Hapten, artificial antigen of avermectin b1a and / or its structural analogs and application thereof

Abstract

The application discloses a hapten of abamectin B1a and / or its structural analog, an artificial antigen and application thereof. The application connects a spacer to abamectin after hydrolysis to obtain a hapten of abamectin B1a and / or its structural analog, and then connects a carrier protein to prepare an artificial antigen, and further prepare a monoclonal antibody. A chemiluminescence immunoassay method is established according to specific recognition of the antibody and the antigen, and abamectin residues in a sample are detected. The method can be used for trace detection of abamectin residues, and can avoid a complicated pretreatment step. The detection limit is 0.03 ng / mL, the IC 50 (50% inhibitory concentration) is 0.1 ng / mL, and the linear range IC 20 ~IC 80 is 0.04-0.20 ng / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, more particularly, to a hapten of avermectin B1a and / or its structural analog, artificial antigen and application thereof. BACKGROUND

[0002] Avermectin, also known as Amdinocid, Amicarbate and Amdenocide, is a kind of macrolide drug produced by fermentation of Streptomyces avermitilis, which was discovered by the team of Dr. Tomisaku Kuwano of Kitasato University in Japan in the late 1970s, and was industrialized by the cooperation with Merck in the late 1980s. Avermectin has high efficiency, broad spectrum of insecticidal, acaricidal and nematocidal ability. Avermectin widely used biological pesticides and veterinary drugs plays an important role in the pest control system of agricultural planting and livestock breeding in China. However, avermectin has hepatotoxicity and neurotoxicity, and long-term intake will cause harm to health. Animal toxicology studies show that the oral median lethal concentration (LD 50 ) of rats is 10 mg / kg, which is a highly toxic compound.

[0003] Currently, the detection of avermectin residues mainly relies on instrumental methods, which have the advantages of high sensitivity, good reproducibility and high degree of automation, and are the most widely used analysis method. However, instrumental analysis method also has obvious shortcomings, such as high technical threshold, requiring trained professionals to operate; complex and strict sample pretreatment, some even need to be derivatized to meet the requirements of instrumental analysis; high cost of purchasing and maintaining advanced analytical instruments; stable environment is required to ensure the accuracy of the analysis results, which cannot be used for on-site rapid screening. Immunoassay is based on the principle of specific binding of antigen-antibody, and outputs the analysis results through other signal conversion. It has high specificity, good sensitivity, convenience, simplicity, and can complete on-site detection without special operation.

[0004] The prior art discloses an avermectin enzyme-linked immunosorbent assay kit and application, which can be used for detecting ivermectin and avermectin, and the detection limit of avermectin in milk is 1.5 μg / kg, which has good sensitivity. However, the sample pretreatment of this method needs centrifugation, blow-drying and re-dissolving operations, and long-time incubation and color development are required during detection, the test time is not less than 45 minutes, the operation steps are more, and the operation precision is required to be high. In addition, some sample matrix particles (such as protein and pigment) may be adsorbed or precipitated in the plastic micropore, causing the absorbance value to be high, resulting in inaccurate determination results. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art, and provides a hapten of avermectin B1a and / or its structural analog, artificial antigen and application thereof.

[0006] The first object of the present application is to provide a hapten of avermectin B1a and / or its structural analogues.

[0007] The second object of the present application is to provide the use of the hapten in the preparation of artificial antigen of avermectin B1a and / or its structural analogues.

[0008] The third object of the present application is to provide a hapten of avermectin B1a and / or its structural analogues.

[0009] The fourth object of the present application is to provide the use of the artificial antigen in the preparation of antibody of avermectin B1a and / or its structural analogues.

[0010] The fifth object of the present application is to provide the use of the hapten and / or the artificial antigen in the detection of avermectin B1a structural analogues for non-diagnostic purposes.

[0011] The sixth object of the present application is to provide the use of the hapten and / or the artificial antigen in the preparation of kit for detecting avermectin B1a structural analogues.

[0012] The seventh object of the present application is to provide a kit for detecting avermectin B1a and / or its structural analogues.

[0013] The eighth object of the present application is to provide a method for detecting avermectin B1a and / or its structural analogues.

[0014] In order to achieve the above objects, the present application is implemented by the following technical solutions:

[0015] A hapten of avermectin B1a and / or its structural analogues, the structural formula of which is as shown in formula (I)

[0016]

[0017] Preferably, the avermectin B1a structural analogues are one or more of ivermectin, emamectin benzoate and abamectin.

[0018] The preparation method of the hapten is as follows: avermectin B1a is reacted with 10% sulfuric acid in tetrahydrofuran to sufficiently hydrolyze and remove the sugar chain group to obtain intermediate 1; ethyl acetate and water are used for extraction, the organic layer is rotary evaporated, and intermediate 1 is obtained by silica gel column purification.

