CN100408163C - Method for Purifying Abamectin Drugs and Its Immunoaffinity Chromatography Column - Google Patents

Abstract

The invention discloses a method for purifying abamectin drugs and an immunoaffinity chromatographic column thereof. The immunoaffinity chromatographic column for purifying avermectin is loaded with an immunoaffinity adsorbent, which is composed of a solid phase carrier and an avermectin polyclonal antibody or monoclonal antibody coupled thereto; the avermectin The abamectin polyclonal antibody or abamectin monoclonal antibody is obtained by using the conjugate of abamectin hapten and carrier protein as an immunogen; "-OH is esterified with succinic anhydride to obtain the succinic acid derivative of abamectin, which is the abamectin hapten. The purification method of the present invention is combined with chromatography to detect the content of abamectin efficiently, which makes up for the simple immunoassay technology The lack of information, poor quantitative accuracy, or low selectivity of physical and chemical methods for direct measurement of samples reflects the complementarity of immunological techniques and conventional physical and chemical techniques in the analysis mechanism.

Description

Method for Purifying Abamectin Drugs and Its Immunoaffinity Chromatography Column

technical field

The present invention relates to a kind of purifying avermectins (Aermectins, AVMs), comprise avermectin (Aermectin, AVM), doramectin (Doramectin, DOR), ebimectin (Eprinomectin, EP) and i The method of Ivermectin (IVM) and its special immunoaffinity column.

Background technique

With the development of life sciences, people have become more and more interested in the substances and their changes in living organisms, and the analysis of biological samples has become a necessary means to explore and discover the mysteries of life. Due to the complex components of biological samples, the concentration of analytes is low, and most of the sample volumes are small, which puts forward higher requirements for the selectivity and sensitivity of the analytical method. Immunoaffinity chromatography (IAC, immunoaffinity chromatography) is an analytical method that combines an immune response with a chromatographic analysis method. Its high selectivity and high affinity undoubtedly simplify the analysis process. In the analysis of veterinary drug residues, the simplest and most effective application of IAC is as a sample purification method for physical and chemical determination techniques (such as HPLC, GC). , speed and sensitivity are complementary, and avoid many shortcomings of immunoassay methods (such as ELISA, RIA) to directly measure samples. At present, this method is widely used in the analysis of antibodies, hormones, peptides, enzymes, recombinant proteins, receptor viruses and small molecule compounds.

AVMs include Abamectin (Avermectin, AVM), Ivermectin (Ivermectin, IVM), Doramectin (Doramectin, DOR), Eprinomectin (Ebmectin, , EP), moxidectin (Moxidectin, MOX) and selamectin (Selamectin, SEL) and other drugs, wherein the consensus structure of AVMs is shown in Figure 2.

Avermectin (AVM), doramectin, ebimectin and ivermectin belong to macrolide broad-spectrum anti-parasitic antibiotics, and the anti-insect spectrum of this class of drugs includes cattle and sheep. A variety of nematodes such as tuberculosis columbia, tuberculosis radiata and their larvae, trichostrongylus, haemaela, parafilaria bovis, various round worms of equines, paraascaris, pinworms, haemaela, coil Cercoids, pig esophageal nematodes, lungfilariae, Trichinella spiralis and its migratory larvae, cysticercus, kidney worms, dog roundworms, pinworms, heartworms, hookworms and external parasites of various animals such as mites , ticks, lice, flies and fly larvae, daily itchy scrub, etc. The anti-insect spectrum of this class of drugs includes a variety of nematodes of cattle and sheep, such as tuberculosis columbia, tuberculosis radiata and its larvae, trichostrongylus, blood soft Nematodes of the genus Bovine, parafilariae bovine, various round worms of animals of the genus equine, pararoundworms, pinworms, haematobium, onchocerciasis, oesophageal nematodes, lungfilariae, trichinella and their migratory larvae and cysts of pigs Larvae, kidney worms, roundworms of dogs, pinworms, heartworms, hookworms and ectoparasites of various animals such as mites, ticks, lice, flies and fly larvae, daily itching scrub bugs, etc. Many countries have established methods for the detection of residues of abamectin drugs and made regulations on residue limits. As a fat-soluble compound, AVMs has a long residual time in animals, so WHO lists it as a highly toxic compound. The detection of residues in animal-derived foods has attracted great attention from various countries, and the maximum residue limit has been established.

