A universal pretreatment method for rapid immunoaffinity detection

Abstract

The present application relates to the technical field of drug residue detection, and in particular to a general pre-treatment method based on immunoaffinity rapid detection. A general pre-treatment method based on immunoaffinity rapid detection, comprising the following steps: using reagent 1, reagent 2, reagent 3, reagent 4, reagent 5 to process the sample to be tested in sequence, wherein the reagent 1 is ammonium citrate buffer; the reagent 2 is β-glucuronidase; the reagent 3 is arylsulfatase; the reagent 4 is a mixture of acetonitrile, methanol, ethanol and an organic acid; the organic acid is citric acid or formic acid; the reagent 5 is N-propylethylenediamine. The pre-treatment method of the present application is applicable to the pre-treatment of the residue detection of multiple major drugs involved in veterinary drug residues in current animal food, and solves the problem that food safety detection usually requires the determination of multiple drugs on the same sample, and multiple pre-treatment operations are required, and the time, equipment and reagent consumption are large.

Description

Technical Field

[0001] The present application relates to the technical field of drug residue detection, and in particular to a universal pretreatment method based on rapid immunoaffinity detection. Background Art

[0002] The level of veterinary drug residues in animal foods is an important part of food safety inspection. Excessive veterinary drug residues not only pose a serious threat to public health, but also affect the import and export trade of animal foods. Countries around the world attach great importance to it. Since drug residue detection is an analysis of trace organic substances in complex mixtures, and the analysis volume is large, it is very necessary to adopt effective, fast and popular purification methods. Immunoaffinity technology has developed rapidly in recent years and its application in veterinary drug residue detection has become more and more extensive. Immunoaffinity technology is a technical means of detecting target compounds by using the specific reaction of antigens and antibodies. The commonly used methods are enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatographic assay (GICA).

[0003] With the development of science and technology, convenient and highly accurate rapid detection methods for veterinary drug residues have gradually become the main means of daily risk monitoring. At present, more and more rapid immunoaffinity detection kits have gradually emerged on the market, greatly improving detection efficiency.

[0004] Regarding the above-mentioned related technologies, the applicant believes that the following problems exist: the sample pretreatment methods in the instructions of each test kit or test paper strip are different, and food safety testing usually requires the determination of multiple types of drugs on the same sample, which requires multiple pretreatment operations, consuming a lot of time, equipment and reagents.

[0005] To address the above-mentioned issues, the following constraints must be considered: various veterinary drugs have widely varying physical and chemical properties, significant polarity differences, and significant solubility differences. A single extraction reagent cannot meet the extraction efficiency requirements for multiple target analytes. Target analytes remaining in animal products exist in different forms and must be present as free, pure compounds to be extracted and detected. Some target analytes have unique residual forms, including those containing phenolic hydroxyl groups, such as salbutamol, which form covalent conjugates with compounds in the animal body at high rates within animal tissues. Some target analytes, such as tetracyclines, fluoroquinolones, and macrolides, have the property of chelating with divalent metal ions to form insoluble precipitates.

[0006] Therefore, it is very necessary to overcome the above constraints to quickly realize the pre-treatment of different drugs and improve the detection efficiency. Summary of the Invention

[0007] In order to be applicable to the pretreatment of residue detection of various major drug categories involved in veterinary drug residues in animal foods, the present application provides a universal pretreatment method based on rapid immunoaffinity detection.

[0008] The above technical objectives of this application are achieved through the following technical solutions:

[0009] A universal pre-treatment method based on immunoaffinity rapid detection comprises the following steps:

[0010] S1. Take the animal product to be tested, homogenize it, and obtain the test sample;

[0011] S2. Add reagent 1 to the sample obtained in S1 and mix well, then add reagent 2 and reagent 3 in sequence and mix well to obtain an initial mixed solution;

[0012] S3. The initial mixed solution was enzymatically hydrolyzed for 110-130 min to obtain an enzymatic hydrolyzate;

[0013] S4. Centrifuge the enzymatic hydrolyzate obtained in S3 and collect the supernatant and precipitate respectively;

[0014] S5. Add reagent 4 to the precipitate obtained in S4, mix, centrifuge, and collect the supernatant;

[0015] S6. The supernatant obtained in S4 is combined with the supernatant obtained in S5 to obtain a mixed solution;

[0016] S7. Add reagent 5 to the mixture obtained in S6, mix, oscillate, centrifuge, and retain the supernatant;

[0017] S8. The supernatant obtained in S7 was concentrated by nitrogen blowing and then diluted and reconstituted with the sample diluent of the kit;

[0018] S9. Freeze and centrifuge the diluted and reconstituted solution obtained in S8, and take the supernatant for testing;

[0019] The reagent 1 is ammonium citrate buffer;

[0020] The reagent 2 is β-glucuronidase;

[0021] The reagent 3 is arylsulfatase;

[0022] The reagent 4 is a mixture of acetonitrile, methanol, ethanol and an organic acid; the organic acid is citric acid or formic acid;

[0023] The reagent 5 is N-propylethylenediamine.

