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

Abstract

The application discloses an ethiprole monoclonal antibody and application thereof, and belongs to the technical field of biology. The monoclonal antibody contains a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are both composed of a complementarity determining region and a framework region, the complementarity determining region is composed of CDR1, CDR2 and CDR3, wherein the amino acid sequences of CDR1, CDR and CDR3 of the heavy chain are respectively shown as SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, and the amino acid sequences of CDR1, CDR and CDR3 of the light chain are respectively shown as SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10. The monoclonal antibody can be used for preparing an ethiprole detection product. The ethiprole colloidal gold detection test strip provided by the application has the characteristics of high sensitivity, good specificity and strong stability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the heavy and light chain variable regions of an acetonitrile monoclonal antibody and its applications. Background Technology

[0002] Ethiprole, chemically known as 5-amino-1-(2,6-dichloro-p-trifluoromethylphenyl)-4-ethylsulfinylpyrazole-3-acrylonitrile, is a novel pyrazole insecticide. Ethiprole suspension concentrate can be used on various crops such as rice and vegetables, effectively controlling a variety of chewing and piercing-sucking pests. With the widespread application of acetiprole, its degradation and residue status in the environment have become a focus of attention.

[0003] The extensive use of acetaminophen in agricultural production has led to its widespread residues in crops and the environment. Acetaminophen and its metabolites possess neurotoxicity and endocrine-disrupting effects, and can be transferred and accumulated through the food chain, posing a potential threat to human health. my country's National Food Safety Standard for Maximum Residue Limits of Pesticides in Food (GB 2763-2026) clearly stipulates the maximum residue limits (MRLs) for acetaminophen in grains, vegetables, fruits, and other foods. However, due to the slow degradation and long half-life of acetaminophen in the environment, and its high control efficacy against various pests, its illegal use and excessive application in actual production occur frequently, leading to frequent incidents of acetaminophen residue exceeding the limits in agricultural products, seriously threatening food safety and public health safety. Therefore, establishing a sensitive, rapid, and efficient method for detecting acetaminophen residues is of significant practical importance for ensuring food safety, strengthening pesticide residue monitoring, and standardizing agricultural production.

[0004] Currently, the main methods for determining acetaminophen are high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While these instrumental analytical methods offer high sensitivity and accuracy, they suffer from drawbacks such as high cost, complex operation, expensive equipment, high technical skill requirements, and cumbersome sample pretreatment, making them unsuitable for rapid on-site screening of large batches of samples. Immunoassay methods, on the other hand, offer advantages such as low cost, high efficiency, high sensitivity, ease of operation, and low technical skill requirements, making them suitable for rapid detection and on-site screening of large numbers of samples. The purpose of this invention is to provide a monoclonal antibody with high affinity and detection sensitivity for acetaminophen, laying the foundation for establishing a colloidal gold test strip detection method and providing technical support for the supervision of agricultural product quality and safety. Summary of the Invention

[0005] Therefore, the present invention provides a heavy chain and light chain variable region of an acetonitrile 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 an acetonitrile 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; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2.

[0009] 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.

[0010] 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; and the amino acid sequence of CDR3 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7.

[0011] 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; and the amino acid sequence of CDR3 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 10.

[0012] Secondly, the above-mentioned acetamiprid monoclonal antibody is characterized in that: the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID No. 3; and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody is as shown in SEQ ID No. 4.

[0013] Thirdly, the present invention provides the application of acetonitrile monoclonal antibody in the preparation of detection products for detecting acetonitrile.

[0014] This antibody can be used as a raw material for immunoassay. In this embodiment, a colloidal gold immunochromatographic assay method based on this antibody is further established.

[0015] The present invention has the following advantages:

[0016] The monoclonal antibody described in this invention possesses high specificity and sensitivity, and can be used as a raw material for ELISA and colloidal gold immunoassay. This invention lays the foundation for the research, development, and promotion of ELISA and colloidal gold test strips. Attached Figure Description

[0017] 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 described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0018] Figure 1 This is a synthetic route diagram of the acetonitrile hapten of the present invention;

[0019] Figure 2 This is a synthetic route diagram of the acetonitrile artificial antigen of the present invention;

[0020] Figure 3 This is an SDS-PAGE image of the acetonitrile monoclonal antibody provided by the present invention;

[0021] Figure 4 The standard curve diagram provided for this invention;

[0022] Figure 5 Homology comparison diagram of the heavy chain gene sequence of the acetonitrile monoclonal antibody provided by the present invention;

[0023] Figure 6 This is a homology comparison diagram of the heavy chain amino acid sequence of the acetonitrile monoclonal antibody provided by the present invention;

[0024] Figure 7 Homology comparison diagram of the light chain gene sequence of the acetonitrile monoclonal antibody provided by the present invention;

[0025] Figure 8 This is a homology comparison diagram of the light chain amino acid sequence of the acetonitrile monoclonal antibody provided by the present invention;

[0026] Figure 9 The image shows the interpretation results of the test strip provided by this invention. Detailed Implementation

[0027] 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.