At present, the methods for detecting abamectin, doramectin, ebimectin and ivermectin mainly include high performance liquid chromatography-fluorescence (HPLC-FLD), liquid-mass spectrometry (LC-MS-MS) and Enzyme-linked immunoassay (ELISA), etc. The pretreatment of these methods utilizes liquid-liquid distribution, conventional SPE column purification and separation, all of which have shortcomings such as cumbersome treatment process, poor purification effect, waste of organic solvents, and long time required to varying degrees. Immunoaffinity technology is a new technology that has been applied in the field of analysis in the 1990s, but there is no report on the use of immunoaffinity columns to purify abamectin, doramectin, ebemectin and ivermectin in the matrix. There are no commercially available IAC columns for sale.

Contents of the invention

The object of the present invention is to provide a method for purifying avermectin and/or ivermectin and/or doramectin and/or ebemectin and a special-purpose immunoaffinity chromatographic column thereof.

The immunoaffinity adsorbent for purifying avermectin and/or doramectin and/or ebamectin and/or ivermectin provided by the present invention is composed of a solid phase carrier and the avermectin coupled thereto Composition of abamectin polyclonal antibody or monoclonal antibody; The avermectin polyclonal antibody or avermectin monoclonal antibody is obtained by using a conjugate of abamectin hapten and carrier protein as an immunogen; Abamectin hapten is the 4″-hydroxy-succinoyl anhydride obtained by linking succinic anhydride at the C4″ position of Abamectin .

Abamectin is a small molecular substance, which has only immunoreactivity, but no immunogenicity, and cannot induce the body to produce an immune response. It must be coupled with a macromolecular carrier protein to be immunogenic. In the invention, abamectin is acylated with succinic anhydride, and then coupled with carrier protein to obtain immunogen. Too low or too high binding ratio of hapten to carrier protein is unfavorable to immunity. The binding molar ratios of hapten to ovalbumin (OVA) and bovine serum albumin (BSA) are 31.6:1 and 23:1, respectively.

The solid phase carrier can be cellulose, dextran gel, polyacrylamide gel, porous glass, agarose gel, polyacrylamide-agarose gel, etc., preferably Sepharose 4B.

The Abamectin polyclonal antibody can be a mouse source, a horse source, a sheep source, a rabbit source or a guinea pig source antibody, and the Abamectin monoclonal antibody is preferably an Abamectin mouse monoclonal antibody, and the Abamectin monoclonal antibody is preferably a mouse monoclonal antibody. Vermectin polyclonal antibody is preferably abamectin rabbit polyclonal antibody.

The abamectin mouse monoclonal antibody is preferably a monoclonal hybridoma cell strain A-1-4CGMCC No. that has a cross reaction to abamectin, ivermectin, doramectin and ebemectin. 1606.

The monoclonal hybridoma cell line A-1-4CGMCC No.1606, which has cross-reactivity to avermectin, ivermectin, doramectin and ebimectin, was deposited on February 9, 2006 In the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short).

The carrier protein can be commonly used carrier proteins such as bovine serum albumin or ovalbumin.

The immunoaffinity adsorbent can be loaded into a column to make an immunoaffinity chromatographic column, which also belongs to the protection scope of the present invention.

Kits containing the above-mentioned immunoaffinity adsorbent or immunoaffinity chromatography column also belong to the protection scope of the present invention.

The kit also includes an eluent, which can be methanol.

The kit also includes washing solution I, washing solution II, washing solution III and preservation solution; the preservation solution can be 0.01M, pH 7.4 phosphate buffer, the 0.01M, pH 7.4 phosphate buffer The washing solution can be a solution containing 0.2g KH ₂ PO ₄ , 0.2g KCl, 2.9g Na ₂ HPO ₄ .12H ₂ O, 8.8g NaCl, 0.02g NaN ₃ in 1L; Methanol with a content of 20%, 0.2gKH ₂ PO ₄ , 0.2g KCl, 2.9g Na ₂ HPO ₄ .12H ₂ O, and 29.3g NaCl, a solution of pH 7.4; the washing solution II is 20% by volume % methanol solution; the washing solution III may be a 50% methanol solution by volume.

The immunoaffinity adsorbent and the chromatographic column containing the immunoaffinity adsorbent are based on immune reaction and chromatographic reaction, and are suitable for purifying abamectin, doramectin from biological samples (such as muscle, liver, lung, kidney plasma). , ebimectin and ivermectin for easy residue analysis. In this immunoaffinity adsorbent, the coupling rates of Abamectin mouse monoclonal antibody and Abamectin rabbit polyclonal antibody to cyanogen bromide-activated Sepharose 4B were 99.7%, 98.1%, respectively, and the coupling ratios of Abamectin and Abamectin rabbit polyclonal antibodies The immunoaffinity chromatographic column for avermectin mouse monoclonal antibody has a dynamic column capacity of 3884ng/mL, 3896ng/mL, 3612ng/mL, 3897ng/mL, the absolute column capacity is 545ng/mg IgG, 549ng/mg IgG, 506ng/mg IgG, 542ng/mgIgG respectively; the dynamic column capacity after 15 uses is 1300-1500ng/mL.

