CN117946262B - Avermectin monoclonal antibody, test strip and application - Google Patents

Avermectin monoclonal antibody, test strip and application Download PDF

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CN117946262B
CN117946262B CN202410349711.3A CN202410349711A CN117946262B CN 117946262 B CN117946262 B CN 117946262B CN 202410349711 A CN202410349711 A CN 202410349711A CN 117946262 B CN117946262 B CN 117946262B
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monoclonal antibody
avermectin
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test strip
amino acid
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CN117946262A (en
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杨晓霞
赵荣茂
李川敏
巩玉洁
赵方圆
陈娟
袁婷婷
张琼林
王紫娟
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Beijing Nabai Bio Tech Co ltd
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Abstract

The invention discloses an avermectin monoclonal antibody, a test strip and application thereof, and belongs to the field of rapid biotechnology detection. The monoclonal antibody contains a heavy chain variable region named V H and a light chain variable region named V L, both V H and V L consisting of a complementarity determining region and a framework region; the complementarity determining regions are each composed of CDR1, CDR2 and CDR 3. The monoclonal antibody can be used for preparing a kit and a test strip for detecting avermectin residues. The avermectin colloidal gold quantitative detection test strip provided by the invention is used for quantitatively detecting avermectin, greatly improves the detection sensitivity, reduces the detection cost and shortens the detection time, and is suitable for screening and on-site monitoring of a large number of samples.

Description

Avermectin monoclonal antibody, test strip and application
Technical Field
The invention relates to the field of rapid biotechnology detection, in particular to an abamectin monoclonal antibody, a kit and application.
Background
Avermectin (AVM) is used as a novel sixteen-membered macrolide pesticide, is produced by fermentation of streptomyces avermitilis in streptomyces, has novel chemical structure, unique action mechanism (the nerve physiological activity of an insect body is interfered, gamma-aminobutyric acid is stimulated to be released, thereby inhibiting nerve conduction of arthropods), and strong insecticidal activity, has the characteristics of broad spectrum, high efficiency, lasting effect, relative safety and the like, meets the requirements of modern ecological pesticides, and is widely applied to crops such as vegetables, fruit trees, cotton and the like to prevent and treat various pests. At present, abamectin products are widely popularized and used in the whole world. However, as the resistance of pests increases, the dosage of avermectin increases, and the damage to the environment, human beings, animals and plants becomes increasingly apparent, and the damage is mainly manifested by drug residues in the soil of the field, central and peripheral nerve symptoms of the human beings and animals, such as tremors, ataxia, mental depression, high coma and even death, and embryotoxicity. They are classified as highly toxic compounds according to the world health organization class 5 classification standard. Therefore, it is necessary to detect the residual amount of avermectin in food.
The detection of the avermectin medicine residue in the food becomes a national key monitoring index, and the detection method of the avermectin residue also becomes key research content. At present, most of methods for detecting the residual amount of avermectin are instrument methods, and have the defects of complex instrument and equipment, complex sample pretreatment and measurement operation, are not suitable for on-site monitoring and large-amount sample screening, have high cost, and are limited in popularization and use. Therefore, the invention provides a monoclonal antibody of abamectin, and the monoclonal antibody is applied to detection products capable of rapidly detecting abamectin and rapidly obtaining results.
Disclosure of Invention
The technical problem solved by the invention is to provide an avermectin monoclonal antibody, a test strip and application, wherein the monoclonal antibody has strong binding force with avermectin and good sensitivity; the detection limit of the kit is lower.
In order to solve the above problems, the embodiment of the present invention provides the following technical solutions:
In a first aspect, the present invention provides an avermectin monoclonal antibody, characterized in that: the monoclonal antibody contains a heavy chain variable region named V H and a light chain variable region named V L;
the amino acid sequence of the V H of the monoclonal antibody is shown as SEQ ID No. 1;
The amino acid sequence of the V L of the monoclonal antibody is shown as SEQ ID No. 3;
Each of V H and V L consists of a complementarity determining region and a framework region, the complementarity determining regions each consisting of CDR1, CDR2, and CDR 3;
The amino acid sequence of CDR1 of V H of the monoclonal antibody is shown as 31 st-37 th position in SEQ ID No. 1;
The amino acid sequence of CDR2 of V H of the monoclonal antibody is shown as 61-92 th position in SEQ ID No. 1;
The amino acid sequence of CDR3 of V H of the monoclonal antibody is shown as 125 th-126 th position in SEQ ID No. 1;
The amino acid sequence of CDR1 of V L of the monoclonal antibody is shown as 13 th-28 th position in SEQ ID No. 3;
the amino acid sequence of CDR2 of V L of the monoclonal antibody is shown as 43-58 th position in SEQ ID No. 3;
The amino acid sequence of CDR3 of V L of the monoclonal antibody is shown as 106-113 th position in SEQ ID No. 3.