[0019] Intermediate 1 is reacted in a solution of succinic anhydride in dichloromethane, extracted with ethyl acetate and water, the organic layer is rotary evaporated, and the final product is obtained by column purification.

[0020] The application also relates to the use of the hapten in the preparation of an artificial antigen of abamectin B1a and / or its structural analogues.

[0021] The application also relates to a hapten of abamectin B1a and / or its structural analogues, which has the structural formula (II),

[0022]

[0023] Preferably, the abamectin B1a structural analogues are one or more of ivermectin, emamectin benzoate and abamectin.

[0024] The application also relates to the use of the artificial antigen in the preparation of an antibody of abamectin B1a and / or its structural analogues.

[0025] The application also relates to the use of the hapten and / or the artificial antigen in the detection of abamectin B1a and / or its structural analogues for non-diagnostic purposes.

[0026] The application also relates to the use of the hapten and / or the artificial antigen in the preparation of a kit for detecting abamectin B1a and / or its structural analogues.

[0027] Preferably, the abamectin B1a structural analogues are one or more of ivermectin, emamectin benzoate, abamectin and doramectin.

[0028] The application also relates to a kit for detecting abamectin B1a and / or its structural analogues, which comprises an anti-abamectin antibody and the artificial antigen, wherein the anti-abamectin antibody is prepared by immunizing animals with the artificial antigen.

[0029] Preferably, the anti-abamectin antibody is a monoclonal antibody, which is labeled with alkaline phosphatase; and the artificial antigen is coupled with magnetic beads.

[0030] More preferably, the kit further comprises acetonitrile.

[0031] More preferably, the kit further comprises one or more of a luminescent solution, a washing solution or PBS.

[0032] The preparation method of the artificial antigen coupled with magnetic beads is as follows: the artificial antigen of abamectin with the structural formula (II) is coupled with magnetic beads with a surface-modified functional group (such as a p-toluenesulfonyl group or a carboxyl group).

[0033] The specific method is as follows: the magnetic beads with p-toluenesulfonyl group on the surface are washed with ammonium sulfate-boric acid buffer (ammonium sulfate concentration is 100 mmol / L and boric acid buffer concentration is 30 mmol / L), mixed with the artificial antigen of avermectin with structural formula as shown in formula (II) in 10 mL ammonium sulfate-boric acid buffer, fully reacted, then blocked, the supernatant is discarded, and the magnetic beads are separated, thereby obtaining the product.

[0034] As a specific example, the kit contains:

[0035] The freeze-dried microbeads containing the anti-avermectin monoclonal antibody labeled with alkaline phosphatase, wherein the anti-avermectin monoclonal antibody is prepared by using the artificial antigen of avermectin with structural formula as shown in formula (II) as an immunogen;

[0036] The freeze-dried microbeads containing the artificial antigen conjugate of avermectin with structural formula as shown in formula (II) coupled with magnetic beads;

[0037] The sample treatment solution (acetonitrile), the reaction buffer (0.01 mol / L PBS solution), the washing solution (0.6 g / L Tris solution, and hydrochloric acid is used to adjust the pH to 9.5), and the luminescence solution (containing 3-(2-spiro adamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane disodium salt);

[0038] The freeze-dried microbeads are prepared by liquid nitrogen freeze-drying, and the entire kit is stored at 2-8℃.

[0039] The use method is as follows:

[0040] The milk sample is mixed with acetonitrile, and then mixed with the reaction buffer to obtain the test solution;

[0041] The fruit and vegetable sample is extracted with acetonitrile, and the liquid is used as the test solution

[0042] The test solution is fully mixed with the freeze-dried microbeads containing the anti-avermectin monoclonal antibody labeled with alkaline phosphatase, and incubated;

[0043] Then, the freeze-dried microbeads containing the artificial antigen conjugate of avermectin with structural formula as shown in formula (II) coupled with magnetic beads are fully mixed and incubated;

[0044] The magnetic beads are separated, washed with the washing solution, and placed in the luminescence solution to read the luminescence value.

[0045] A method for detecting avermectin B1a and / or its structural analogs, which utilizes an anti-avermectin antibody as a detection antibody and the artificial antigen as a detection antigen for detection, wherein the anti-avermectin antibody is prepared by immunizing animals with the artificial antigen.

[0046] Preferably, the anti-avermectin antibody is a monoclonal antibody, which is labeled with alkaline phosphatase; and the detection antigen is coupled with magnetic beads.

