A uniconazole monoclonal antibody and its application

Abstract

This invention discloses a uniconazole monoclonal antibody and its application, relating to the field of biochemistry. The amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain of the monoclonal antibody are shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively; the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the light chain are shown in SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10, respectively. This invention also provides a gene encoding the above amino acid sequences, as well as uniconazole hapten and uniconazole antigen for preparing the monoclonal antibody; this invention further provides the application of the antibody in the detection of uniconazole. The beneficial effect of this invention is that it discloses a highly sensitive and specific uniconazole monoclonal antibody, exhibiting superior detection sensitivity and specificity for uniconazole.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a uniconazole monoclonal antibody and its applications. Background Technology

[0002] Uniconazole, a triazole compound, is a plant growth regulator with fungicidal, herbicidal, lodging-resistant, drought-resistant, and yield-enhancing effects. It is commonly used in crops such as rice, wheat, soybeans, peanuts, and rapeseed. However, reports indicate that uniconazole has certain side effects on the endocrine system of humans and animals, posing a risk of birth defects and even causing organ mutations. Therefore, the safety of uniconazole use is a major concern. Many countries have established residue limits for uniconazole. For example, Japan stipulates a residue limit of 0.05–0.5 mg / kg for uniconazole in some fruits and vegetables; my country's food safety standard GB2763-2021 specifies a maximum residue limit of 0.1 mg / kg for uniconazole in brown rice, and a maximum residue limit of 0.05 mg / kg for uniconazole in wheat, soybeans, peanuts, and rapeseed, with a daily allowable intake of 0.02 mg / kg.

[0003] Currently, the main methods for detecting uniconazole both domestically and internationally include instrumental analysis methods such as gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. These methods are cumbersome, time-consuming, and labor-intensive, requiring highly skilled personnel and are not suitable for on-site testing. Immunoassay, on the other hand, is a detection method based on the specific binding mechanism between antigens and antibodies. This method has advantages such as rapid operation, high sensitivity, and strong specificity, and has been widely used in the detection of harmful substances such as pesticide residues and mycotoxins. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a uniconazole monoclonal antibody and its application. Specifically, it provides a uniconazole monoclonal antibody, an encoding gene, a uniconazole hapten, a uniconazole antigen, and its application. The uniconazole monoclonal antibody of this invention has high sensitivity and specificity to uniconazole and can be used for the immunological analysis of uniconazole pesticide residues through indirect competition.

[0005] The technical solution of the present invention is as follows:

[0006] A first aspect of the present invention provides a uniconazole monoclonal antibody, the uniconazole monoclonal antibody comprising a heavy chain variable region and a light chain variable region;

[0007] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively.

[0008] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0009] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1.

[0010] Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 2.

[0011] A second aspect of the invention provides a gene encoding the aforementioned uniconazole monoclonal antibody, wherein,

[0012] The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 are shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively;

[0013] The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 are shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively.

[0014] Preferably, the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively;

[0015] A third aspect of the present invention provides a uniconazole hapten for preparing the aforementioned uniconazole monoclonal antibody, the uniconazole hapten having the following structural formula:

[0016] .

[0017] A fourth aspect of the present invention provides a method for preparing a uniconazole hapten, comprising the following steps:

[0018] S1. 1,2,4-1H-triazole was reacted with 5-bromo-3,3-dimethyl-4-oxovalerate, and the mixture was purified to obtain compound 1.

[0019] S2. The compound 1 is reacted with acetic anhydride, potassium carbonate and 4-chlorobenzaldehyde, and purified to obtain the uniconazole hapten;

[0020] Compound 1 has the following structural formula:

[0021] .

[0022] Preferably, the ratio of 1,2,4-1H-triazole to 5-bromo-3,3-dimethyl-4-oxovalerate is 1.5g~2.5g:4mL~6mL;

[0023] The ratio of compound 1, acetic anhydride, potassium carbonate and 4-chlorobenzaldehyde is 0.5g~1.5g: 4mL~6mL: 1.5g~2.5g: 0.6g~1.0g.

[0024] A fifth aspect of the present invention provides a uniconazole antigen for preparing the aforementioned uniconazole monoclonal antibody, the uniconazole antigen having the following structural formula:

[0025] ;

[0026] Wherein, BSA is bovine serum albumin and OVA is ovalbumin.