The method for purifying Abamectin and/or Ivermectin and/or Doramectin and/or Ebamectin provided by the present invention may further comprise the steps:

1) Pretreatment of samples:

Take the homogenate of animal tissue and add 15mL of methanol in an amount of 5±0.01g, mix well, then shake on the shaker at a medium speed for 30min, and centrifuge at 3000rpm for 10min, and the supernatant obtained is the sample solution;

2) Mix 3ml of the sample solution with 20mL of the above-mentioned preservation solution, pass through the above-mentioned immunoaffinity chromatography column, then wash with 20mL of washing solution I, 15mL of washing solution II and 3mL of washing solution III, and then elute with 4mL of eluent, The eluate is collected to obtain purified avermectin and/or ivermectin and/or doramectin and/or ebimectin solutions.

The immunoaffinity chromatographic column of the present invention has high selectivity, greatly simplifies the sample pretreatment process, is especially suitable for the pretreatment of trace abamectin drugs in muscle, liver and blood plasma, and improves the analysis quality. The high selectivity of immunoaffinity adsorbent makes the detection limit of abamectin, ivermectin, ebimectin and doramectin analysis methods mainly depend on the sampling volume, which is difficult to achieve by pure physical and chemical means; The immunoaffinity chromatographic column of the present invention has strong retention and concentration capabilities for the components to be measured. As long as the amount of sample added does not exceed the column capacity, the retention capacity of the immunoaffinity adsorbent to the components is hardly affected by the sample under actual sample conditions. Effect of volume or component concentration. The method of the present invention provides qualitative information while purifying the components. The method of the invention is operated in water phase, has simple operation and good purification effect, and the immunoaffinity chromatographic column can be reused, can save a large amount of organic solvents, and reduce analysis cost and environmental pollution. The purification method of the present invention combines the chromatographic method to efficiently detect the content of abamectin, ivermectin, ebimectin and doramectin, which makes up for the lack of information and poor quantitative accuracy of the direct determination of samples by simple immunoassay technology. , or low selectivity of physical and chemical methods, reflecting the complementarity of immunological techniques and conventional physical and chemical techniques in the analysis mechanism.

Description of drawings

Figure 1 is the liquid chromatograms of AVMs standard, blank muscle tissue and added samples

Figure 2 is a common structure diagram of AVMs

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified.

Embodiment 1, the preparation of the immunochromatographic column of purifying Abamectin and/or Ivermectin and/or Ebamectin and/or Doramectin

1. Preparation of Abamectin Rabbit Polyclonal Antiserum

The synthesis of abamectin hapten is divided into three steps:

(1) Synthesis of 5-ot-BuMe ₂ Si-AVM-4″-OH: 1.0g of abamectin was placed in a 25mL three-neck heart-shaped bottle, dissolved in 6mL of N,N-dimethylamide (DMF), and added 0.47g imidazole, mixed. Under mechanical stirring, 0.52g t-BuMe ₂ SiCl (tert-butyldimethylchlorosilane) diluted with 2mL DMF was added dropwise, and reacted at 30°C for 2h. Added 100mL ethyl acetate (EtoAc) to the reaction solution and mixed. The mixed solution was washed 3 times with 50 mL of water. The EtoAc layer was separated, dried over MgSO ₄ , and concentrated under reduced pressure to obtain a slightly yellow viscous substance. The slightly yellow viscous substance was dissolved with CH ₂ Cl ₂ , and the product was separated by silica gel column chromatography, and after drying 5-ot- _BuMe2Si -AVM-4"-OH is obtained.