[0047] As a specific example, the detection antibody is lyophilized microbeads containing anti-avermectin monoclonal antibody labeled with alkaline phosphatase, and the anti-avermectin monoclonal antibody is prepared by using an avermectin artificial antigen with the structural formula as shown in formula (II) as an immunogen;

[0048] The detection antigen is lyophilized microbeads containing avermectin artificial antigen conjugate with the structural formula as shown in formula (II) coupled with magnetic beads;

[0049] The specific detection method is as follows:

[0050] The milk sample is mixed with acetonitrile, and then mixed with a reaction buffer to obtain a test solution;

[0051] The fruit and vegetable sample is extracted with acetonitrile, and then the liquid is taken as the test solution

[0052] The test solution is mixed with the detection antibody and incubated;

[0053] Then the detection antigen is added, mixed thoroughly, and incubated;

[0054] The magnetic beads are separated, washed with a washing solution, and placed in a luminescence solution to read the luminescence value.

[0055] Compared with the prior art, the present application has the following beneficial effects:

[0056] The present application connects a spacer to avermectin after hydrolysis to obtain a hapten of avermectin B1a and / or its structural analog, and then couples the hapten with a carrier protein to prepare an artificial antigen, and further prepares a monoclonal antibody. According to the specific recognition of the antibody and the antigen, a chemiluminescence immunoassay method is established to detect avermectin residues in a sample. The method can eliminate the cumbersome pretreatment steps for trace detection of avermectin residues, the detection limit is 0.03 ng / mL, the IC 50 (50% inhibitory concentration) is 0.1 ng / mL, and the linear range IC 20 ~ IC 80 is 0.04-0.20 ng / mL. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 is the mass spectrum identification of the avermectin hapten.

[0058] Figure 2 is the hydrogen spectrum result of the avermectin hapten.

[0059] Figure 3 is the carbon spectrum result of the avermectin hapten.

[0060] Figure 4 is the UV absorption chart of the artificial antigen of abamectin.

[0061] Figure 5 is the identification chart of the magnetic beads coupled with abamectin.

[0062] Figure 6 is the standard curve of the chemiluminescence detection of abamectin.

[0063] Figure 7 is the accelerated test chart of the chemiluminescence kit of abamectin. DETAILED DESCRIPTION

[0064] The present application will be further described below in conjunction with the drawings and specific examples of the specification, which are only used to explain the present application and are not used to limit the scope of the present application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0065] Example 1 Synthesis of abamectin hapten

[0066] I. Experimental methods

[0067] S1. (2S, 2a'E, 2a1'S, 4'E, 5S, 6R, 6'S, 7'S, 8'E, 11'R, 15'S, 17a'R, 20'R, 20a'R)-6-((S)-sec-butyl)-2a1', 20'-dihydroxy-7'-(((2R, 4S, 5S, 6S)-5-(((2S, 4S, 5S, 6S)-5-hydroxy-4-methoxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-methoxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-5, 6', 8', 19'-tetramethyl-2a1', 5, 6, 6', 7', 10', 11', 14', 15', 17a', 20', 20a'-dodecahydro-2'H, 17'H-spiro[pyrane-2, 13'-[11, 15]methano[1, 5]dioxocin[9, 8, 7-cd]benzofuran]-17'-one (abamectin B1a, CAS No: 65195-55-3) (5.00 g 5.726 mmol) was taken into a single-necked flask, then tetrahydrofuran 94 mL was added for dissolution, and an aqueous solution containing 10% sulfuric acid 94 mL was measured, and then it was added dropwise at room temperature, and after the dropwise addition was completed, the temperature was raised to 65°C and reacted for 24 h. After the reaction was completed, an appropriate amount of water was added, and extraction was performed three times with ethyl acetate, and the organic phase was combined and dried and concentrated to obtain intermediate 1, and the reaction formula is as follows:

[0068]

[0069] S2. Intermediate 1 (951.3 mg 1.627 mmol), succinic anhydride (163 mg 1.627 mol), DMAP (1.988 g 16.268 mmol) were added to a single neck flask, followed by dry dichloromethane 10 mL. After stirring homogenously, triethylamine (3.29 g 32.537 mmol 4.52 mL) was added. The reaction was carried out at 50 °C for 3 h. After the reaction was completed, the solvent was removed by rotary evaporation, water and ethyl acetate were added, the aqueous phase was adjusted to weakly acidic with hydrochloric acid. The ethyl acetate was extracted 3 times, the organic phase was combined and washed with water 3 times. The organic phase was dried and concentrated, and the yellow foamy solid final product was obtained by column. The reaction scheme is as follows:

[0070]

[0071] II. Experimental results

[0072] The mass spectrum results of the final product are shown in Figure 1 ;

