

[0002] T-2 toxin is a type A trichothecene mycotoxin produced by various Fusarium species, with the molecular formula C2. 24 H 34 O9. It is one of the most dangerous compounds among natural toxins, known for its strong cytotoxicity and ability to inhibit protein synthesis. T-2 toxin exhibits extremely high chemical and thermal stability, resisting high temperatures: ordinary cooking temperatures (such as boiling and baking) cannot decompose it; it only begins to slowly degrade at temperatures above 200°C. It is also resistant to acids and alkalis: very stable in weakly acidic and neutral environments, only being destroyed under strongly alkaline conditions. This exceptional stability means that once food is contaminated, it is very difficult to remove during processing, making it a persistent threat to food safety. T-2 toxin is extremely toxic and can cause serious harm to humans and animals through ingestion, inhalation, and skin contact. Its primary function is to strongly inhibit protein synthesis in eukaryotic cells. It inhibits protein synthesis by binding to the 60S subunit of the ribosome, inhibiting the activity of peptidyl transferase. Inhibition of protein synthesis triggers a cellular stress response, leading to apoptosis in rapidly proliferating cells (such as skin, mucous membranes, immune cells, and hematopoietic cells). It severely damages the immune system, inhibiting antibody production and white blood cell proliferation, making the body more susceptible to infection. It can damage vascular endothelial cells, increase the fragility of blood vessel walls, and lead to bleeding in tissues and organs.

[0003] The core issue of T-2 toxin is "mold contamination," which primarily occurs during crop planting, harvesting, storage, and feed ingredient storage. Grain raw materials used in feed, such as corn, wheat, wheat bran, and soybean meal, can naturally produce T-2 toxin if contaminated with toxin-producing Fusarium in the field or if they become moldy during storage due to improper humidity and temperature control. Using contaminated raw materials during feed production or poor management of finished feed during storage can also lead to toxin accumulation. Therefore, establishing a safe and efficient biological detection method for T-2 toxin is imperative.

[0004] Immunoassay methods offer advantages such as low cost, high efficiency, high sensitivity, and relatively low skill requirements, making them suitable for rapid detection of large numbers of samples. The purpose of this invention is to provide a monoclonal antibody with extremely high affinity and specificity for T-2 toxin, laying the foundation for establishing a colloidal gold test strip detection method and developing and promoting colloidal gold test strip products. Summary of the Invention

[0005] Therefore, the present invention provides a heavy chain and light chain variable region of a T-2 toxin monoclonal antibody and its application.

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

[0007] In a first aspect, the present invention provides a T-2 toxin monoclonal antibody, characterized in that: the monoclonal antibody comprises a heavy chain and a light chain;

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

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

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

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

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

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

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

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

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

[0017] Secondly, the present invention provides the T-2 toxin monoclonal antibody described above, characterized in that:

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

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

[0020] Thirdly, the present invention provides the application of a T-2 toxin monoclonal antibody in the preparation of a detection product for detecting T-2 toxin.

[0021] Preferably, the test strip is a colloidal gold test strip;

[0022] The present invention has the following advantages:

[0023] This invention provides a T-2 toxin monoclonal antibody and its applications. The prepared monoclonal antibody exhibits high specificity, high sensitivity, and good stability, 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 the heavy and light chain variable regions of the T-2 toxin monoclonal antibody and their detection applications, laying the foundation for the research and development and promotion of ELISA, colloidal gold test strips, and fluorescent immunoassay strips. Attached Figure Description

[0024] 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.

[0025] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

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

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

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

[0029] Example 1: Synthesis of T-2 toxin artificial antigen

[0030] 1. Preparation of T-2 toxin immunogen

[0031] (1) T-2 toxin solution: Weigh 2.0 mg of T-2 toxin hydrochloride, dissolve it in 0.2 mL of anhydrous DMF to prepare a stock solution of 10 mg / mL, and vortex to completely dissolve it.

[0032] (2) Protein solution: Weigh 10 mg BSA and dissolve it in 1.0 mL of coupling buffer (such as 0.1 M MES, pH 6.0) to ensure complete dissolution.

[0033] (3) EDC / NHS solution: Weigh 6.0 mg EDC and 3.6 mg NHS and dissolve them together in 0.1 mL of coupling buffer.