(2) Synthesis of 5-ot-BuM ₂ Si-AVM-4″-o-succinoyl: Take 0.50g of 5-ot-BuM ₂ Si-R-4″-OH in a 50mL three-necked chicken heart bottle, add 12mLCH ₂ Cl ₂ to make it dissolve, add 0.28g 4-dimethylaminopyridine (DMAP), 0.45g triethylamine and 0.92g succinic anhydride successively, reflux in water bath for 2.5 hours, the solution changes from colorless to brown, black (with the reaction raw material irrelevant). After evaporating CH ₂ Cl ₂ to dryness under reduced pressure, add 100 mL of diethyl ether, remove insoluble matter by filtration, transfer to a 250 mL separatory funnel, wash the diethyl ether layer with 100 mL of 3.6% (mass percentage) HCl twice, and then wash with 100 mL of water for 2 Second-rate. MgSO ₄ was dried and concentrated to obtain a light yellow viscous substance, which was dissolved in CH ₂ Cl ₂ and separated by thin-layer chromatography (TLC) (the volume ratio of the components of the developer was CH ₂ Cl ₂ : tetrahydrofuran (THF): _CH3OH =95:5:5). Three zones appeared on TLC, the largest zone in the middle was scraped off, rinsed with CH ₂ Cl ₂ and CH ₃ OH (volume ratio 1:1), concentrated under reduced pressure, and dried in vacuo for 24 hours (protected from light) to obtain 5-ot _-BuM2Si -AVM-4″-o-succinoyl.

(3) Remove the protective group 5-ot-BuM ₂ Si-AVM from the product obtained in step 2) to obtain 4″-o-succinoyl-AVM, and the succinic acid derivative of this abamectin is abamectin hapten.

Preparation of immunogen: In this experiment, the N-hydroxysuccinimide ester method (NHS) was used to couple the avermectin hapten to the carrier protein bovine serum albumin (BSA) or ovalbumin (OVA) to obtain BSA- AVM conjugates or OVA-AVM conjugates are immune antigens.

The specific steps of immunogen synthesis are as follows:

1) Put 12mg of abamectin hapten in a 5mL round bottom flask, add 0.4mL of dimethylformamide (DMF) to dissolve the abamectin hapten, then add 2.7mg of N-hydroxysuccinimide ester and 4.7mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl), after mixing, stir at room temperature (18-20°C) in the dark for 10 hours.

2) Under stirring, add the reaction solution obtained in step 1) dropwise to 6 mL of borate buffer (0.2 M, pH 9.4) containing 30 mg of BSA or OVA and 0.6 mL of DMF within 1 h. The solution was slightly turbid at the beginning, and finally a small amount of precipitate appeared. Continue to stir for 1 hour, then transfer to 4°C and stir for 10-12 hours, and immediately carry out the following dialysis purification.

3) The reaction solution obtained in step 2) was transferred into a dialysis bag (molecular weight cut-off 10,000 Da) under stirring at 4°C, and dialyzed for 2 days, during which time PBS was changed three times. The dialyzate was centrifuged at 1000 rpm for 5 min to remove the precipitate, and the obtained supernatant was the immunogen BSA-AVM conjugate or OVA-AVM conjugate.

Animal immunization: New Zealand white rabbits were used as immunized animals, and the immunization dose was 1 mg·ml ^-1 BSA-AVM conjugate or 1 mg·ml ^-1 OVA-AVM conjugate. New Zealand rabbits were bred for several weeks and then immunized. The first immunization was emulsified with 1ml complete Freund's adjuvant, and the booster immunization was emulsified with 1ml incomplete Freund's adjuvant. The interval between each immunization is 3-4 weeks, a total of 5 immunizations, the last direct intramuscular injection without adjuvant, and blood sampling 7-10 days after the last immunization. After measuring the serum titer and cross-reaction, the carotid artery was bled and the serum was collected.

2. Preparation of Abamectin Mouse Monoclonal Antibody

Animal immunization: BALA/C mice were used as immunized animals, and the above-mentioned avermectin hapten and protein carrier conjugate BSA-AVM conjugate or OVA-AVM conjugate was used as the immunogen, and the immunization dose was 50 μg/ Only (0.1ml in volume) was emulsified with an equal volume of complete Freund's adjuvant for the first immunization. One month later, take the same amount of immune antigen plus incomplete Freund's adjuvant, emulsify, and carry out booster immunization, and then carry out booster immunization again in the same way after another month, and collect blood 10 days after the second immunization, and measure the antibody titer and cross-reaction , take splenocytes.

Cell fusion: Splenocytes were fused with SP2/0 myeloma cells at a ratio of 5:1.

Hybridoma cell cloning: The hybridoma cells were screened by the limiting dilution method until completely homogeneous monoclonal antibodies and a stable monoclonal hybridoma cell line A-1-4CGMCC No.1606 were obtained. Abamectin, ivermectin, doramectin, and ebimectin all have cross-reactivity. The monoclonal hybridoma cell line A-1-4CGMCCNo.1606 with cross-reactivity to avermectin, ivermectin, doramectin and ebimectin was deposited in China Microbiology on February 9, 2006 General Microbiology Center of Culture Collection Management Committee (CGMCC for short).