[0028] Example 1: Synthesis of acetonitrile hapten and artificial antigen

[0029] 1. Synthesis of acetonitrile hapten

[0030] Ethylenenitrile is a small molecule compound (molecular weight 397.20) and is not immunogenic on its own; it must be coupled with a large molecular weight carrier protein to elicit an immune response in animals. To preserve the characteristic structure of ethylenenitrile (phenylpyrazole ring and ethyl sulfinyl group) and maintain the integrity of its antigenic determinant, this invention introduces a carboxyl-containing linker arm at the amino site of the ethylenenitrile molecule to prepare an ethylenenitrile hapten. The carboxyl group of this hapten can form a stable amide bond with the amino group on the carrier protein under the action of an activator, thereby achieving coupling between the hapten and the carrier protein through the terminal carboxyl group of the linker arm. The synthetic route is as follows: Figure 1 As shown.

[0031] The specific synthesis steps are as follows:

[0032] (1) Weigh 1.0 g of acetonitrile (2.52 mmol, Mw=397.20 g / mol) and dissolve it in 40 mL of anhydrous tetrahydrofuran (THF). Add 0.338 g of glutaric anhydride (2.96 mmol, 1.2 eq) and 0.30 g of triethylamine (2.96 mmol, 1.2 eq). Stir the mixture magnetically at 45 °C for 8 h under nitrogen protection. Monitor the reaction progress by thin-layer chromatography (TLC) until the starting material spot disappears.

[0033] (2) After the reaction is completed, THF is removed by rotary evaporation under reduced pressure. The residue is dissolved in dichloromethane (DCM), washed twice with 1 mol / L dilute hydrochloric acid and once with saturated brine, and dried with anhydrous sodium sulfate.

[0034] (3) After filtration, the sample was evaporated under reduced pressure and purified by silica gel column chromatography with dichloromethane / methanol = 20:1 (v / v). The target component was collected and evaporated to obtain acetonitrile hapten.

[0035] 2. Preparation of acetonitrile immunogenicity antigen

[0036] The above-mentioned acetonitrile hapten was coupled to the carrier protein bovine serum albumin (BSA) using the active ester method to prepare the acetonitrile immunoantigen. The synthetic route of the acetonitrile immunoantigen is as follows: Figure 2 As shown.

[0037] The specific steps are as follows:

[0038] (1) Activation of hapten: Accurately weigh 8.0 mg (15.6 μmol) of acetonitrile hapten and dissolve it in 0.5 mL of anhydrous N,N-dimethylformamide (DMF). Then add 4.5 mg (23.4 μmol, 1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 2.7 mg (23.4 μmol, 1.5 eq) of N-hydroxysuccinimide (NHS) in sequence. Stir the mixture magnetically for 4 hours at room temperature and in the dark to obtain the hapten activation solution.

[0039] (2) Coupling reaction: Weigh 20 mg of bovine serum albumin (BSA) and dissolve it in 3.0 mL of 0.01 mol / L phosphate buffer (PBS, pH 7.4). Under ice bath and magnetic stirring, slowly add the above hapten activation solution dropwise to the protein solution. After the addition is complete, place it at 4℃ and stir slowly for 12 hours.

[0040] (3) Purification and dialysis: The reaction solution was transferred to a pretreated dialysis bag (molecular weight cutoff 14 kDa). Dialysis was performed at 4°C using 0.01 mol / L PBS (pH 7.4). The dialysis solution was changed 2-3 times a day for 3 consecutive days. After dialysis, the liquid in the bag was collected and centrifuged at low speed to remove any small amount of precipitate that might be generated. The supernatant was ethiprole-BSA, which was aliquoted and stored at -20°C for later use. The conjugation ratio was determined by UV-Vis spectrophotometry (in this example, the conjugation ratio of hapten to BSA was approximately 8:1 to 12:1).