[0027] The sixth aspect of the present invention provides the use of the described uniconazole monoclonal antibody, the described gene, the described uniconazole hapten, or the described uniconazole antigen in the detection of uniconazole.

[0028] Preferably, the content of uniconazole in the test sample is detected by indirect competitive ELISA using the uniconazole monoclonal antibody.

[0029] This invention has at least one of the following beneficial effects:

[0030] This invention first prepares a uniconazole hapten through chemical synthesis; then, by coupling a carrier protein to the uniconazole hapten, a uniconazole antigen is obtained; finally, uniconazole monoclonal antibodies are prepared by immunizing animals with the uniconazole antigen.

[0031] Experimental results show that the uniconazole monoclonal antibody provided by this invention has high sensitivity and specificity for uniconazole. The uniconazole monoclonal antibody can be used for immunological analysis of uniconazole pesticide residues in an indirect competitive manner, realizing highly sensitive, accurate and rapid detection of uniconazole residues in food, which is of great significance for ensuring food safety. Attached Figure Description

[0032] Figure 1 is a flowchart of the synthesis route of the immunogenic antigen and detection antigen chemically synthesized in Example 1;

[0033] Figure 2 is an SDS-PAGE electrophoresis image of the uniconazole monoclonal antibody in Example 2;

[0034] Figure 3 is a standard curve of indirect competitive ELISA based on uniconazole monoclonal antibody in Example 3. Detailed Implementation

[0035] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0036] Example 1: Preparation of uniconazole artificial antigen

[0037] Add 5 g of potassium carbonate to 20 mL of acetone and stir at room temperature. Then add 2 g of 1,2,4-1H-triazole to the solution and stir for 30 minutes. Add 5 mL of cold 5-bromo-3,3-dimethyl-4-oxovalerate to the mixture and stir at room temperature for 2.5 h. Then add 10 mL of water to terminate the reaction, and then add 70 mL of water to dissolve the potassium carbonate. After removing acetone by rotary evaporation, acidify with hydrochloric acid, and then extract with ethyl acetate (60 mL × 3). Wash the organic layer with saturated brine solution (15 mL × 3) and dry with anhydrous sodium sulfate. Filter and concentrate the product under reduced pressure, and purify by hexane-ethyl acetate (7:3) silica gel column chromatography to obtain compound 1.

[0038] Add 5 mL of acetic anhydride to 1 g of compound 1, then add 2 g of potassium carbonate and 0.8 g of 4-chlorobenzaldehyde and stir at room temperature for 3.5 h. Terminate the reaction by adding 20 mL of water, acidify with hydrochloric acid, and then add 10 mL of ethyl acetate to dissolve the product. Extract the organic layer with ethyl acetate (2 mL × 3), wash with water (20 mL × 3), and dry with anhydrous sodium sulfate. After concentration under reduced pressure, purify by silica gel column chromatography with hexane-ethyl acetate (4:1). Dissolve the purified substance in 10 mL of ethyl acetate and irradiate with 365 nm ultraviolet light for 5 h. Concentrate the reaction solution under reduced pressure to obtain compound 2, i.e., the uniconazole hapten.

[0039] 1 mg EDC and 2 mg NHS were added to 100 μL of 10 mg / mL uniconazole hapten (DMF solution), and the mixture was activated at room temperature for 8 h to obtain the activated hapten. Carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) were prepared with PBS at concentrations of 3.68 mg / mL and 2.5 mg / mL, respectively. 1.5 mL of the carrier protein solution was taken, and the activated hapten was added to each. The mixture was stirred at 4 °C for 12 h, and then dialyzed against PBS for 3 days to obtain the immunogenic antigen uniconazole-BSA and the detection antigen uniconazole-OVA.