Massive growth and purification of monoclonal antibodies: BALB/c mice were intraperitoneally injected with sterilized paraffin oil by in vivo induction method, and 7-14 days later, intraperitoneal injection was effective against avermectin, ivermectin, doramectin and avermectin. Monoclonal hybridoma cell line A-1-4CGMCC No. 16065×10 ⁵ -10 ⁶ cells/only with cross-reactivity to bibactin, ascites was collected 7-10 days later.

Storage of monoclonal antibodies: Store in liquid nitrogen at -20°C, and quickly thaw in a water bath at 37°C before use.

3. Purification of IgG:

Antiserum or ascitic fluid was purified by saturated ammonium sulfate method (SAS) and DEME cellulose ion exchange chromatography. First, IgG was crudely extracted and concentrated by saturated ammonium sulfate method, and then IgG was further purified by DEAE52 cellulose anion exchange method. . The specific steps are as follows:

Crude extraction: take 3mL of abamectin polyclonal antibody serum or monoclonal hybridoma cell line A-1-4CGMCC with cross-reactivity to abamectin, ivermectin, doramectin and ebimectin Mix No.1606 ascites with 3mL PBS (0.01M, pH 7.0), absorb 6mL of saturated ammonium sulfate solution, add it slowly and dropwise while stirring to serum or ascites solution, so that the saturation of ammonium sulfate solution reaches 50%. After standing at 4°C for 60 minutes, centrifuge at 3000 g for 15 minutes. The resulting precipitate was redissolved in 3 mL of PBS (0.01 M, pH 7.0), and 1.6 mL of saturated ammonium sulfate solution was slowly added dropwise to make the saturation of the ammonium sulfate solution reach 35%. After standing at 4°C for 20 minutes, centrifuge at 3000g for 15 minutes. Precipitate again by the above method in ammonium sulfate solution with a saturation of 35%.

Desalting: Dissolve the precipitate in (0.0175M, pH 6.7) PBS, put it into a dialysis bag, and place the dialysis bag in 1000mL of PBS (0.0175M, pH 6.7) solution and dialyze for 24 hours at 4°C. During the process, the buffer was replaced 3 times to obtain a crude IgG solution.

Purification: Weigh 2g of DE-52 (DEAE-52) cellulose powder, put it in a beaker filled with double distilled water, stir well, remove the excess solution after standing, then add 0.5mol/L NaOH solution, stir evenly, 1h Suction filtration with a Buchner funnel, followed by washing with double distilled water until neutral. Then use 0.5mol/L HCl solution to treat in the same way. Finally, it was treated once with 0.5mol/L NaOH solution, washed with water until neutral, and then the treated DEAE-52 was loaded into a chromatographic column (2.0×20cm), and equilibrated in 0.0175M PBS with pH 6.7. Drop the crudely extracted IgG solution obtained by salting out into the chromatography column. After all the samples enter the column, close the lower port of the column, and cover the column with 3-5cm 0.0175M, pH 6.7 PBS for elution, connect the elution bottle, The flow rate was controlled at 1.0 mL/min, and the protein fraction was collected with a UV detector. The eluate containing the antibody protein was precipitated with 50% saturated ammonium sulfate, and after desalination of the obtained precipitate, the abamectin rabbit multi-reactive compound with cross-reactivity to ivermectin, ebimectin and doramectin was obtained. Cloned antibody or the monoclonal antibody secreted by the monoclonal hybridoma cell line A-1-4CGMCC No.1606 which has cross-reactivity to avermectin, ivermectin, doramectin and ebimectin, the antibody Aliquot and store at -20°C.

4. Preparation of immunochromatographic column (IAC)

Matrix preparation: take cyanogen bromide-activated Sepharose 4B dry freeze powder and expand it in a _G3 funnel filled with 1.0mmol 1 ^-1 HCl.

Coupling reaction: After equilibrating the gel with 0.1mol/L NaHCO ₃ solution, mix it with 20 mg of the above-mentioned purified antibody (dissolved in 5 mL 0.1 mol/L NaHCO ₃ solution), and stir at 4°C for 20 h.

The reaction solution was transferred to a G3 funnel, and 100mL of 0.01M, pH 7.4 phosphate buffer (1L solution contained 0.27g of potassium dihydrogen phosphate, 2.86g of disodium hydrogen phosphate 12 hydrate, 0.2g of potassium chloride, sodium chloride 8.8g) wash, collect the washing solution, identify by ultraviolet light, and calculate the coupling rate. The formula for calculating the coupling rate is:

The test results of the coupling rate showed that the rabbit polyclonal antibody and the monoclonal hybridoma cell line A-1-4CGMCC No The coupling rates of monoclonal antibody secreted by .1606 to Sepharose 4B activated by cyanogen bromide were 99.7±1.5%, 98.1±1.6%, respectively.