[0041] 3. Preparation of acetonitrile detection antigen (coating antigen)

[0042] Following the above method for preparing immunoantigens, the carrier protein was replaced with ovalbumin (OVA), and the remaining steps were exactly the same, to prepare ethiprole-OVA, which is used as a coating antigen in immunological detection.

[0043] Example 2: Preparation of acetonitrile monoclonal antibody

[0044] 1. Animal immunization

[0045] Three 6-8 week old female BALB / c mice were immunized with 20 μg of ethiprole-BSA artificial antigen per mouse. For the first immunization, the ethiprole-BSA artificial antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. Immunizations were repeated every two weeks for a total of three immunizations. For the second and third immunizations, incomplete Freund's adjuvant was used for antigen emulsification, and the dosage and method of immunization remained unchanged. One week after the third immunization, blood was collected from the tail vein of the mice, and serum was obtained. Serum titers were determined using indirect ELISA, and the competitive inhibitory effect of serum on ethiophene was evaluated using indirect competitive ELISA. The results are shown in Table 1.

[0046] Table 1 Serum titers and inhibition detection in immunized mice

[0047]

[0048] Note: The concentration of acetonitrile added was 1000 ppb; PBS was a negative control, and its OD value has been adjusted for background. "—" indicates that it is not applicable.

[0049] The formula for calculating the inhibition rate is: Inhibition rate (%) = [1 - (OD)] 450 OD (Original Discharge) 450 [Valence] × 100%.

[0050] As shown in Table 1, the serum titer of mouse #1 was the highest after immunization (still with a high OD value at 1:64000), and the inhibition rate against acetonitrile reached as high as 59.50%. Therefore, mouse #1 was selected for booster immunization. 40 μg of Ethiprole-BSA artificial antigen was diluted with 1×PBS to 200 μL and injected intraperitoneally into mice for booster immunization. Cell fusion was performed three days later.

[0051] 2. Cell fusion

[0052] (1) Preparation of spleen cells: Take the spleen of the 1# mouse after booster immunization, remove it under sterile conditions, grind it with DMEM medium to prepare a single cell suspension, filter it through a 200 mesh nylon net, centrifuge at 1500 r / min for 5 min, discard the supernatant, and resuspend the cell pellet in DMEM medium for later use.

[0053] (2) Preparation of myeloma cells: SP2 / 0 myeloma cells in the logarithmic growth phase were collected by centrifugation at 1000 r / min for 5 min, washed twice with DMEM medium and resuspended for later use.

[0054] (3) Cell fusion: Spleen cells and SP2 / 0 cells were mixed at a ratio of 10:1, centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the cell clumps were gently broken up. The mixture was placed in a 37 ℃ water bath, and 1 mL of PEG (molecular weight 1450) fusion agent was slowly added dropwise over 1 min while gently rotating the centrifuge tube. Then, 9 mL of preheated DMEM medium was added dropwise over 3 min to terminate the fusion, and the mixture was centrifuged at 800 r / min for 5 min, and the supernatant was discarded.

[0055] (4) Plating: Gently resuspend the fused cells in HAT selective medium and evenly spread them in a 96-well cell culture plate containing feeder cells, 100 μL / well, and incubate at 37 ℃ in a 5% CO2 incubator.

[0056] 3. Screening and subcloning of positive hybridoma cells

[0057] Seven to ten days after cell fusion, cell growth was observed. When the hybridoma cell clusters grew to about 1 / 3 to 1 / 2 of the bottom of the well, the cell supernatant was collected, and the titer was detected by indirect ELISA using Ethiprole-OVA (1 μg / mL) as the coating antigen. Immunized mouse serum was used as a positive control, and pre-immunization serum was used as a negative control. Positive hybridoma cell wells were screened, and the results are shown in Table 2.

[0058] Table 2. Titer and Inhibition Detection of Fusion Monoclonal Cells

[0059]

[0060] As shown in Table 2, clone 1D2H6 exhibited the highest inhibition rate against acetonitrile (73.81%) and a moderate OD value. Therefore, wells with 1D2H6 were selected for limiting dilution subcloning. The subcloned positive wells were further cultured and, after 3-5 consecutive subcloning and stability tests, a hybridoma cell line stably secreting acetonitrile-specific monoclonal antibodies was finally obtained and named 1D2H6.