[0073] The hydrogen spectrum results of the final product are shown in Figure 2 ; 1H NMR (600 MHz, Methanol-d4) δ 5.89 (dd, J = 14.7, 11.3 Hz, 1H), 5.83 (dt, J = 11.3, 2.4 Hz, 1H), 5.75 (dd, J = 9.9, 1.8 Hz, 1H), 5.70 (dd, J = 14.7, 10.0 Hz, 1H), 5.61 (q, J = 1.6 Hz, 1H), 5.54 (dd, J = 9.9, 2.6 Hz, 1H), 5.48 (dq, J = 4.2, 1.3 Hz, 1H), 5.44 (ddt, J = 11.4, 4.9, 1.6 Hz, 1H), 5.03 (tt, J = 11.4, 4.6 Hz, 1H), 4.64 (dd, J = 14.2, 2.5 Hz, 1H), 4.56 (dd, J = 14.2, 2.3 Hz, 1H), 3.95 (d, J = 5.8 Hz, 2H), 3.88 (tdd, J = 10.8, 4.9, 2.1 Hz, 1H), 3.47 (dd, J = 9.9, 1.8 Hz, 1H), 3.26 (q, J = 2.4 Hz, 1H), 2.68 - 2.58 (m, 5H), 2.32 - 2.23 (m, 3H), 2.20 (ddd, J = 12.0, 4.6, 1.9 Hz, 1H), 1.97 - 1.91 (m, 1H), 1.74 (s, 3H), 1.67 - 1.62 (m, 1H), 1.57 - 1.52 (m, 4H), 1.49 - 1.44 (m, 1H), 1.38 - 1.31 (m, 2H), 1.14 (d, J = 7.0 Hz, 3H), 0.98 (t, J = 7.4 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 7.2 Hz, 3H).

[0074] The carbon spectrum results of the final product are as shown in Figure 3 13 C NMR (151 MHz, Methanol-d4) δ 175.86, 173.87, 172.89, 140.63, 140.54, 138.62, 136.64, 132.90, 129.28, 126.14, 123.22, 122.42, 118.13, 97.24, 81.85, 79.66, 78.31, 76.18, 71.90, 70.21, 69.97, 68.75, 46.91, 42.09, 41.48, 37.38, 36.58, 35.20, 31.77, 29.97, 29.81, 28.70, 19.70, 19.49, 16.80, 14.71, 13.31, 12.45;

[0075] In summary, the structural formula of the final product is as shown in formula (I),​

[0076]

[0077] Preparation of artificial antigen of abamectin

[0078] I. Experimental method

[0079] Using the abamectin hapten with structural formula as shown in formula (I) prepared in Example 1, the carrier protein (bovine serum albumin BSA and lactoferrin LF) was coupled by active ester method, and the specific steps were as follows:

[0080] Under stirring, the N, N-dimethylformamide solution of the hapten with structural formula as shown in formula (I), 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide were mixed, and stirred at room temperature for 4 h in dark to obtain A liquid.

[0081] The carrier protein was dissolved in carbonic acid buffer to obtain B liquid, and the concentration of the carrier protein was 10 mg / mL.

[0082] Under stirring in ice bath, A liquid was added dropwise into B liquid, and the pH was adjusted to 9.5-9.6 by NaOH solution or carbonic acid buffer, and reacted for 12 h in dark. After dialysis and purification, the artificial antigen with structural formula as shown in formula (II) was obtained.

[0083] The molar ratio of the abamectin hapten with structural formula as shown in formula (I) prepared in Example 1, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide was 1:1.2:1.5.

[0084]

[0085] II. Experimental results

[0086] The absorption light of the abamectin hapten with structural formula as shown in formula (I) prepared in Example 1, the abamectin artificial complete antigen (Protein was BSA or LF) with structural formula as shown in formula (II) and the carrier protein solution (bovine serum albumin BSA or lactoferrin LF) in ultraviolet region (200-400 nm) was scanned respectively, and the results were shown in Table 1. Figure 4 The characteristic peak of ultraviolet absorption of the conjugate had a certain degree of red shift or blue shift relative to the original carrier protein, which proved that the artificial antigen was successfully prepared.

[0087] Preparation of abamectin antibody

[0088] I. Experimental method

[0089] 1. Animal immunization

[0090] Take 250 μL prepared and diluted to 1 mg / mL of the artificial antigen of abamectin with structural formula as shown in formula (II) (Protein is LF) and equal amount of immunoadjuvant (Freund's complete adjuvant for the first immunization, and Freund's incomplete adjuvant for the subsequent booster immunization) to emulsify uniformly, immunize animals, and immunize 6-7 week old Balb / c mice by various injection methods such as back subcutaneous injection, subcutaneous injection in various parts, intraperitoneal injection and foot injection, with an immunization amount of 100 μL per mouse, and the second immunization is performed after 2 weeks, and the booster immunization is performed every 2 weeks thereafter. The mouse tail blood is taken 1 week after the third booster immunization, and the titer and inhibition rate of the antiserum are determined by using indirect competitive ELISA. When the titer no longer increases, the mouse with better titer and inhibition rate is selected for the next test: 100 μL of the abamectin immunization antigen with a concentration of 1 mg / mL is injected intraperitoneally for impact immunization, and the cell fusion is performed within 48-72 hours after the injection.