In a second aspect, the abamectin monoclonal antibody provided by the invention is characterized in that:
the nucleotide sequence of V H for encoding the monoclonal antibody is shown as SEQ ID No. 2;
The nucleotide sequence of V L for encoding the monoclonal antibody is shown in SEQ ID No. 4.
In a third aspect, the use of a monoclonal antibody to avermectin in the preparation of a test kit and/or test strip for detecting avermectin.
Preferably, the test strip is a colloidal gold test strip, or a fluorescent microsphere test strip, or a latex microsphere test strip;
Preferably, the kit competes for an ELISA detection kit.
The fourth aspect is an avermectin colloidal gold quantitative detection test strip, which is characterized in that the colloidal gold quantitative detection test strip contains the colloidal gold labeled monoclonal antibody of the avermectin.
Compared with the prior art, the invention has the following beneficial effects:
According to the invention, mice are immunized by the avermectin artificial antigen, and monoclonal antibodies with higher affinity and detection sensitivity to the avermectin are obtained through the hybridoma technology screening, so that a foundation is laid for the research and development and popularization of colloidal gold test strips. The monoclonal antibody has the characteristics of high specificity, high sensitivity and the like, and the colloidal gold test strip has the advantages of high sensitivity, high specificity, low cost, simplicity in operation, short detection time, suitability for various units, simplicity in storage and long shelf life. The method for detecting avermectin residues by using the test strip is simple, convenient, quick, visual and accurate, has wide application range, low cost and easy popularization and use.
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In order 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 described below. It will be apparent to those skilled in the art from this disclosure that the drawings described below are merely exemplary and that other embodiments may be derived from the drawings provided without undue effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the scope of the invention.
FIG. 1 is a graph of a RIDA SOFT four-parameter method R 2 for detecting sensitivity of an avermectin monoclonal antibody by adopting an indirect competition ELISA method provided by the embodiment of the invention;
FIG. 2 is a graph of a RIDA SOFT four-parameter method IC 50 for detecting sensitivity of an avermectin monoclonal antibody by adopting an indirect competition ELISA method provided by the embodiment of the invention;
fig. 3 is a structural diagram of an avermectin test strip provided by the embodiment of the invention;
fig. 4 is a standard graph of an avermectin test strip provided by the embodiment of the invention;
fig. 5 is a diagram of a specific result of an avermectin test strip provided by the embodiment of the invention.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 Synthesis of Avermectin Artificial antigen
2.0G of AVM was placed in a 50mL round bottom flask, 10mL of tetrahydrofuran was dissolved, 0.9g of imidazole was added and mixed. 5mL of tetrahydrofuran-dissolved 1.0g t-BuMe2SiCl was added dropwise with mechanical stirring and reacted at 30℃for about 4 hours. After the reaction is completed, 50mL of ethyl acetate is added into the reaction solution to be mixed, the mixed solution is washed with 80mL of water for 3 times to separate an ethyl acetate layer, an organic layer is collected, dried by anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a yellowish sticky 5' -O-t-BuMe2Si-AVM.
1.0G of 5' -O-t-BuMe2Si-AVM is placed in a 100mL round bottom flask, 30mL of dichloromethane is added to dissolve the AVM, 0.5g of 4-dimethylaminopyridine, 1.5mL of triethylamine and 0.6g of succinic anhydride are sequentially added to react for 2.5h in the absence of light, and the solution turns from colorless to brown and black. After removal of dichloromethane under reduced pressure, 100mL of ethyl acetate was added, the insoluble material was removed by filtration and transferred to the liquid funnel, the ethyl acetate layer was washed 2 times with 100mL of 3.6% HCl, and then with water 2 times, dried over MgSO 4, and concentrated to give a pale yellow viscous material, 5'-O-t-BuMe2Si-R-4' HS-AVM, IT-TOF, for molecular weight identification.