[0061] 4. Ascites preparation and antibody purification

[0062] After collecting expanded-cultured hybridoma cells 1D2H6, they were resuspended in sterile PBS and intraperitoneally injected into mice pretreated with liquid paraffin. Ascites fluid was collected 7-10 days later, when the mice's abdomens were significantly distended. The collected ascites fluid was centrifuged at 8000 r / min for 20 min at 4 ℃, and the clear intermediate layer was carefully aspirated. The clear ascites fluid was purified using a Protein G affinity chromatography column, and the purity of the purified antibody was identified by SDS-PAGE. The results are shown below. Figure 3 As shown, the purity of the purified antibody is approximately 95%.

[0063] Example 3: Specificity and sensitivity detection of acetonitrile monoclonal antibody

[0064] 1. Specificity

[0065] The sensitivity and specificity of acetamiprid monoclonal antibody were detected using an indirect competitive ELISA method. The ethiprole-OVA artificial antigen was coated at a concentration of 1 μg / mL. The monoclonal antibody 1D2H6 was serially diluted at a concentration of 1 mg / mL, and competitive inhibition assays were performed using 50 ppb of acetamiprid, fipronil, and tebufenozide standards, respectively. The sensitivity and specificity of the monoclonal antibody were verified, and the results are shown in Table 3.

[0066]

[0067] Table 3 shows that the inhibition rate of acetamiprid against the monoclonal antibody gradually increases with decreasing antibody concentration. When the antibody concentration is 0.0156 μg / mL, the inhibition rate reaches 88.36%, indicating that the monoclonal antibody has good detection sensitivity against acetamiprid. Meanwhile, fipronil and tebufenozide showed no significant inhibitory effect at any antibody concentration (inhibition rates were all less than 1%), indicating that the monoclonal antibody has no cross-reactivity with fipronil or tebufenozide.

[0068] To further determine the half-maximal inhibitory concentration (IC50) of the antibody 50 The sensitivity and specificity of monoclonal antibodies were systematically analyzed using an indirect competitive ELISA method. Competitive inhibition assays were performed using acetamiprid and its structural analogues—fipronil and flufiprole—at the optimal working concentration of the antibody. The half-maximal inhibitory concentration (IC50) of each competitor was determined. 50 And calculate the cross-reactivity rate (CR) using the following formula:

[0069] Cross-reactivity rate (CR, %) = (acetonitrile IC50) 50 / Competitor IC under test 50 ) × 100%

[0070] The specificity of the monoclonal antibody 1D2H6 was evaluated using an indirect competitive ELISA method. Using ethiprole-OVA as the immobilized coating antigen, and under the same antigen concentration and antibody working concentration, acetamiprid, fipronil, and tebufenozide were used as free competitors, and the IC50 of each competitor was determined. 50 And calculate the cross-reactivity rate.

[0071] Table 4. Cross-reactivity identification results of acetamiprid monoclonal antibodies

[0072]

[0073] Table 4 shows that the IC50 of the purified monoclonal antibody against acetonitrile is... 50 The IC50 concentration for fipronil and tebufenozide is 50 ng / mL. 50 All values ​​were greater than 1000 ng / mL, and the cross-reactivity rates were all less than 0.1%, indicating that the monoclonal antibody had no significant cross-reactivity with fipronil and tebufenozide, and was highly specific for acetamiprid, and could be used for the specific immunological detection of acetamiprid.

[0074] 2. Sensitivity

[0075] Competitive ELISA detection was further performed using acetonitrile standards at different concentrations. A standard curve was plotted with the logarithm of the standard concentration on the x-axis and the inhibition rate on the y-axis. Figure 4 ), calculate the half-maximal inhibitory concentration (IC50).50 The results are shown in Table 5.

[0076] Table 5 Sensitivity detection of acetonitrile monoclonal antibody competitive ELISA

[0077]

[0078] Table 5 shows the half-maximal inhibitory concentration (IC50) of acetonitrile monoclonal antibody. 50 The concentration was 50 ng / mL, indicating that the monoclonal antibody can be used for competitive immunoassay of acetonitrile.

[0079] Example 4: Cloning of the variable region gene of acetonitrile monoclonal antibody

[0080] Total RNA was extracted from hybridoma cells, and cDNA was synthesized by reverse transcription. The variable regions of the antibody light and heavy chains were amplified using universal primers for mouse immunoglobulins. The PCR products were recovered by agarose gel electrophoresis, cloned into a vector, transformed into competent cells, and positive clones were screened for sequencing.