Blocking of the activation site: Transfer the above-mentioned coupled gel into Tris-HCl buffer containing 0.1mol/L, pH 8.0, mix, and stir slowly at 4°C for 2 hours to block the uncoupled activation site. location.

Washing: The gel was alternately washed 3 times with 5 times the volume of 0.1 mol/L, pH 4.0 acetate buffer and 0.1 mol/L, pH 8.0 Tris-HCl buffer. Equilibrated with 0.01mol/L, pH7.4 PBS (1L solution contains 0.27g of potassium dihydrogen phosphate, 2.86g of disodium hydrogen phosphate 12 hydrate, 0.2g of potassium chloride, 8.8g of sodium chloride), and drained The gel was transferred to 0.01mol/L, pH7.4 containing 0.1% NaN ₃ phosphate buffer (1L solution contained 0.27g of potassium dihydrogen phosphate, 2.86g of disodium hydrogen phosphate 12 hydrate, 0.2g of potassium chloride, chlorine sodium chloride 8.8g, NaN ₃ 1g), and stored at 4°C for later use.

Column packing: Monoclonal hybridoma cell line A-1 that is conjugated with abamectin rabbit polyclonal antibody or has cross-reactivity to avermectin, ivermectin, doramectin and ebimectin - The immunosorbent of the monoclonal antibody secreted by 4CGMCC No.1606 was transferred to the glass column containing the _G3 filter plate to make a rabbit polyclonal antibody or abamectin, ivermectin, dora Both bacterin and eibectin have immunochromatographic columns (IAC columns) of monoclonal antibodies secreted by cross-reactive monoclonal hybridoma cell line A-1-4CGMCC No.1606.

5. Determination of IAC column capacity

The monoclonal hybridoma cell line A-1-4CGMCC No. prepared in step 4 is conjugated with rabbit polyclonal antibody or has cross-reactivity to abamectin, ivermectin, doramectin and ebimectin .1606 The immunochromatographic column of the monoclonal antibody secreted by 1606 was washed with the preservation solution and equilibrated. Gently shake the IAC column up and down to drive away the air bubbles in the column. 2mL containing 2000ng·ml ^-1 avermectin (AVM), 2000ng·ml ^-1 ivermectin (IVM), 2000ng·ml ^-1 ebimectin (EP) and 2000ng·ml ^-1 doramectin The preservation solutions of the mixed standard substances of the four drugs were continuously added to the immunoaffinity chromatographic column respectively, and flowed out under natural gravity. When the column reaches saturation (the concentration of the sample in the effluent is the same as that of the sample solution), successively wash with 20mL washing solution I (1L contains 20% methanol by volume, 0.2g KH ₂ PO ₄ , 0.2g KCl, 2.9 g Na ₂ HPO ₄ .12H ₂ O, and 29.3g NaCl, pH 7.4), 15mL wash solution II (20% by volume in methanol solution) and 3mL wash solution III (50% by volume in methanol solution) to wash the immunoaffinity column to remove interfering impurities. Finally, the avermectins were eluted with 4ml eluent (methanol), flowed out under natural gravity, collected, dried, and carried out HPLC determination. The dynamic and absolute column capacities are calculated. Dynamic column capacity refers to the maximum absorption value of the analyte per milliliter of immunosorbent (or column bed volume). The absolute column capacity (specific column capacity) refers to the maximum binding capacity of the analyte per mg of immobilized antibody. The results show that there is a monoclonal antibody secreted by the monoclonal hybridoma cell line A-1-4CGMCC No.1606 which has cross-reactivity to abamectin, ivermectin, doramectin and ebimectin. The dynamic column capacity and absolute column capacity of the immunochromatographic column are 3884ng/mL, 545ng/mg IgG (abamectin); 3896ng/mL, 549ng/mg IgG (ivermectin); 3612ng/mL, 506ng/mg IgG (epamectin); 3897 ng/mL, 542 ng/mg IgG (doramectin).