1.0G of 5'-0-t-BuMe2Si-R-4' -HS-AVM was placed in a 250mL round bottom flask, 50mL of methanol was added for dissolution, a methanol solution containing 1% of p-toluenesulfonic acid (C 7H8O3) was added dropwise with stirring at room temperature, stirring was continued for 30min, and the reaction solution was washed with ethyl acetate and transferred into a 250mL separating funnel. 2% NaHCO 3 solution, washed 3 times with water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was dissolved in an appropriate amount of ethyl acetate and purified by TLC. Developing agent (ethyl acetate: hexane, 3:2, v/v, adding one drop of glacial acetic acid into each 2.5mL of eluent), collecting the third component, concentrating under reduced pressure to obtain hapten 4' -HS-AVM, IT-TOF identification molecular weight.
The active groups of hapten 4' -HS-AVM are carboxyl groups, and the carboxyl groups of the hapten and the amino groups of protein are respectively connected by EDC method to prepare the AVM-BSA conjugate, namely the immunogen.
EXAMPLE 2 preparation of monoclonal antibodies to avermectin
1. Immunization of animals
(1) Preparation of antigens for immunization
The immunogen AVM-BSA prepared in example 1 was used to immunize mice at an antigen level of 10 μg per mouse, the immunogen was diluted to the desired concentration with sterile PBS, equal amounts of adjuvant and immunogen were separately aspirated into two 2.5mL disposable syringes, and then inserted into the communicating vessel and pushed 8500 times with the emulsifier. The immunization procedure employed one basic immunization and several booster immunizations.
(2) Immunization protocol and serum monitoring
Selecting 3 healthy Balb/c female mice with the age of 6-8 weeks and the weight of 18-20g, performing subcutaneous multipoint injection on the antigen mice emulsified by equivalent Freund complete adjuvant for the first time, performing boosting immunization on the antigen emulsified by Freund incomplete adjuvant every 14 days (21 days from the first time to the second time), taking blood from the tail tip of the 7 th-10 th day after three times immunization, diluting the antigen with 1 XPBS by 100 times, centrifuging to obtain supernatant, detecting serum titer and inhibition, performing 1 time of impact immunization when the titer reaches more than 1:100000, namely, directly injecting 0.5mL of immunogen solution into abdominal cavity, fusing spleen cells with myeloma cells after three days, and screening positive holes. Cloning the positive hole by utilizing a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the abamectin monoclonal antibody.
2. Preparation of avermectin monoclonal antibody
(1) Hybridoma cell preparation: SP2/0 myeloma cells were conditioned to a cell density of 1X 10 6/mL using RPMI-1640 basal medium. Three days after the mice are subjected to impact immunization, the feeder spleen cells are taken, the feeder spleen cells are regulated to 1X 10 5/mL, PEG is added to fuse the two cells, then hybridoma positive holes are screened, and positive holes are cloned by a limiting dilution method, so that hybridoma cell strains which stably secrete avermectin monoclonal antibodies are obtained and established.
(2) Preparing ascites: the monoclonal antibody is prepared by adopting an in-vivo induction method, each mouse is injected with 0.5mL of Freund's incomplete adjuvant to purify the abdominal cavity, the hybridoma cell number in the logarithmic growth phase is regulated to 1X 10 6/mL after 7d, and each mouse is inoculated with 0.5mL. The ascites production in mice was observed daily at intervals of about 7 d. When the abdomen of the mouse is obviously enlarged, the spirit is worsened and the mouse is dying, collecting ascites, centrifuging for 5min at 10000r/min, removing surface fat, and carefully sucking the supernatant for ammonium sulfate precipitation.