[0081] The amino acid sequence of the heavy chain variable region of the acetonitrile monoclonal antibody obtained by sequencing 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; the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 3, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 4. The specific sequences are as follows:

[0082] EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYRMNWVRQSPEKGLEWVAEIRLRSNYYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTREGPSYYNFFDYWGQGTTLTVSS (SEQ ID No. 1)

[0083] DIQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDETVKLLIYYTSRLHSGVPSRFRGSGSGTNYSLTISNVEQEDIATYFCQQGDTLPTFGGGTKLEIK (SEQ ID No. 2)

[0084] GAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACAGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAGATCTAATTATTATGCAACACATTA TGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGGGAGGGACCCTCCTATTATAACTTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ IDNo.3)

[0085] GATATCCAGATGACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAACAATTATTTAAACTGGTATCAGCAGAAACCAGATGAAACTGTTAAACTCCTGATCTATTACACCTCAAGATT ACACTCAGGAGTCCCGTCAAGGTTCCGTGGCAGTGGGTCTGGAACAAATTATTCTCTCACCATTAGCAACGTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTGATACGCTTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID No.4)

[0086] Homology analysis of the variable region nucleotide and amino acid sequences: Comparison analysis was performed in the NCBI database. The results showed that the heavy and light chain variable region gene sequences of this antibody had high homology with known mouse immunoglobulin sequences, but no sequences completely identical to those of this invention were found in the publication.

[0087] The monoclonal antibody heavy chain variable region gene sequence showed the highest homology with the mouse immunoglobulin heavy chain variable region mRNA (Sequence ID: JX492521.1), with a homology of 282 / 285 and a homology percentage of 99%. Figure 5As shown. The amino acid sequence of the variable region of the monoclonal antibody heavy chain has the highest homology with the amino acid sequence of the variable region of the mouse immunoglobulin heavy chain (Sequence ID: WVW31995.1), with a homology of 110 / 122, representing 90% homology. Figure 6 As shown.

[0088] The gene sequence of the light chain variable region of this monoclonal antibody showed the highest homology with the mRNA of the light chain variable region of mouse immunoglobulin κ (Sequence ID: L41836.1), with a homology of 309 / 318 and a homology percentage of 97%. Figure 7 As shown. The amino acid sequence of the variable region of the monoclonal antibody light chain has the highest homology with the mouse immunoglobulin κ light chain (Sequence ID: CA06190980.1), with a homology of 100 / 106 and a homology percentage of 94%. Figure 8 As shown.

[0089] The sequences of the light chain variable region and the heavy chain variable region were submitted to an online CDR analysis tool to obtain their complementarity-determining regions (CDRs). The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID No. 5–SEQ ID No. 7, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID No. 8–SEQ ID No. 10, respectively. The specific sequences are as follows:

[0090] GFTFSNYR (SEQ ID No. 5);

[0091] IRLRSNYYAT (SEQ ID No. 6);

[0092] TREGPSYYNFFDY(SEQ ID No.7);

[0093] QDINNY (SEQ ID No. 8);

[0094] YTSR (SEQ ID No. 9);

[0095] QQGDTLPT (SEQ ID No. 10).

[0096] Example 5: Preparation of acetonitrile colloidal gold test strip

[0097] 1. Preparation of colloidal gold solution

[0098] Take a 1 L Erlenmeyer flask, add 495 mL of ultrapure water, add 5 mL of 1% chloroauric acid, heat to boiling, then add 5-7 mL of 1% trisodium citrate solution, continue stirring and heating until the solution color turns completely transparent purple-red, maintain for 5 min, then stop heating, add water to the original volume, cool to room temperature, and the colloidal gold solution is obtained.

[0099] 2. Preparation of acetonitrile monoclonal antibody-colloidal gold label

[0100] (1) Take 40 mL of colloidal gold solution and adjust the pH to 8.5 with 0.25 mol / L K2CO3;

[0101] (2) Add 4 mL of protein solution containing 0.3 mg of antibody protein dropwise under electromagnetic stirring, and react for 10 min;

[0102] (3) Add 4 mL of 10% BSA dropwise and continue stirring for 10 min;

[0103] (4) Centrifuge the gold-labeled antibody solution at 1500 r / min for 10 min and discard the precipitate;

[0104] (5) Centrifuge the supernatant at 12000 r / min for 20 min, discard the supernatant and collect the precipitate;

[0105] (6) The precipitate was diluted to 1 mL with gold-labeled antibody diluent to obtain acetonitrile monoclonal antibody colloidal gold-labeled product.

[0106] 3. Preparation of colloidal gold binding pads

[0107] The colloidal gold-labeled acetonitrile monoclonal antibody was uniformly sprayed onto a glass cellulose membrane at a concentration of 5 μL / cm using a membrane coating instrument, and dried at 37°C for 3 h to obtain the colloidal gold conjugate pad.