[0040] Example 2: Preparation of uniconazole monoclonal antibody

[0041] 1. Animal immunization

[0042] Three 6-8 week old female Balb / c mice were selected. Uniconazole-BSA was mixed with an equal volume of Freund's adjuvant as the immunogen, and the mixture was vortexed until the immunogen was completely emulsified. The mice were then immunized via multiple subcutaneous injections into the abdomen. The initial immunization used Freund's complete adjuvant at a dose of 120 μg / mouse. Subsequent immunizations used Freund's incomplete adjuvant, with the immunogen dose decreasing each time, immunized every 3 weeks for a total of 3 immunizations. One week after the second immunization, tail blood was collected from the mice, and the serum antibody titer and inhibition rate were measured using an indirect ELISA method. Three days before fusion, a booster immunization was administered directly to the mice with the highest titer at a dose of 40 μg / mouse.

[0043] 2. Preparation and screening of hybridoma cells

[0044] Selected immunized mice were euthanized by cervical dislocation. The spleens were removed and ground in a clean bench. Mouse myeloma cells SP2 / 0 and spleen cells were mixed at a 1:10 ratio, and preheated 50% PEG was added for cell fusion. The mixture was then added to 96-well cell culture plates containing feeder cells. Cell status was observed under a microscope after fusion. A semi-quantitative medium change was performed on day 5 post-fusion, and culture continued until day 7, after which the entire medium was replaced.

[0045] Approximately 7-10 days after cell fusion, the cell supernatant from the cell culture plate was aspirated, and the amount of secreted antibodies in the supernatant was determined using an indirect ELISA method. Positive cells were screened in the wells, and subcloning was performed using a limiting dilution method. The cells were coated with tebuconazole-OVA; PBS was used as a blank control well; culture medium was used as a negative control; and serum from the ocular blood of immunized mice was used as a positive control. When the selected monoclonal cells showed a 100% positivity rate in a 96-well cell culture plate, they were considered positive monoclonal cells and were cryopreserved and expanded for further culture as appropriate.

[0046] 3. Preparation and purification of monoclonal antibody ascites fluid

[0047] Eight-week-old Balb / c mice were pre-stimulated with liquid paraffin one week in advance to promote the secretion and accumulation of nutrients in the peritoneal cavity. The resulting hybridoma cell line was expanded to the required number, centrifuged, carefully washed, resuspended in sterile 75% physiological saline, and injected intraperitoneally into the mouse abdomen. After approximately one week, when the mouse abdomen showed significant distension, ascites fluid was collected, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected as the monoclonal antibody ascites fluid.

[0048] The collected ascites fluid was purified using a Protein G affinity chromatography column. Before loading, the ascites fluid was filtered through a microporous membrane and used as the loading solution. The Protein G column was equilibrated with binding buffer (0.15M NaCl, 20mM Na2HPO4, pH 7.4). The filtered ascites fluid was then loaded onto the chromatography column. After equilibration, elution was performed using elution buffer (0.1M citric acid, pH 2.5–3.0), and the antibody-rich eluent was collected. The eluent was acidic and should be immediately adjusted to neutral using neutralization buffer (1M Tris-HCl, pH 9.0) to prevent antibody inactivation. Subsequent SDS-PAGE electrophoresis was used for identification, yielding the tebuconazole monoclonal antibody. (See [link to relevant documentation]). Figure 2 ,Depend on Figure 2 It can be seen that the band size of the tebuconazole monoclonal antibody is approximately 160 kDa (non-reduced), while the band sizes of the heavy chain and light chain are 50 kDa and 22 kDa (reduced), respectively. The band sizes are similar to the molecular weight, which is in line with expectations.

[0049] 4. Determination of the variable regions of the heavy and light chains of uniconazole monoclonal antibody