(3) Antibody purification: ascites (ml) name, volume was recorded. 3mL of PH=4.0.06M sodium acetate buffer solution is added, evenly mixed for 5min, 10 mu L of octanoic acid is added, and stirring is carried out for 15min at 4 ℃. The mixture was filtered through cotton wool once with a syringe. Centrifugation was performed at 12000rpm for 15min at 4 ℃. Taking the supernatant, adding saturated ammonium sulfate with the same volume to a final concentration of 50%, stirring while adding, and standing at 4 ℃ for 3 hours. Centrifugation was performed at 12000rpm for 15min at 4 ℃. The supernatant was discarded, and 1.8mL of 0.01M PBS (pH 7.2) was added to the centrifuge tube until the pellet was completely dissolved. After the total volume was determined, 1/2 volume of saturated ammonium sulfate was added to a concentration of 33% and stirred overnight at 4 ℃. Centrifugation was performed at 12000rpm for 15min at 4 ℃. The supernatant was discarded and the pellet was dissolved by adding 0.45mL of 0.01M PBS. Treating the dialysis bag (boiling for 5min, cleaning with pure water, and detecting leakage). The dissolved solution was placed in a dialysis bag and dialyzed overnight against 0.01M PBS. Changing the dialysate for 2-3 times, collecting antibody, measuring concentration, packaging, and storing in refrigerator at-80deg.C.
Example 3 identification of Avermectin monoclonal antibodies
1. Identification of Avermectin monoclonal antibody subclasses
The monoclonal antibody prepared in example 2 was of IgG1 type as determined by using Catalog No. PK20002 mouse monoclonal antibody subtype assay kit (Proteintech product).
2. Sensitivity identification of avermectin monoclonal antibody
Sensitivity of monoclonal antibody detection by indirect ELISA method.
And drawing a standard curve by using a RIDA SOFT four-parameter method by taking each concentration of avermectin (0,0.2,0.4,0.8,1.6,3.2,6.4, 12.8 ng/mL) as an abscissa and taking an OD 450nm value corresponding to each concentration of avermectin as an ordinate, and calculating half inhibition concentration (IC 50). The results indicated that the curve R 2=0.9962,IC50 = 3.273ng/mL, as in figures 1 and 2, with a detection limit of 0.12ng/mL.
TABLE 1 sensitivity detection
3. Specific detection of avermectin monoclonal antibody
The indirect ELISA method is used for detecting the cross reactivity of the avermectin monoclonal antibody and the avermectin derivative of AVM, doramectin and the structural analogue erythromycin and leukomycin, and the result shows that the cross reactivity rate of the antibody and the medicine is less than 0.01 percent (see table 2), which shows that the avermectin monoclonal antibody prepared in the embodiment 2 has stronger specificity.
TABLE 2 specificity detection
EXAMPLE 4 monoclonal antibody light chain and heavy chain variable region Gene cloning of Avermectin
1. Hybridoma cell culture and total RNA extraction:
Hybridoma cells were cultured 1X 10 7 with RPMI 1640 complete medium at 37℃and 5% carbon dioxide. Cell total RNA was extracted from cultured cell total RNA extraction kit (purchased from Tiangen).
Synthesis of first strand cDNA
TIANSCRIPT II cDNA first Strand Synthesis kit (purchased from Tiangen) cDNA was synthesized.
3. Gene amplification
The Lambda strand, kappa strand, heavy strand downstream primer and upstream universal primer were designed.
Primer: f (SEQ ID NO. 5) AAGCAGTGGTATCAACGCAGAG;
Rκ（SEQ ID NO. 6）:ACATTGATGTCTTTGGGGTAGAAG；
Rλ（SEQ ID NO. 7）:ATCGTACACACCAGTGTGGC；
RH（SEQ ID NO. 8）:GGGATCCAGAGTTCCAGGTC。
and carrying out PCR (polymerase chain reaction) by taking the first strand of the cDNA as a template, wherein the reaction system is 50 mu L.
PCR reaction system: template 3 [ mu ] L, respectively 2.5 [ mu ] L, 2X Pfu PCR MasterMix [ mu ] L, ddH 2 O17 [ mu ] L of an upstream primer and a downstream primer (10 [ mu ] M).