[0108] 4. Preparation of nitrocellulose membranes

[0109] Goat anti-mouse IgG antibody and acetonitrile-OVA detection antigen were diluted to 1 mg / mL and sprayed onto nitrocellulose membranes at a concentration of 1 μL / cm using a coating instrument. These were used as the control line (C line) and the detection line (T line), respectively, and dried at 37℃ for 6 h.

[0110] 5. Test strip assembly

[0111] The sample absorption pad, colloidal gold binding pad, nitrocellulose membrane, and absorbent pad are sequentially pasted onto a PVC base plate and cut into 4 mm wide test strips to obtain the acetonitrile colloidal gold test strip.

[0112] Example 6: Application of acetonitrile colloidal gold test strips

[0113] 1. Sensitivity

[0114] Different concentrations of acetonitrile standard solutions were taken and tested using the acetonitrile colloidal gold immunochromatographic test strip prepared in this invention according to the detection method described in Example 5. The visual detection limit of the test strip for acetonitrile was determined by visually interpreting the color intensity of the test strip's detection line (T line). The results are shown in Table 6.

[0115] Table 6. Sensitivity Detection Results of Acetaminophen Colloidal Gold Immunochromatographic Test Strips

[0116]

[0117] Note: "++" indicates that the color intensity of the T line is the same as that of the C line; "++" indicates that the color intensity of the T line is weaker than that of the C line but still clearly visible; "+" indicates that the color intensity of the T line is significantly weaker than that of the C line; "±" indicates that the T line shows very little color; "-" indicates that the T line does not show any color.

[0118] The results showed that the visual cut-off threshold of the test strip was 10 ng / mL.

[0119] 2. Specificity

[0120] The test strip was used to detect standard solutions of acetamiprid (50 ng / mL), fipronil (100 ng / mL), and tebufenozide (100 ng / mL), with a negative control included. Results are shown in [Figure number missing]. Figure 9 .

[0121] The results showed that only the test line (T line) for acetamiprid was significantly weaker than the control line (C line) or showed no color at all, indicating a positive result (+). For fipronil and tebufenozide, the T line color intensity was consistent with the C line (i.e., the T line color intensity was not weaker than the C line), indicating a negative result (-). This demonstrates that the acetamiprid colloidal gold immunochromatographic test strip prepared in this invention has no cross-reactivity with fipronil and tebufenozide, exhibiting good specificity.

[0122] 3. Stability

[0123] The acetonitrile colloidal gold immunochromatographic test strips prepared according to this invention were stored at 4℃ and 37℃, respectively. The test strips were taken out at 0 d, 7 d, 14 d, and 28 d, respectively, and tested with 0 ng / mL (i.e., blank negative control) acetonitrile standard solution. The signal values ​​of the T line and C line were read by a colloidal gold reader to examine the stability of the test strips. The results are shown in Table 7.

[0124] Table 7. Stability test results of acetonitrile colloidal gold immunochromatographic test strips

[0125]

[0126] As shown in Table 7, the acetaminophen colloidal gold test strip prepared by this invention, after being stored at 4℃ for 28 days, maintained an average T / C ratio between 0.800 and 0.810, with a CV value of 0.54% to 1.95%. After being stored at 37℃ under accelerated aging conditions for 28 days, the average T / C ratio remained stable between 0.776 and 0.812, with a CV value of 0.54% to 2.63%. Under both storage conditions, the T / C values ​​at each time point showed no significant decrease compared to 0 days, and the coefficient of variation (CV) was much less than 5%, indicating that the acetaminophen colloidal gold test strip provided by this invention exhibits good stability under both 4℃ and 37℃ conditions for 28 days.

[0127] 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 monoclonal antibody against acetonitrile, characterized in that, It includes a heavy chain variable region and a light chain variable region. The amino acid sequences of 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 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 acetonitrile 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; the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

3. The use of the acetonitrile monoclonal antibody as described in claim 1 in the preparation of products for detecting acetonitrile.

4. The application according to claim 3, characterized in that, The product in question is a test strip.

5. A test strip for detecting acetonitrile, characterized in that, The test strip includes a base plate, a sample absorption pad, a colloidal gold binding pad, a nitrocellulose membrane, and an absorbent pad; The colloidal gold conjugate pad is loaded with the colloidal gold-labeled monoclonal antibody as described in claim 1 or 2; The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with acetonitrile-carrier protein conjugate, and the control line is coated with anti-mouse IgG antibody.

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