[0091] The specific method for determining the serum titer by indirect competitive ELISA is as follows:

[0092] S1. The artificial antigen of abamectin with structural formula as shown in formula (II) (Protein is LF) is diluted to 0.25 μg / mL in a pH 7.4 0.01 mol / L phosphate buffer solution, and is added to an enzyme-labeled plate, with 100 μL per well, and is coated at 37°C; the liquid in the well is removed, the plate is washed twice, and the liquid in the well is shaken dry; 5% skimmed milk powder is added for blocking, the liquid in the well is removed, and the plate is dried for standby;

[0093] S2. 50 μL of the sample to be tested (blank, negative and positive well controls are simultaneously prepared) is added to the enzyme-labeled plate in S1; the serum is diluted with a pH 7.4 0.01 mol / L phosphate buffer solution, and is added for full reaction, the plate is washed, and the liquid in the well is removed;

[0094] S3. 100 μL of HRP-goat anti-mouse diluted 5000 times is added to the enzyme-labeled plate in S2 for full reaction, and the plate is washed as in S2;

[0095] S4. TMB color developing liquid and 0.1% hydrogen peroxide solution (v / v) are mixed in equal amounts, and are added to the wells of the enzyme-labeled plate in S3 for full color development, and the termination liquid is added per well;

[0096] S5. The absorbance value of each well in S4 at A450 nm is determined by using an enzyme-linked immunosorbent detector.

[0097] 2. PEG (polyethylene glycol) is used for cell fusion three days after impact immunization, and the specific steps are as follows:

[0098] a, Collecting mouse spleen cells: the mice were executed by cervical dislocation method, and immediately immersed in 75% alcohol for sterilization. The spleen of the mouse was taken out under aseptic operation, put into a 200-mesh cell screen, and gently ground with the rubber head of a sterile syringe. The spleen cell suspension was obtained by washing with basal medium, collected, centrifuged (1000 rpm, 7 min), and washed with basal medium three times. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and reserved for use;

[0099] b, Collecting SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 myeloma cells were cultured in a 5% CO2 incubator with complete medium. The number of SP2 / 0 tumor cells was required to reach 1-4 x 10 7 Before fusion, the SP2 / 0 tumor cells were in the logarithmic growth phase. The myeloma cells were collected and suspended in basal medium for cell counting;

[0100] c, Mixing the two cells according to the ratio of spleen cells: SP2 / 0 = 5:1, centrifuging, discarding the supernatant, and obtaining the mixed cells deposited at the bottom of the centrifuge tube;

[0101] d, Fusion: in the first minute, 1 mL of PEG was slowly added to the cells at the bottom of the centrifuge tube; in the second minute, the centrifuge tube was shaken evenly; in the third minute, 1 mL of preheated basal medium was added; in the fourth minute, 3 mL of preheated basal medium was added; in the fifth minute, 8 mL of preheated basal medium was added; in the sixth minute, 8 mL of preheated basal medium was added; centrifuging (1000 rpm, 7 min), discarding the supernatant, and resuspending in HAT-containing screening medium, adding 200 μL / well to a 96-well cell plate, and culturing in a 37°C, 5% CO2 incubator;

[0102] 3, Cell screening and cell strain establishment

[0103] On the 5th day of cell fusion, the fusion cells were semi-replaced with HT medium, and on the 8th day, they were fully replaced. On the 10th day, the cell supernatant was taken, screened by ic-ELISA, and the positive cells were determined for potency inhibition effect. The cell wells that had good inhibition on the standard avilamycin were selected, subcloned by limiting dilution, detected by the same method, repeated 4-5 times, and the cell strain was obtained

[0104] 4, Preparation and identification of monoclonal antibody

[0105] Take several 10-week-old Balb / c mice, and inject each mouse with 500 μL of liquid paraffin into the abdominal cavity; 7 days later, inject each mouse with about 1 x 10 6The hybridoma cells were collected after 7 days when the mouse abdomen was swollen. The ascites were collected and purified by chromatography column. The column was filled with 1 mL protein G packing. 50 mL ascites diluted by PBS buffer was added. The flow-through was repeatedly loaded for 7-8 times. Then, glycine (0.1 mol / L) was used for elution. The eluate was timely adjusted to neutral by Tris-HCL. The eluate was dialyzed and desalted. Finally, the purified abamectin monoclonal antibody was obtained. After being labeled with alkaline phosphatase, the antibody was reconstituted with lyophilization protectant (60 g / L mannitol, 40 g / L polyethylene glycol, 10 g / L casein, 20 g / L glycine, 12 g / L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and the pH was adjusted to 7.4). After being adjusted to 25 ng / mL, the antibody was prepared into lyophilized microbeads with a specification of 15 μL / bead.