[0034] (4) Add 200 μL of T-2 toxin solution (containing 2 mg of T-2 toxin) to a clean glass vial, slowly add 100 μL of freshly prepared EDC / NHS mixture dropwise, gently vortex mix, and react at room temperature in the dark for 1-2 hours.

[0035] (5) Slowly and dropwise add the activated T-2 toxin reaction solution to 1 mL of protein solution (containing 10 mg BSA) while continuously and gently stirring with a magnetic force to ensure uniform mixing. After the addition is complete, stir the reaction mixture slowly at room temperature in the dark for 4-6 hours.

[0036] (6) Transfer the reaction mixture into a dialysis bag and dialyze to purify it, thus obtaining T-2 toxin immunogen (T-2-BSA).

[0037] 2. Preparation of T-2 toxin detection antigen

[0038] (1) T-2 toxin solution: Weigh 2.0 mg of T-2 toxin hydrochloride, dissolve it in 0.2 mL of anhydrous DMF to prepare a stock solution of 10 mg / mL, and vortex to completely dissolve it.

[0039] (2) Protein solution: Weigh 10 mg OVA and dissolve it in 1.0 mL of coupling buffer (such as 0.1 M MES, pH 6.0) to ensure complete dissolution.

[0040] (3) EDC / NHS solution: Weigh 6.0 mg EDC and 3.6 mg NHS and dissolve them together in 0.1 mL of coupling buffer.

[0041] (4) Add 200 μL of T-2 toxin solution (containing 2 mg of T-2 toxin) to a clean glass vial, slowly add 100 μL of freshly prepared EDC / NHS mixture dropwise, gently vortex mix, and react at room temperature in the dark for 1-2 hours.

[0042] (5) Slowly and dropwise add the activated T-2 toxin reaction solution to 1 mL of protein solution (containing 10 mg BSA) while continuously and gently stirring with a magnetic force to ensure uniform mixing. After the addition is complete, stir the reaction mixture slowly at room temperature in the dark for 4-6 hours.

[0043] (6) Transfer the reaction mixture into a dialysis bag and dialyze to purify it, thus obtaining the T-2 toxin detection antigen (T-2-OVA).

[0044] Example 2: Preparation of T-2 toxin monoclonal antibody

[0045] 1. Mouse immunization

[0046] Three 6-8 week old female Balb / c mice were immunized with 25 μg of T-2-BSA artificial antigen each. For the first immunization, the T-2-BSA artificial antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. Immunizations were repeated every two weeks for a total of three immunizations. For the second and third immunizations, Freund's incomplete adjuvant was used for antigen emulsification, and the dosage and method of immunization remained unchanged. One week after the third immunization, blood was collected from the tail vein of the mice, and serum was analyzed using an indirect ELISA method to determine its titer and inhibition. The results are shown in Table 1. The serum titer of mouse #1 was the highest after immunization, reaching 1:3.2×10⁻⁶. 4 The inhibition rate can reach up to 62.70%. Take 50 μg of T-2-BSA artificial antigen, dilute it with 1×PBS to 200 μL, and inject it intraperitoneally to boost the immunization of mice. Cell fusion can be performed three days later.

[0047] Table 1 Serum titers and inhibition detection in immunized mice

[0048]

[0049] 2. Culture of SP2 / 0 myeloma cells

[0050] One vial of SP2 / 0 myeloma cells, frozen in liquid nitrogen, was immediately transferred to a 37°C water bath. The cryovial was gently agitated periodically until the cells reached a semi-ice crystal state. Under sterile conditions, the SP2 / 0 cells were transferred to a 50 mL sterile centrifuge tube. 10 mL of preheated RPMI 1640 complete culture medium was slowly added dropwise to the centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell clumps were gently dispersed, and the cells were resuspended in 5 mL of culture medium and transferred to a T75 cell culture flask. An additional 5 mL of culture medium was added, and the flask was agitated in a cross-hatching motion before being placed in a CO2 cell culture incubator at 37°C. The cell condition was observed under a microscope. When the cell density reached approximately 80%, the SP2 / 0 cells were passaged.