Example 2, Preparation of a kit containing an immunochromatographic column coupled to a rabbit polyclonal antibody or a mouse monoclonal antibody and its purification effect on avermectins

1. Preparation of the test kit of Abamectin

The kit mainly consists of a box body, an immunochromatographic column (IAC column), abamectin standard solution, ivermectin standard solution, doramectin standard solution, ebimectin standard solution, washing solution I, washing solution II, washing solution III, eluting solution, preservation solution, and sponge bracket are composed of holes and grooves on the sponge bracket. The groove of the sponge bracket is filled with abamectin standard solution, ivermectin standard solution, doramectin standard solution, ebimectin standard solution, washing solution I, washing solution II, washing solution III, The eluent, the reagent bottle of the preservation solution, and the hole of the sponge bracket are equipped with IAC columns. Wherein the immunochromatographic column is the monoclonal hybridoma cell strain A that is coupled with rabbit polyclonal antibody prepared in Example 1 or has cross-reactivity to abamectin, ivermectin, doramectin and ebimectin - Immunochromatographic column of monoclonal antibody secreted by 1-4CGMCCNo.1606.

The eluent was methanol.

The preservation solution is 0.01M, pH 7.4 phosphate buffer solution, that is, a solution containing 0.2gKH ₂ PO ₄ , 0.2g KCl, 2.9gNa ₂ HPO ₄ .12H ₂ O, 8.8gNaCl, 0.02g NaN ₃ in 1L;

Washing solution I is a solution containing 20% by volume of methanol, 0.2gKH ₂ PO ₄ , 0.2gKCl, 2.9gNa ₂ HPO ₄ .12H ₂ O, and 29.3gNaCl in 1L, with a pH of 7.4;

Washing solution II is a 20% methanol solution by volume;

The washing solution III is a 50% methanol solution by volume.

Secreted monoclonal hybridoma cell line A-1-4CGMCC No.1606 containing rabbit polyclonal antibody conjugated or cross-reactive to abamectin, ivermectin, doramectin and ebimectin The mAb immunochromatographic column kits were placed at 4 °C.

2. Purification and purification effect experiment of avermectins

The principle of IAC extraction is that the specific antibody Abamectin rabbit polyclonal antibody or the monoclonal hybridoma cell line A -1-4 Monoclonal antibody secreted by CGMCC No.1606 was coupled with an inert matrix to prepare an immunosorbent and packed into a column. When the mixture containing abamectin and/or ivermectin and/or doramectin and/or ebimectin flows through the IAC column, the immobilized antibody selectively binds abamectin and/or ivermectin Doramectin and/or doramectin and/or ebimectin, other unrecognized sample impurities flow out of the IAC column unhindered, after washing, the antigen-antibody complex is dissociated and eluted, Avi The decontamination of ivermectin and/or ivermectin and/or doramectin and/or ebimectin. The IAC column can be reused after regeneration.

Processing of test samples: animal tissue samples: take pig muscle, bovine liver, lung, muscle, heart, kidney tissue samples homogenate 5.0±0.01g, put in 50ml plastic centrifuge tube, each sample is 10ng/g Add avermectin, ivermectin, doramectin and ebimectin standard substances respectively, let stand for 15 minutes, add methanol 15mL, mix well, shake on the shaker for 30 minutes, centrifuge at 3000rpm for 10 minutes, take 3 mL of the supernatant was mixed with 20 mL of the preservation solution as the sample solution.

Monoclonal hybridoma cell line A-1-4CGMCC No.1606 which is coupled with rabbit polyclonal antibody IgG or has cross-reactivity to abamectin, ivermectin, doramectin and ebimectin, respectively The immunoaffinity chromatographic column of the secreted monoclonal antibody is equilibrated to room temperature, then the above-mentioned sample solution is passed through the column, flows out under natural gravity, and the immunoaffinity chromatography is washed successively with 20mL washing solution I, 15mL washing solution II and 3mL washing solution III The column was finally eluted with 4 mL of eluent, and the eluate was collected and blown dry with nitrogen.

Add 100 μL of derivatization reagent A solution (composed of 1-methylimidazole and acetonitrile with a volume ratio of 3:4), vortex for 0.5 minutes, and then add 100 μL of derivatization reagent B solution (composed of acetic anhydride with a volume ratio of 2:5 and acetonitrile), vortexed for 0.5min, airtight, derivatized at 96°C for 100 minutes, distilled to 2.5mL with acetonitrile, passed through a 0.45μm syringe filter, and analyzed by HPLC (chromatographic conditions: C ₁₈ reverse phase chromatographic column , Inertsil ODS-3 (4.6mm×250mm, particle size 5μm); mobile phase is 97% methanol solution by volume; flow rate is 1mL/min; injection volume is 20μL; excitation wavelength is 365nm, emission wavelength is 475nm ). The IAC column was equilibrated with 20ml of preservation solution and stored in a 4°C refrigerator for later use. The results show that sample purification with IAC does not interfere with the drug chromatographic peak and can be completely separated, indicating that the non-specific adsorption of IAC prepared by the present invention is extremely small, wherein abamectin, ivermectin, and doramectin in pig muscle and ebilamectin purification effect as shown in Figure 1, in Figure 1, A is the standard solution containing each 10ng/mL of abamectin, ivermectin, doramectin and ebilamectin; B is Add 10ng/g avermectin, 10ng/g ivermectin, 10ng/g doramectin and 10ng/g ebimectin standard pig muscle sample concentration; C is no abamectin, ivermectin Pig muscle tissue of vermectin, doramectin and ebimectin; in Figure 1, 1 is ebimectin B1a, 2 is abamectin B1a, 3 is doramectin, and 4 is ivermectin Prime B1a.