The PCR reaction conditions were: 95 ℃ for 5min; the temperature is 95 ℃ for 30s, the temperature is 63.5 ℃ to 57.5 ℃ for 30s, the temperature is reduced by 0.5 ℃ each time until the temperature reaches 58 ℃, and the cycle is 12 times; 72 ℃ for 1min; cycling for 24 times at 95 ℃ for 30s,56 ℃ for 30s and 72 ℃ for 1min; 7min at 72 ℃.
Cloning and screening of PCR amplified products
The PCR products were subjected to 2% agarose gel electrophoresis, antibody Kappa chain, lambda chain and heavy chain fragments were recovered using a PCR product recovery kit (Beijing day root), the fragments were inserted into pLB vector using a pLB zero background rapid cloning kit (Beijing day root), transformed into DH 5. Alpha. Competent cells (ampicillin resistance), and recombinant positive clones were screened for sequencing.
The PCR product is subjected to 2% agarose gel electrophoresis, and the result shows that the Lambda chain is not provided with a strip, the amino acid sequence of the variable region of the Heavy chain is shown as SEQ ID NO.1, and the gene sequence is shown as SEQ ID NO. 2; the amino acid sequence of the Kappa chain variable region is shown as SEQ ID NO. 3, and the gene sequence is shown as SEQ ID NO. 4.
The Heavy chain variable region amino acid sequence (SEQ ID No. 1):
EVKLEESGPELVKPGASMKMSCKASGYTFTSYIIHWLKQKSSYVMSWVRQTPEKRLEWVASSFSLKIFRLYIKSISSGGSTYYPDSVKGFSKRFTISRDNARNTLYLQMSSLRSEDTALYYISSGINAEYMGPFAAIGGSQWKIDVLLL.
the Heavy chain variable region gene sequence (SEQ ID NO. 2):
GAAGTGAAACTGGAAGAAAGCGGCCCGGAACTGGTGAAACCGGGCGCGAGCATGAAAATGAGCTGCAAAGCGAGCGGCTATACCTTTACCAGCTATATTATTCATTGGCTGAAACAGAAAAGCAGCTATGTGATGAGCTGGGTGCGCCAGACCCCGGAAAAACGCCTGGAATGGGTGGCGAGCAGCTTTAGCCTGAAAATTTTTCGCCTGTATATTAAAAGCATTAGCAGCGGCGGCAGCACCTATTATCCGGATAGCGTGAAAGGCTTTAGCAAACGCTTTACCATTAGCCGCGATAACGCGCGCAACACCCTGTATCTGCAGATGAGCAGCCTGCGCAGCGAAGATACCGCGCTGTATTATATTAGCAGCGGCATTAACGCGGAATATATGGGCCCGTTTGCGGCGATTGGCGGCAGCCAGTGGAAAATTGATGTGCTGCTGCTG.
kappa chain variable region amino acid sequence (SEQ ID NO. 3):
YSPSFQGQVTISADKSINTAYLQWSSLKASDQKPGTSPKLWIYSTNLASGVPDRSRQNEVQSLRARFSGSGLSHSEEDHVFPSSGTSYSLTISRMEAEDAATYYCQRTSYPFTFGSGTKLEIK.
Kappa chain variable region gene sequence (SEQ ID NO. 4):
TATAGCCCGAGCTTTCAGGGCCAGGTGACCATTAGCGCGGATAAAAGCATTAACACCGCGTATCTGCAGTGGAGCAGCCTGAAAGCGAGCGATCAGAAACCGGGCACCAGCCCGAAACTGTGGATTTATAGCACCAACCTGGCGAGCGGCGTGCCGGATCGCAGCCGCCAGAACGAAGTGCAGAGCCTGCGCGCGCGCTTTAGCGGCAGCGGCCTGAGCCATAGCGAAGAAGATCATGTGTTTCCGAGCAGCGGCACCAGCTATAGCCTGACCATTAGCCGCATGGAAGCGGAAGATGCGGCGACCTATTATTGCCAGCGCACCAGCTATCCGTTTACCTTTGGCAGCGGCACCAAACTGGAAATTAAA.