[0106] II. Experimental results

[0107] The anti-serum detection results are shown in Table 1. The results show that the immunogen can induce immune response reaction of the mouse to produce antibodies. The inhibition effect of the No. 3 mouse is only 4% different from that of the No. 2 mouse. The titer is doubled. Therefore, the No. 3 mouse is selected for subsequent experiments.

[0108] Table 1. Anti-serum identification results

[0109]

[0110] Example 4. Preparation of abamectin artificial antigen-magnetic bead conjugate

[0111] I. Experimental method

[0112] The abamectin artificial antigen with the structural formula shown in formula (II) prepared in Example 2 was coupled with magnetic beads modified with p-toluenesulfonyl groups (Japan / JSR Life science, 1002TA-01). The specific steps are as follows: the magnetic beads were mixed at room temperature for 1 h. 1 mL of Tosyl magnetic beads (100 mg / mL) was taken into a 50 mL centrifuge tube. The magnetic beads were separated and the supernatant was discarded. 10 mL of 100 mmol / L ammonium sulfate-boric acid buffer (100 mmol / L ammonium sulfate and 30 mmol / L boric acid buffer solution) was added for washing twice. 5 mL of ammonium sulfate-boric acid buffer and 100 μL of artificial antigen (1 mg / mL) were added and mixed. The mixture was mixed at 37°C for 18 h. 5 mL of 10% BSA blocking solution was added and mixed at 37°C for 12 h. The magnetic beads were separated and the supernatant was discarded. 20 mL of lyophilization protectant (60 g / L mannitol, 40 g / L polyethylene glycol, 10 g / L casein, 20 g / L glycine, 12 g / L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and the pH was adjusted to 7.4) was added for reconstitution. After being adjusted to 1 mg / mL, the mixture was lyophilized into lyophilized microbeads with a volume of 15 μL / bead.

[0113] ELISA assay of the conjugated magnetic beads with the conjugated magnetic beads and the unconjugated artificial antigen, the specific steps are as follows:

[0114] S1. Dilute the magnetic beads to 0.1 mg / mL with 0.01 mol / L phosphate buffer, take 50 μL into a 0.5 mL centrifuge tube, then add 50 μL of the abamectin monoclonal antibody prepared in Example 3 diluted 20000 times with 0.01 mol / L phosphate buffer, mix well, and react at 37°C;

[0115] S2. Separate the magnetic beads with a magnetic stand, and wash 5 times with 0.01 mol / L phosphate buffer containing 0.05% Tween (PBST);

[0116] S3. Add HRP-goat anti-mouse diluted 5000 times again, mix well, and react at 37°C. Separate the magnetic beads and wash as in S2;

[0117] S4. Mix equal volumes of TMB color developing solution and 0.1% hydrogen peroxide solution (v / v), and add into the centrifuge tube in S3, develop color fully, and then add 100 μL of stop solution;

[0118] S5. Take 150 μL of the solution after stopping in S4, and add into a microplate, and determine the absorbance of each well at A450 nm with an enzyme-linked immunodetection instrument.

[0119] II. Experimental results

[0120] The conjugated magnetic beads, the conjugated magnetic beads and the unconjugated artificial antigen are determined by ELISA experiment, and the results are shown in Table 1. Figure 5 The conjugated magnetic beads can be recognized by the antibody, indicating that the artificial antigen-magnetic bead conjugate is successfully prepared.

[0121] Example 5. Establishment of abamectin direct competition chemiluminescence standard curve

[0122] I. Experimental method

[0123] The freeze-dried microbeads (25 ng / mL, 15 μL / bead) containing the anti-abamectin monoclonal antibody labeled with alkaline phosphatase prepared in Example 3 and the freeze-dried microbeads (1 mg / mL, 15 μL / bead) containing the abamectin artificial antigen-magnetic bead conjugate prepared in Example 4 are used for direct competition chemiluminescence immunoassay. The specific steps are as follows:

[0124] 1 bead of lyophilized microbead containing anti-ivermectin monoclonal antibody labeled with alkaline phosphatase prepared in Example 3 was incubated with 60 μL of gradient diluted ivermectin B1a (CAS No. 65195-55-3) standard solution (concentrations were 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, 0.125 ng / mL, 0.0625 ng / mL, 0.032 ng / mL, 0.016 ng / mL, 0.008 respectively) at 37°C for 150 seconds.

[0125] 1 bead of lyophilized microbead containing ivermectin artificial antigen-magnetic bead conjugate prepared in Example 4 was added and incubated at 37°C for 300 seconds.