[0051] 3. Cell fusion

[0052] (1) Blood was collected from the orbital cavity of mice after booster immunization and placed in EP tubes. After standing at 37°C for 2 hours, the tubes were centrifuged at 4000 r / min for 10 minutes. The serum was collected as a positive control for subsequent screening of monoclonal antibodies. The mice were euthanized by cervical dislocation and then disinfected by soaking in 75% alcohol.

[0053] (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 r / min for 5 minutes. Discard the supernatant. Gently break up the cell clumps by hand. Resuspend the cells in 30 mL of DMEM culture medium and centrifuge again. Discard the supernatant, gently break up the cell clumps by hand, and resuspend the cells in 10 mL of DMEM culture medium.

[0054] (3) Cell fusion: Centrifuge at 1000 r / min for 5 minutes to collect well-grown SP2 / 0 cells 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 r / min for 5 minutes, 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 minute. At this time, the cells are red, homogeneous, and quicksand-like, and rotating the tube wall feels like frosted glass.

[0055] (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 minute, the second stage is to add 1 mL in the first minute, and the third stage is to add the remaining 7 mL of medium in the first 3 minutes. Then, let it stand in a 37°C water bath for 5 minutes to stabilize, and then centrifuge at 800 r / min for 5 minutes.

[0056] (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.

[0057] 4. Screening of positive hybridoma cells

[0058] Seven days after cell fusion, when the cell clusters were relatively large, the cell supernatant was analyzed using an indirect ELISA method. The T-2-OVA artificial antigen (1 μg / mL) was used as the detection antigen; the positive control was serum from fused mice, and the negative control was serum from mice immunized with PBS. Wells with the strongest chromogenic reaction were selected as positive wells. The selected positive hybridoma cells were subcloned using a limiting dilution method. The hybridoma cell lines that stably secreted monoclonal antibodies, identified after subcloning, were expanded and cultured in T75 cell flasks. When the cell count reached approximately 80%, the cells were collected for ascites preparation.

[0059] 5. Preparation of ascites

[0060] Add 10 mL of sterile 1×PBS to the cell culture flask, blow off the cell layer, resuspend it, and transfer it to a 15 mL centrifuge tube. Centrifuge at 1000 r / min for 10 minutes. Discard the supernatant, resuspend the precipitate in 1 mL of sterile 1×PBS, mix well, and aspirate using a 1 mL syringe. Inject approximately 500 μL of the cell suspension into each mouse, monitoring the mouse's growth. Collect ascites fluid one week later when the mouse's abdomen is swollen. Collect the mouse ascites fluid into a centrifuge tube, centrifuge at 8000 r / min for 20 minutes, aspirate the middle ascites layer, and purify the collected ascites fluid.

[0061] 6. Purification of monoclonal antibodies

[0062] The collected ascites fluid was purified. SDS-PAGE was used to assess the purity of the purified monoclonal antibody, which was approximately 94%. (See attached image). Figure 1 .

[0063] Example 3: Specificity and sensitivity detection of T-2 toxin monoclonal antibody

[0064] The sensitivity and specificity of T-2 toxin monoclonal antibodies were detected using an indirect, competitive ELISA method. T-2-OVA artificial antigen was coated at a concentration of 1 μg / mL, and 1 mg / mL of monoclonal antibody 3B5-1F6 was diluted to verify the antibody's sensitivity. The results are shown in Table 2. Monoclonal antibody 3B5-1F6 showed a sensitivity of up to 1:60000 dilution, with an inhibition rate of up to 70.17%. Aflatoxin B1 artificial antigen, aflatoxin M1 artificial antigen, and zearalenone artificial antigen were coated at a concentration of 1 μg / mL, and 1 mg / mL of monoclonal antibody 3B5-1F6 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 aflatoxin B1 artificial antigen, aflatoxin M1 artificial antigen, or zearalenone artificial antigen, indicating good specificity of the purified monoclonal antibody.

[0065] Table 2. Monoclonal antibody sensitivity validation

[0066]

[0067] Table 3. Validation of Monoclonal Antibody Specificity

[0068]

[0069] Example 4: Cloning of the variable region gene of T-2 toxin monoclonal antibody

[0070] 1. Hybridoma cell culture and total RNA extraction

[0071] Hybridoma cells 3B5-1F6 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).