EXAMPLE 5 variable region Gene and amino acid sequence homology analysis
The comparison analysis is carried out in NCBI database, and the analysis result shows that the abamectin monoclonal antibody light chain variable region gene Sequence has the highest homology with the synthesized single-chain antibody ScFv gene Sequence (Sequence ID: EU 589245.1), the homology is 99/114, and the homology percentage is 87%; the amino acid Sequence of the light chain variable region of the avermectin monoclonal antibody has the highest homology with the light chain (Sequence ID: AAB 05147.1) of the immune group 5A12.A10, the homology is 69/118, and the homology percentage is 58%. The amino acid Sequence of the heavy chain variable region of the avermectin monoclonal antibody has the highest homology with TPA (IGHV 5-6-5X 01) (Sequence ID: DBA 12656.1), the homology percentage is 84/125, the homology percentage is 67%, the amino acid Sequence of the heavy chain variable region of the avermectin monoclonal antibody has the highest homology with the mRNA Sequence of the muscle strain BALB/c anti-BaP 1 metalloproteinase (Sequence ID: KF 724934.1), the homology percentage is 102/114, and the homology percentage is 89%. The results of the homology analysis of the gene sequences and amino acid sequences of the light chain and heavy chain variable regions of the avermectin-encoding monoclonal antibodies show that the sequences identical to the present invention are not found.
The heavy chain variable region and the light chain variable region sequences were analyzed at https:// www.novopro.cn/tools/cdr.htmL using the Kabat protocol to derive the CDR regions.
The sequences of the 3 Complementarity Determining Regions (CDRs) of the heavy chain variable region of the avermectin monoclonal antibody are respectively:
CDR-H1（SEQ ID NO. 9）:SYIIHWL；
CDR-H2（SEQ ID NO. 10）:SSFSLKIFRLYIKSISSGGSTYYPDSVKGFSK；
CDR-H3:GI。
The sequences of the 3 Complementarity Determining Regions (CDRs) of the light chain variable region of the avermectin monoclonal antibody are respectively:
CDR-L1（SEQ ID NO. 11）:ADKSINTAYLQWSSLK；
CDR-L2（SEQ ID NO. 12）:STNLASGVPDRSRQN；
CDR-L3（SEQ ID NO. 13）:QRTSYPFT。
example 6 preparation of Abamectin colloidal gold quantitative detection test strip
The test strip (shown in figure 3) consists of absorbent paper, a nitrocellulose membrane (NC membrane), a sample pad and a bottom plate; the nitrocellulose membrane (NC membrane) was scored with a scoring instrument in two parallel bands, with a 6.5mm spacing between the bands, and a band width of about 1mm. The first one is a quality control line and is close to the water absorption paper end, and the second one is a detection line and is close to the sample pad end. Spraying goat anti-mouse (IgG) and one ten thousandth blue pigment on the quality control line, wherein the concentration is 0.5-1 mg/ml; the concentration of the AVW-OVA antigen sprayed on the second strip is 0.1-0.5 mg/ml. Obtaining the chromatographic membrane, drying at 40 ℃ for 16 hours, sealing, and preserving at normal temperature. Sequentially adhering water absorbing paper, NC film and sample pad on the bottom plate, wherein the initial end of the sample pad is connected with the tail end of the NC film, the initial end of the NC film is connected with the water absorbing pad, the tail end of the sample pad is aligned with the tail end of the bottom plate, and the initial end of the water absorbing pad is aligned with the initial end of the bottom plate. The glued plastic plate was cut longitudinally into test strips 4.5mm wide by a slitter. The microporous reagent is provided with a microporous plug, and the microporous reagent is freeze-dried with an AVM monoclonal antibody-colloidal gold marker. 8 cut test strips and 1 microporous reagent are taken and put into a reagent bucket for sealing, and the test strips and the 1 microporous reagent are preserved at the temperature of 2-8 ℃.
Example 7 definition of detection limits and specificity of Avermectin detection kit
1. Detection method
Weighing a blank sample of 5.0g plus or minus 0.02g, homogenizing, sieving with a 20-mesh sieve, and respectively adding an avermectin standard substance into the blank sample until the mass concentration is 0, 20, 40, 80, 160 and 320 mug/kg to obtain samples with different avermectin concentrations. Transferring 200 mu L of liquid to be detected into a microporous reagent by using a micropipette, slowly pumping until a sample solution and the microporous reagent are fully and uniformly mixed, placing the mixture on an incubator, and performing incubation reaction at 40 ℃ for 3 min; after incubation, immediately inserting the test paper strip into the microporous reagent, enabling the end of the water absorbing paper to be upward, enabling the other end to be downward, fully immersing the test paper strip into the solution, and reacting for 5min; after the reaction is finished, the test strip is taken out, a sample pad at the lower end of the test strip is removed, and a homogeneous phase immunity detector is immediately used for reading the detection result. Care was taken not to touch or damage the central test area of the test strip and the results were read within 1 min.