[0126] The magnetic beads were separated with a magnetic stand, washed twice with washing solution (0.6 g / L Tris solution, pH adjusted to 9.5 with hydrochloric acid), and 60 μL of luminous solution (main component was 3-(2-spiro adamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxetane disodium salt) was added. The luminescence value was read with a chemiluminescence instrument.

[0127] The IC50value of the inhibition curve was calculated with the four-parameter fitting module of Origin 8.5. 10 , IC 20 , IC 50 , IC 80 value. The ivermectin B1a drug concentration was taken as the abscissa, and the RLU / RLU0ratio was taken as the ordinate. A scatter plot was drawn in Origin software and a logistic function was fitted to obtain the standard curve.

[0128] II. Experimental results

[0129] The results are shown in Table 1. Figure 6 The IC 10 (limit of detection) of this method for ivermectin was 0.03 ng / mL, the IC 50 (half-inhibitory concentration) was 0.1 ng / mL, and the linear range IC 20 ~ IC 80 was 0.04~0.20 ng / mL.

[0130] Example 6 A chemiluminescence detection kit for detecting ivermectin

[0131] I. Composition

[0132] Lyophilized microbeads containing anti-ivermectin monoclonal antibody labeled with alkaline phosphatase prepared in Example 3 (25 ng / mL, 15 μL / bead);

[0133] The freeze-dried microbeads containing the abamectin artificial antigen-magnetic bead conjugate prepared in Example 4 were prepared (1 mg / mL, 15 μL / bead).

[0134] Sample treatment solution (acetonitrile), reaction buffer (0.01 mol / L PBS solution), washing solution (0.6 g / L Tris solution, pH adjusted to 9.5 with hydrochloric acid), and light-emitting solution (Hai Xing New Enzyme Biotechnology Co., Ltd., item number: HH7601-01, main component: 3-(2-spiro adamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxolane disodium salt).

[0135] The freeze-dried microbeads were prepared by liquid nitrogen freeze-drying, and the entire kit was stored at 2-8°C.

[0136] II. Method of use

[0137] (1) After the kit is taken out, it is balanced at room temperature or in an automatic chemiluminescence instrument.

[0138] (2) Sample pretreatment

[0139] Milk sample: 60 μL of the milk sample was mixed with 60 μL of the sample treatment solution, shaken for 30 seconds, 100 μL of the shaken solution was mixed with 900 μL of the reaction buffer, shaken for 30 seconds, and the resulting solution was the test solution.

[0140] Fruit and vegetable sample: 2 g of leaf surface or fruit peel was cut into pieces, 8 mL of the sample treatment solution was added, and shaken for 1 min, the supernatant was taken after standing for 3 min, and the resulting solution was the test solution.

[0141] (3) 150 μL of the test solution was mixed with 1 freeze-dried microbead containing the anti-abamectin monoclonal antibody labeled with alkaline phosphatase prepared in Example 3, shaken for 30 seconds, and incubated at 37°C for 150 seconds.

[0142] (4) Then, 1 freeze-dried microbead containing the abamectin artificial antigen-magnetic bead conjugate prepared in Example 4 was added, shaken for 30 seconds, and incubated for 300 seconds.

[0143] (5) The magnetic beads were separated using a magnetic stand, and the magnetic beads were washed twice with the washing solution.

[0144] (6) The magnetic beads were placed in 60 μL of the light-emitting solution, and the light-emitting value was read.

[0145] Example 7: Detection of abamectin in milk

[0146] I. Detection object

[0147] Selecting UHT sterilized milk, pasteurized milk, 10 times diluted sheep milk powder as negative samples, adding 1 μg / kg, 2 μg / kg, 4 μg / kg level of abamectin B1a standard to them respectively.

[0148] II. Detection method

[0149] The kit of Example 6 was used for detection.

[0150] III. Detection results

[0151] The detection results of the three samples are shown in Table 1 below. The recovery rate of the chemiluminescence immunoassay established by the application is 89% to 101%, the coefficient of variation is in the range of 1.3% to 6.9%, which is within the acceptable range, indicating that the pre-treatment operation effect of the gradual dilution method is good, which can eliminate the centrifugal precipitation, blow-drying and re-dissolving operations in the usual sample pre-treatment, saving the detection time; the pre-treatment operation relying on shaking and blowing is very suitable for application on the automatic platform, realizing the integration of pre-treatment to data reading, reducing the influence of human operation.

[0152] Table 1 CLEIA actual milk sample addition recovery (n=3)

[0153]

[0154]

[0155] Example 8 Detection of abamectin in fruit and vegetable samples

[0156] I. Detection object

[0157] Taking lettuce, cucumber and jujube as negative samples, adding 20 μg / kg, 50 μg / kg, 100 μg / kg level of abamectin B1a standard to them respectively,

[0158] II. Detection method

[0159] The kit of Example 6 was used for detection.