[0072] 2. Synthesis of the first strand of cDNA

[0073] 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.

[0074] 3. Gene amplification

[0075] Design downstream primers and upstream universal primers for the Lambda, Kappa, and Heavy chains.

[0076] Primer: F (SEQ ID No. 11): AAGCGTGGTATCAACGCAGA

[0077] Rκ (SEQ ID No. 12):AACATTGATGTCTTTGGGGTAGAA

[0078] Rλ (SEQ ID No.13):AATCGTACACACCAGTGTGTGGG

[0079] R H (SEQ ID No.14):AGGGATCCAGAGTTCCAGGT

[0080] 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.

[0081] The landing PCR reaction conditions were as follows: 98℃ for 30 seconds; 98℃ for 15 seconds, 64℃-58℃ for 30 seconds, decreasing by 0.5℃ each time until reaching 58℃, for 10 cycles; 72℃ for 30 seconds; 98℃ for 15 seconds, 56℃ for 30 seconds, 72℃ for 30 seconds, for 15 cycles; and the program was terminated at 72℃ for 7 minutes.

[0082] 4. Cloning and screening of PCR amplification products

[0083] 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.

[0084] 5. The T-2 toxin monoclonal antibody gene sequence and amino acid sequence of this embodiment are as follows:

[0085] (1) Heavy chain gene sequence (SEQ ID No. 3):

[0086]

[0087] (2) Heavy chain amino acid sequence (SEQ ID No.1):

[0088] EVKLVESGGGLVKPGGSLSLSCAASGFTFSSYALSCVRQTPEKRLEWVASISNWGRTYYSDSVKGRFTISRDNARNILYLQMSSLTSEDTAMYLCAGFISMAEGYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMNTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLVITLTPKVTCVVVDISKDDPEVQFSWFVDSVEVHTAQTQPREEQFNATFRSVSELPIMHQDWLNGKVFKCRVNSAAFPAPIEKTISKTDKGRPKAPQVYTIPSPKEQMAKDKVSLTCMITDFFPEDITVEWEWNGQPAENPKNTQPIMDTDASYFVYSKLNVQKSNWEVGNTFTCSVLHEGLHNEHTEFSLPHSPGK.

[0089] (3) Light chain gene sequence (SEQ ID No.4):

[0090] GAGATCGTGCTGACCCAGAGCCCCGCCATCGTGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGTACTGGTACCAGCAGAAGCCCGGCAGCAGCCCCAGGCTGTGGATCTACGACACCAGCAACCTGGTGAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCAGGAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTTCAGCGGCTACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGAGGGCCGACGCCGCCCCCACCGTGAGCATCTTCCCCCCCAGCAGCGAGCAGCTGACCAGCGGCGGCGCCAGCGTGGTGTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGATCGACGGCAGCGAGAGGCAGAACGGCGTGCTGAACAGCTGGACCGACCAGGACAGCAAGGACAGCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGAGGCACAACAGCTACACCTGCGAGGCCACCCACAAGACCAGCACCAGCCCCATCGTGAAGAGCTTCAACAGGAACGAGTGC。

[0091] (4)Light chain amino acid sequence (SEQ ID No.2)

[0092] EIVLTQSPAIVSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLWIYDTSNLVSGVPARFSGSRSGTSYSLTISSMEAEDAATYYCQQFSGYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0093] 6. Variable region amino acid sequence and homology analysis

[0094] 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 3B5-1F6 had the highest homology with the synthetic construct HC / T2A / N6 (Sequence ID: OK564404.1), with a homology of 1172 / 1336 (88%). The amino acid sequence of the heavy chain variable region of monoclonal antibody 3B5-1F6 had the highest homology with the amino acid sequence of the mouse immunoglobulin heavy chain variable region (Sequence ID: AAB59665.2), with a homology of 311 / 325 (96%). The light chain variable region gene sequence of monoclonal antibody 3B5-1F6 had the highest homology with the mouse immunoglobulin light chain variable region (Sequence ID: AJ784038.2), with a homology of 293 / 318 (92%). The amino acid sequence of the light chain variable region of monoclonal antibody 3B5-1F6 showed the highest homology with the amino acid sequence of the light chain variable region of mouse immunoglobulin (Sequence ID: AAL04476.1), with a homology of 173 / 183 and a homology percentage of 95%.