[0050] Hybridoma cell lines capable of stably secreting tebuconazole monoclonal antibodies were cultured to 1×10⁻⁶ cells / year. 6Cells / mL, discard the cell waste supernatant, add 1 mL Trizol reagent, gently vortex, and incubate on ice for 10 min for lysis. Add 200 μL chloroform, invert and vortex for 30 s, and incubate on ice for 10 min. Centrifuge at 4℃, 12000g for 15 min, transfer the supernatant to a new centrifuge tube, add an equal volume of pre-chilled isopropanol, gently invert, and incubate on ice for 10 min. Centrifuge at 4℃, 12000g for 10 min, discard the supernatant, add 1 mL 75% ethanol, invert to suspend the precipitate in ethanol, incubate for 5 min, centrifuge at 4℃, 12000g for 5 min, discard the supernatant, and air dry in a clean bench for 10 min. Resuspend the precipitate in 100 μL of enzyme-free sterile water to obtain total RNA. Use 5 μg of total RNA as a template for reverse transcription according to the instructions of the Hifair III 1st Strand cDNA Synthesis Kit. Upstream and downstream primers for the variable regions of the heavy and light chains of monoclonal antibodies were designed: ggggatatccaccatgracttcgggytgagctkggtttt (heavy chain upstream primer), ctttacccggagaccgggagatggtcT (heavy chain downstream primer), gacattswgatgacmcagtctcca (light chain upstream primer), and gccgcggcctgcaaagactcactttattga (light chain downstream primer). The cDNA obtained from reverse transcription was amplified using the following reaction parameters: 94℃ for 5 min, 94℃ for 10 s, 60℃ for 20 s, and 72℃ for 30 s, for a total of 25 cycles, followed by 72℃ for 5 min. The amplified products were identified using 1% agarose gel electrophoresis, and their sequences were determined.

[0051] Sequencing revealed that it encodes V HNucleotide sequence: caggtccaactacagcagcctggggctgaactggtgaggcctggggcttcagtgaagttgtcctgcaaggcttctggctacaccttcaccgtctactatatctactgggtgaaacagaggcctggacaaggccttgagtggattggggggattcatcctagaaacggtggttcttattacaatgcgaagttcaggaacagggccacactgactgtagacaaatcctccaacacagcctacatgcaattcagcagcctgacatctgaggactctgcggtctattactgtacaagagggatttactacgatggtaaattcggagagcgtgctatggactactggggtcaaggaacctctgtcaccgtctcctca (SEQ ID No.3);

[0052] Encoding V L Nucleotide sequence: gacattgtgatgacccagactccaaacactttgtctgttaccattggacagccagcttccatttcttgcaagtcaagtcagagcctcttatatagtgatggaaaaacctatttgcattggttattacagagtccaggccagtctccaaagctcctaatctatctggtgtctaaactggaatctggagtccctgacagattcagtggcagtggatcagggacagattttacactgaaaatcagcagagtggaggctgaggatttgggggtttattactgcgtgcaagctgcacatctcccccatacgttcgggtcggggaccaagctggaaataaaa (SEQ ID No.4).

[0053] The nucleotide sequence encoding the amino acid shown in SEQ ID No. 5 is: ggctacaccttcaccgtctactata (SEQ ID No. 11); the nucleotide sequence encoding the amino acid shown in SEQ ID No. 6 is: attcatcctagaaacggtggttct (SEQ ID No. 12); the nucleotide sequence encoding the amino acid shown in SEQ ID No. 7 is: acaagagggatttactacgatggtaaattcggagagcgtgctatggactac (SEQ ID No. 13);

[0054] The nucleotide sequence encoding the amino acid shown in SEQ ID No. 8 is: cagagcctcttatatagtgatggaaaaacctat (SEQ ID No. 14); the nucleotide sequence encoding the amino acid shown in SEQ ID No. 9 is: ctggtgtct (SEQ ID No. 15); and the nucleotide sequence encoding the amino acid shown in SEQ ID No. 10 is: gtgcaagctgcacatctcccccatacg (SEQ ID No. 16).

[0055] The amino acid sequence obtained by Snapgene translation of nucleotide sequences includes the tebuconazole monoclonal antibody containing the name V. H The heavy chain variable region and its name is V L The variable region of the light chain;

[0056] V H Amino acid sequence: QVQLQQPGAELVRPGASVKLSCKASGYTFTVYYIYWVKQRPGQGLEWIGGIHPRNGGSYYNAKFRNRATLTVDKSSNTAYMQFSSLTSEDSAVYYCTRGIYYDGKFGERAMDYWGQGTSVTVSS (SEQID No. 1);

[0057] V L Amino acid sequence: DIVMTQTPNTLSVTIGQPASISCKSSQSLLYSDGKTYLHWLLQSPGQSPKLLIYLVSKLESGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCVQAAHLPHTFGSGTKLEIK (SEQ ID No. 2);

[0058] V H and V LEach region consists of complementary determinant regions and frame regions, and each complementary determinant region consists of CDR1, CDR2 and CDR3.