[0160] III. Detection results

[0161] The detection results of the three samples are shown in Table 2 below. The CLEIA recovery rate of the fruit and vegetable samples is 87% to 97%, the coefficient of variation is in the range of 2.6% to 6.6%, which is within the acceptable range. The pre-treatment method is simple, which can save other instruments and improve the convenience of detection.

[0162] Table 2 CLEIA actual fruit and vegetable sample addition recovery

[0163]

[0164]

[0165] Example 9: Stability of the chemiluminescent detection kit for avermectin

[0166] I. Experimental Methods

[0167] The kit from Example 6 was accelerated at 37°C, and the signal values ​​were tested on days 1, 2, 3, 5, and 7, with a blank control included. The detection steps are as follows:

[0168] (1) After the kit is removed, it should be equilibrated at room temperature or in an automated chemiluminescence analyzer.

[0169] (2) Dilute the avermectin B1a standard solution to 0.15 ng / mL.

[0170] (3) One lyophilized microbead containing an anti-avermectin monoclonal antibody labeled with alkaline phosphatase, prepared in Example 3, was incubated with 60 μL of avermectin B1a standard solution at 37°C for 150 seconds.

[0171] (4) Add one lyophilized microbead containing avermectin artificial antigen-magnetic bead conjugate prepared in Example 4 and incubate at 37°C for 300 seconds.

[0172] (5) Separate the magnetic beads with a magnetic rack and wash the magnetic beads twice with washing solution.

[0173] (6) Place the magnetic beads in 60 μL of luminescent liquid and read the luminescence value.

[0174] II. Experimental Results

[0175] Accelerate stability testing, such as Figure 7 As shown, the results indicate that the kit exhibits good stability during accelerated reaction, with a signal value decrease of approximately 7%. This demonstrates that the chemiluminescent solution used in the kit possesses excellent stability, and the lyophilization protectant formulation used in preparing the kit plays a crucial role in maintaining the effectiveness of antibody and enzyme activity. The lyophilization protectant used retains the activity of antibodies and enzymes to a certain extent, and the kit exhibits good stability characteristics, which can extend its shelf life and ensure its effectiveness.

[0176] Example 10: Specificity of the chemiluminescent detection kit for avermectin.

[0177] I. Experimental Methods

[0178] The kit from Example 6 was used to detect four avermectin B1a structural analogs: ivermectin, acetaminophen, doramectin, and emamectin benzoate, and their IC50 values ​​were obtained. 50Specific procedure as in Example 5, except that the avermectin B1a standard was replaced by ivermectin, doramectin, acetylamino avermectin or emamectin benzoate.

[0179] The cross-reactivity was calculated using the following formula. R (%) = IC50(avermectin B1a) / IC50(avermectin B1a structural analogue) x 100%.

[0180] II. Results

[0181] The results are shown in Table 3. The kit has different degrees of recognition for ivermectin, doramectin, acetylamino avermectin and emamectin benzoate, and has high sensitivity, and can be used for rapid immunoassay of avermectin multi-residues to achieve rapid screening on site.

[0182] Table 3 Kit specificity results

[0183]

[0184]

Claims

1. A hapten of avermectin B1a and / or its structural analogues, characterized in that, Its structural formula is as shown in (I).

2. The use of the hapten of claim 1 in the preparation of artificial antigens of avermectin B1a and / or its structural analogues.

3. An artificial antigen of avermectin B1a and / or its structural analogues, characterized in that, Its structural formula is as shown in (II).

4. The use of the artificial antigen according to claim 3 in the preparation of avermectin B1a and / or its structural analogue antibodies.

5. The use of the hapten of claim 1 or the artificial antigen of claim 3 in the detection of avermectin B1a and / or its structural analogues for non-diagnostic purposes.

6. The use of the hapten of claim 1 or the artificial antigen of claim 3 in the preparation of a kit for detecting avermectin B1a and / or its structural analogues.

7. A kit for detecting avermectin B1a and / or its structural analogues, characterized in that, It contains an anti-avermectin antibody and the artificial antigen of claim 3, wherein the anti-avermectin antibody is prepared by immunizing animals with the artificial antigen of claim 3.

8. The reagent kit according to claim 7, characterized in that, The anti-avermectin antibody is a monoclonal antibody labeled with alkaline phosphatase; the artificial antigen of claim 3 is conjugated with magnetic beads.

9. A method for detecting avermectin B1a and / or its structural analogues, characterized in that, The detection is performed using an anti-avermectin antibody as the detection antibody and the artificial antigen described in claim 3 as the detection antigen, wherein the anti-avermectin antibody is prepared by immunizing animals with the artificial antigen described in claim 3.

10. The method according to claim 9, characterized in that, The anti-avermectin antibody is a monoclonal antibody labeled with alkaline phosphatase; the detection antigen is coupled with magnetic beads.

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