[0095] 7. CDR Area Analysis

[0096] The amino acid sequences of the heavy chain variable region and light chain variable region of monoclonal antibody 3B5-1F6 were analyzed at https: / / www.novopro.cn / tools / cdr.html to obtain its CDR region.

[0097] Antibody heavy chain CDR region:

[0098] CDR-H1 (SEQ ID No.5): SYALS

[0099] CDR-H2 (SEQ ID No. 6): SISNWGRTYYSDSVKG

[0100] CDR-H3 (SEQ ID No.7): FISMAEGY

[0101] Antibody light chain CDR region:

[0102] CDR-L1 (SEQ ID No.8): SASSSVSYMY

[0103] CDR-L2 (SEQ ID No.9): DTSNLVS

[0104] CDR-L3 (SEQ ID No. 10): QQFSGYPLT.

[0105] Example 5: Preparation of T-2 toxin colloidal gold test strip

[0106] 1. Preparation of chloroauric acid

[0107] Weigh 800 mL of ultrapure water into a 1000 mL Erlenmeyer flask, add 8 mL of 1% chloroauric acid to the ultrapure water, and place the flask on a magnetic heating rod stirrer to mix and heat at 500℃ and speed setting 1. After the water boils, increase the speed to speed setting 6. Once the liquid is stirring, quickly add 1 mL of 1.2% reducing agent and adjust the speed to speed setting 5. After heating for 6 minutes and 30 seconds, stop heating and allow it to cool to room temperature. The prepared colloidal gold is pure, transparent, and free of precipitates and floating matter.

[0108] 2. Preparation of T-2 toxin monoclonal antibody-colloidal gold label

[0109] (1) Add bare gold: Mix the colloidal bare gold well and take 1L of colloidal gold solution;

[0110] (2) Add K2CO3: Add 0.2M K2CO3 at a ratio of 5μL / mL colloidal gold and vortex mix well;

[0111] (3) Add antibody: Mix 1 mL of 1 mg / mL T-2 toxin 3B5-1F6 antibody with 4 mL of 10% BSA (IgG), and then add the mixed antibody dropwise to the gold using a 1 mL pipette, and equilibrate for 5 minutes;

[0112] (4) Leveling: Reduce the rotation speed to stabilize the vortex at a drop of about 1 cm, and then rebalance for 1 hour;

[0113] (5) Add BSA: Increase the rotation speed until the vortex drops by about 3cm, pour in 10mL of 10% BSA (Cebao), balance for 3 minutes, reduce the rotation speed to stabilize the vortex at about 1cm, and then balance for another 30 minutes;

[0114] (6) Add PEG: Increase the rotation speed until the vortex drops by about 3cm, pour in 10mL of 10% PEG, and let it balance for 5 minutes;

[0115] (7) Centrifugation: Using a benchtop high-speed centrifuge, place the centrifuge tubes in pairs at opposite corners of the rotor, 12000 r / min, 8 minutes, 4℃;

[0116] (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;

[0117] (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;

[0118] (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.

[0119] (11) Volume adjustment: Finally, adjust the volume to 1 / 10 of the gold solution, i.e. 100 mL, with the blue cap bottle, mix well and set aside.

[0120] 3. Preparation of microporous reagents

[0121] (1) Prepare lyophilization buffer: 0.05M phosphate buffer, 0.5% BSA (Science), 3% trehalose;

[0122] (2) Preparation of microwell reagent solution: Prepare according to the ratio of 10 μL / well T-2 toxin colloidal gold and 50 μL / well lyophilized buffer; after preparation, stir and mix for 20 minutes.

[0123] (3) Microwell reagent coating: Dispense 60 μL / well of microwell reagent solution into 96-well plates;

[0124] (4) Drying of micro-well reagents: The coated 96-well microplates are placed in a vacuum freeze dryer with a preset freeze drying program for processing;

[0125] (5) Microporous reagent packaging: After the freeze-drying process is completed, take out the microporous reagent, put on the rubber cap, put it in an aluminum foil bag with desiccant, and store it for later use.