[0059] V H The amino acid sequence of CDR1 is: GYTFTVYY (SEQ ID No. 5);

[0060] V H The amino acid sequence of CDR2: IHPRNGGS (SEQ ID No. 6);

[0061] V H The amino acid sequence of CDR3 is: TRGIYYDGKFGERAMDY (SEQ ID No. 7);

[0062] V L The amino acid sequence of CDR1: QSLLYSDGKTY (SEQ ID No. 8);

[0063] V L The amino acid sequence of CDR2: LVS (SEQ ID No. 9);

[0064] V L The amino acid sequence of CDR3 is: VQAAHLPHT (SEQ ID No. 10).

[0065] Example 3: Indirect competitive ELISA determination of paclobutrazol

[0066] The detection antigen, tebuconazole-OVA, was diluted to 1 μg / mL with 1×PBS solution and added to a 96-well microplate at 100 μL / well. The plate was coated at 37°C for 2 h. After coating, the liquid in the wells was discarded, and the plate was washed three times with PBST. 5% skim milk was added to the wells at 300 μL / well, and the plate was blocked at 37°C for 2 h. After blocking, the liquid in the wells was discarded, and the plate was washed three times with PBST. The tebuconazole pesticide standard was serially diluted from 100 ng / mL with 5% methanol-PBS solution at 50 μL / well, and 100 ng / mL of tebuconazole monoclonal antibody was added at 50 μL / well. Simultaneously, zero standard wells (with tebuconazole standard replaced by 5% methanol-PBS, all other conditions the same) and blank control wells (with antibody solution replaced by PBS, all other conditions the same) were set up and incubated at 37°C for 30 min. After incubation, the plate was washed 3 times with PBST, and 100 μL / well of HRP-labeled goat anti-mouse IgG antibody (1:5000) was added. The plate was incubated at 37°C for 30 min. After incubation, the liquid in the wells was discarded, the plate was washed 3 times with PBST, and 100 μL / well of TMB chromogenic solution was added. The plate was reacted at 37°C in the dark for 7 min, and then 50 μL / well of 2 M sulfuric acid was added to terminate the reaction. Finally, the plate was quickly placed in a microplate reader, and its OD value was measured at 450 / 630 nm wavelength.

[0067] Please see Figure 3 The above ELISA results show that its half-maximal inhibitory concentration (IC50) is... 50 The concentration of the antibody was 4.1 ng / mL, and the linear range was 0.78-25 ng / mL. The monoclonal antibody described above has high sensitivity and a wide linear range for the detection of uniconazole.

[0068] Example 4: Specific reactions based on monoclonal antibodies

[0069] Based on the monoclonal antibody prepared in Example 2 of this invention, structural and functional analogues of uniconazole (see Table 1) were selected and serially diluted with 5% methanol-PBS. These were then used indirect competitive ELISA detection against the monoclonal antibody to establish a standard curve and obtain the IC50. 50 And calculate its cross-reactivity rate CR (%) according to the formula = IC 50 (Analyte) / IC 50 (Analogous)*100%.

[0070] Table 1 Cross-reactivity

[0071]

[0072] The results of the indirect competitive ELISA cross-reactivity experiment with tebuconazole analogues are shown in Table 1. As can be seen from the results in Table 1, the present invention utilizes... Figure 1 The cross-reactivity rates of the monoclonal antibodies prepared from the hapten to the structural and functional analogues of uniconazole were all less than 0.8%, indicating that the obtained uniconazole monoclonal antibodies can recognize uniconazole with high specificity.

[0073] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A uniconazole monoclonal antibody, characterized in that, The uniconazole monoclonal antibody includes a heavy chain variable region and a light chain variable region; The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively. The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

2. The uniconazole monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

3. A gene encoding the uniconazole monoclonal antibody according to any one of claims 1 to 2, characterized in that, The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 are shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively; The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 are shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively.

4. The gene according to claim 3, characterized in that, The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

5. The use of the uniconazole monoclonal antibody as described in any one of claims 1 to 2 or the gene as described in any one of claims 3 to 4 in the detection of uniconazole.

6. The application according to claim 5, characterized in that, The content of uniconazole in the test sample was detected by indirect competitive ELISA using the uniconazole monoclonal antibody.

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