Claims (12)

1. An immunoaffinity adsorbent for purifying abamectin and/or ivermectin and/or doramectin and/or ebimectin, composed of a solid phase carrier and the abamectin coupled thereto Monoclonal antibody composition; Described avermectin monoclonal antibody is the monoclonal antibody secreted by monoclonal hybridoma cell line A-1-4 CGMCC No.1606. 2. The adsorbent according to claim 1, characterized in that: the solid phase carrier is cellulose, dextran gel, polyacrylamide gel, porous glass, agarose gel or polyacrylamide-agar sugar gel. 3. adsorbent according to claim 2, is characterized in that: described solid phase carrier is Sepharose 4B. 4. The immunoaffinity chromatographic column of filler with the immunoaffinity adsorbent described in any one of claims 1-3. 5. the test kit containing the arbitrary described immunoaffinity adsorbent of claim 1-3. 6. test kit according to claim 5, is characterized in that: also comprise eluent in described test kit, and described eluent is methyl alcohol. 7. according to the described kit of claim 6, it is characterized in that: also comprise washing solution I, washing solution II, washing solution III and preservation solution in the described test kit; Described preservation solution is 0.01M, the phosphoric acid of pH7.4 Salt buffer, the 0.01M, pH7.4 phosphate buffer contains 0.2g KH ₂ PO ₄ , 0.2g KCl, 2.9gNa ₂ HPO ₄ .12H ₂ O, 8.8gNaCl, 0.02g NaN ₃ in 1L Solution; the washing solution I is 1L containing 20% by volume of methanol, 0.2gKH ₂ PO ₄ , 0.2gKCl, 2.9gNa ₂ HPO ₄ .12H ₂ O and 29.3gNaCl, pH 7.4 solution; the washing Liquid II is a 20% methanol solution by volume; the washing solution III is a 50% methanol solution by volume. 8. the test kit containing the immunoaffinity chromatography column described in claim 4. 9. test kit according to claim 8, is characterized in that: also comprise eluent in the described test kit, and described eluent is methyl alcohol. 10. test kit according to claim 9, is characterized in that: also comprise washing solution I, washing solution II, washing solution III and preservation solution in the described test kit; Described preservation solution is 0.01M, the phosphoric acid of pH7.4 Salt buffer, the 0.01M, pH7.4 phosphate buffer contains 0.2g KH ₂ PO ₄ , 0.2g KCl, 2.9gNa ₂ HPO ₄ .12H ₂ O, 8.8gNaCl, 0.02g NaN ₃ in 1L Solution; the washing solution I is 1L containing 20% by volume of methanol, 0.2gKH ₂ PO ₄ , 0.2gKCl, 2.9gNa ₂ HPO ₄ .12H ₂ O and 29.3gNaCl, pH7.4 solution; the The washing liquid II is a methanol solution with a volume percentage of 20%; the washing liquid III is a methanol solution with a volume percentage of 50%. 11. A method for purifying Abamectin and/or Ivermectin and/or Doramectin and/or Ebamectin, comprising the following steps: 1) Pretreatment of samples: Take the homogenate of animal tissue and add 15mL of methanol in an amount of 5±0.01g, mix well, then shake on the shaker at a medium speed for 30min, and centrifuge at 3000rpm for 10min, and the supernatant obtained is the sample solution; 2) Get 3ml sample solution and mix with the preservation solution described in 20mL claim 7, cross the immunoaffinity chromatographic column described in claim 4, then successively use 20mL washing liquid 1 and 15mL right described in claim 7 The washing solution II described in claim 7 and the washing solution III described in 3mL claim 7 are washed, and then eluted with the eluent described in 4mL claim 6, and the eluent is collected to obtain purified Avermella Doramectin and/or doramectin and/or ivermectin and/or ebimectin solution. 12. The method according to claim 11, wherein the animal tissue samples include muscle, liver, lung, heart, kidney and plasma.

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