[0126] 4. Preparation of sample absorption pads

[0127] The sample absorption pad was immersed in 0.1 mol / L phosphate buffer containing 0.5% BSA, pH=7.2 for 2 hours and then dried at 37℃ for 2 hours to obtain the sample absorption pad.

[0128] 5. Preparation of nitrocellulose membranes

[0129] The T-2 toxin artificial antigen was diluted to 10 mg / mL with phosphate buffer and coated onto the detection line T of a nitrocellulose membrane using a Biodot coating instrument, with a coating amount of 1.0 μg / cm. Goat anti-mouse IgG antibody was diluted to 200 μg / mL with 0.01 mol / L, pH 7.2 PBS buffer and coated onto the control line C of a nitrocellulose membrane using a Biodot coating instrument. The coated reaction membrane was dried at 37°C for 16 hours to obtain the nitrocellulose membrane.

[0130] 6. Assembly of T-2 toxin colloidal gold test strip

[0131] The sample absorption pad, nitrocellulose membrane, and absorption pad are sequentially attached to the base plate. The end of the sample absorption pad is connected to the beginning of the nitrocellulose membrane, and 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 base plate, and the end of the absorption pad is aligned with the end of the base plate. This assembly forms a colloidal gold test strip. The strip is then cut into strips at a size of 4.50 mm / strip to obtain the colloidal gold test strip.

[0132] Example 6: Application of T-2 Toxin Colloidal Gold Test Strip

[0133] 1. Test strip detection

[0134] The required test strips and sample diluent should be brought to room temperature (20-25°C). Using a micropipette, pipette 200µL of the sample solution into each well, slowly aspirate and thoroughly mix with the reagent in the well. After incubating at room temperature for 5 minutes, insert the labeled test strip into the well, ensuring it is fully immersed in the solution. After incubating at room temperature 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.

[0135] 2. Interpretation of test results

[0136] Negative (﹣): Both C and T lines show color, with the T line showing stronger color than the C line, indicating that the concentration of T-2 toxin in the sample is below the detection limit.

[0137] Positive (+): C line shows color, T line shows the same color as C line, T line shows weaker color than C line, or T line shows no color, indicating that the concentration of T-2 toxin in the sample is equal to or higher than the detection limit.

[0138] Invalid: No C line appears, indicating incorrect operation or that the test strip has deteriorated and become ineffective. In this case, carefully read the instructions again and retest with a new test strip.

[0139] If the test strips need to be archived, the lower sponge pad should be cut off immediately after interpretation, and the strips should be dried before archiving.

[0140] In addition to naked-eye interpretation, NBReader can be used for result interpretation.

[0141] 3. Sensitivity detection of T-2 toxin colloidal gold test strips

[0142] The concentrations of T-2 toxin standard were diluted to 5 ppb and 2.5 ppb, and feed 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 5 ppb for T-2 toxin standard in feed.

[0143] Table 4. Sensitivity of T-2 Toxin Colloidal Gold Test Strips

[0144]

[0145] 4. Specificity detection of T-2 toxin colloidal gold test strip

[0146] The test strips were tested for aflatoxin B1, aflatoxin M1, and zearalenone at a concentration of 1000 ppb according to the detection method. The results are shown in Table 5. All results were negative, indicating that the test strips provided by this invention do not cross-react with other toxins and drugs, and have good specificity.

[0147] Table 5. Specificity of T-2 Toxin Colloidal Gold Test Strips

[0148]

[0149] 5. Stability testing of T-2 toxin colloidal gold test strips

[0150] The prepared test strips were subjected to accelerated testing at 4℃ and 37℃. They were used to detect T-2 toxin standard (5 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 Table 6. The CV values ​​were all <10%, indicating that the test strips provided by this invention have good stability.

[0151] Table 6. Stability of T-2 Toxin Colloidal Gold Test Strips

[0152]

[0153] 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 T-2 toxin monoclonal antibody, characterized in that: The monoclonal antibody comprises a heavy chain and a light chain; The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID No. 1; The amino acid sequence of the light chain 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 T-2 toxin monoclonal antibody according to claim 1, characterized in that: The gene sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID No. 3; The gene sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID No.

4.

3. The application of the T-2 toxin monoclonal antibody according to claim 1 in the preparation of T-2 toxin detection products.

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

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