2. Analysis of detection results
Drawing a standard curve according to the detection result of the standard substance, and corresponding the detection result of the sample to the standard curve to obtain the content of the avermectin in the detection sample: detecting detection bands and quality control band signals of the quality control products with various concentrations by using a colloidal gold quantitative analyzer, obtaining a T/C value after internal calculation of the instrument, drawing a standard curve by taking the T/C value as an ordinate and the corresponding quality control product concentration as an abscissa, wherein y= -0.00001x2+0.00153x+9.923, and R 2 >0.99, and the standard curve is shown in figure 4.
Collecting a sample to be detected, weighing 5.0 g+/-0.02 g of the sample to be detected, homogenizing, sieving with a 20-mesh sieve, testing, using a colloidal gold quantitative analyzer to read standard curve information on an ID card, and calculating by internal software to obtain the AVM content in the sample.
3. Detection limit definition
Taking 20 blank samples to be detected, detecting by using an avermectin colloidal gold quantitative detection test strip, calculating the average value of the samples, and adding 3 times of standard deviation, namely the lowest detection limit. The detection result is shown in Table 3, and the lowest detection limit of the detection test strip is 2.85 mug/kg.
Table 3 statistical table of blank sample measurement results (μg/kg)
4. Specific detection
Respectively preparing standard substance solutions of (A) blank samples, (B) avermectin, (C) ivermectin, (D) doramectin and structural analogues thereof, (E) erythromycin and (F) white mold medicaments, wherein the concentration of the avermectin standard substance solution is 64 mug/kg, and the concentration of other standard substance solutions is 1000 mug/kg. The assay was performed using an avermectin colloidal gold quantitative test strip, and each sample was repeated three times. As shown in fig. 5, only marked inhibition was produced in the labeling assay for avermectin. The result shows that the test strip prepared by the invention has good specificity to avermectin.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (4)

1. An avermectin monoclonal antibody, which is characterized in that: the monoclonal antibody contains a heavy chain variable region named V H and a light chain variable region named V L;
the amino acid sequence of V H of the monoclonal antibody is shown as SEQ ID No. 1;
The amino acid sequence of V L of the monoclonal antibody is shown as SEQ ID No. 3;
Each of V H and V L consists of a complementarity determining region and a framework region, the complementarity determining regions each consisting of CDR1, CDR2, and CDR 3;
The amino acid sequence of CDR1 of V H of the monoclonal antibody is shown as 31 st-37 th position in SEQ ID No. 1;
The amino acid sequence of CDR2 of V H of the monoclonal antibody is shown as 61-92 th position in SEQ ID No. 1;
The amino acid sequence of CDR3 of V H of the monoclonal antibody is shown as 125 th-126 th position in SEQ ID No. 1;
The amino acid sequence of CDR1 of V L of the monoclonal antibody is shown as 13 th-28 th position in SEQ ID No. 3;
the amino acid sequence of CDR2 of V L of the monoclonal antibody is shown as 43-58 th position in SEQ ID No. 3;
The amino acid sequence of CDR3 of V L of the monoclonal antibody is shown as 106-113 th position in SEQ ID No. 3.
2. The avermectin monoclonal antibody of claim 1, characterized in that:
The nucleotide sequence of V H for encoding the monoclonal antibody is shown as SEQ ID No. 2;
The nucleotide sequence of V L for encoding the monoclonal antibody is shown as SEQ ID No. 4.
3. Use of the avermectin monoclonal antibody of claim 1 in the preparation of an avermectin detection product.
4. An abamectin colloidal gold quantitative detection test strip, which is characterized in that the colloidal gold quantitative detection test strip contains a colloidal gold-labeled monoclonal antibody of abamectin as defined in claim 1.
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