[0024] By adopting the above technical solution, ammonium citrate has a strong divalent metal ion chelating ability. The sample to be tested is first treated with ammonium citrate. Ammonium citrate chelates the divalent metal ions and seizes the chelating sites between target analytes such as tetracyclines, fluoroquinolones and macrolides and the divalent metal ions, thereby preventing the target analytes such as tetracyclines, fluoroquinolones and macrolides from combining with the divalent metal ions to form precipitation, thereby ensuring that the target analytes such as tetracyclines, fluoroquinolones and macrolides are detected in a free state.

[0025] β-glucuronidase and arylsulfatase are used to hydrolyze two common conjugated bonds formed by target analytes in animals, releasing the target analytes from the conjugated form for extraction. Other methods for hydrolyzing conjugated bonds include strong acid hydrolysis and strong base hydrolysis. However, these methods are unsuitable for pretreatment of drugs with poor stability under strong acid and base conditions. For example, tetracyclines are prone to isomerization or ring structure destruction under these conditions. Enzymatic hydrolysis, on the other hand, offers mild reaction conditions, avoiding the issues of poor accuracy and recovery due to drug loss.

[0026] In addition, ammonium citrate buffer can provide a suitable pH environment for β-glucuronidase and arylsulfatase, making the enzymatic catalytic activity of β-glucuronidase and arylsulfatase stronger.

[0027] After sequential treatment with ammonium citrate, β-glucuronidase, and arylsulfatase, followed by centrifugation, the solution contains some highly polar target analytes, while the precipitate contains most of the less polar target analytes and some of the more polar target analytes. Therefore, the precipitate needs to be treated to dissolve the target analytes. Most target analytes are readily soluble in acetonitrile, the most commonly used extraction solvent, and it also has a strong precipitating effect on removing proteins from the sample. However, some target analytes require the more polar methanol and ethanol to improve solubility. These three solvents are miscible and can meet the extraction requirements of various target analytes. Target analytes are mostly amine-containing compounds, which often form a certain degree of binding with proteins in the sample matrix. Therefore, organic acids are added to improve extraction efficiency.

[0028] N-propylethylenediamine, as a commonly used solid phase adsorbent in dispersed solid phase extraction, can effectively adsorb impurities in sample matrices such as small molecule organic acids, pigments and metal ions, reducing interference with detection.

[0029] In the present application, the sample to be tested is first treated with ammonium citrate to eliminate the interference of divalent metal ions on target analytes such as tetracyclines, fluoroquinolones and macrolides; then β-glucuronidase and arylsulfatase are added to hydrolyze the conjugated bond, so that the target analyte is released from the conjugated form for extraction; then the target analyte is dissolved in a solvent with strong solubility and a wide solubility range; finally, N-propylethylenediamine is used to adsorb impurities, and lipids are removed by freeze centrifugation, thereby achieving pretreatment of multiple target analytes and greatly improving detection efficiency.

[0030] Furthermore, the ratio of the sample to be tested to reagent 1 in S2 is 1 g of the sample to be tested corresponding to 1-1.5 ml of reagent 1, the ratio of reagent 1 to reagent 2 is (45-55):1, and the ratio of reagent 1 to reagent 3 is (45-55):1.

[0031] Furthermore, the ammonium citrate buffer in the reagent 1 is an aqueous solution of ammonium citrate with a concentration of 1.20-1.25 g / ml.

[0032] By adopting the above technical solution, the chelating capacity of the ammonium citrate buffer at this concentration meets the chelation requirements of divalent metal ions, and at this concentration, the pH value of the ammonium citrate buffer is 5.2, which can provide a better pH environment for the enzymatic hydrolysis of conjugated bonds by β-glucuronidase and arylsulfatase, thereby improving the enzymatic hydrolysis effect.

[0033] Furthermore, the enzymatic hydrolysis in S3 is carried out under shaking in a 45° C. water bath shaker.

[0034] By adopting the above technical solution, water bath shaking can prevent substrate precipitation, making the enzymatic hydrolysis reaction more thorough.

[0035] Furthermore, the enzymatic hydrolysate is cooled to 25-27° C. before centrifugation in S4, and the centrifugal parameters are: centrifugal force of 4000 g, centrifugal temperature of 4° C., and centrifugal time of 10 min.

[0036] Furthermore, the volume ratio of acetonitrile, methanol and ethanol in the reagent 4 is 2:2:1; the organic acid is citric acid, and the weight of the organic acid is 0.5-0.6% of the total weight of acetonitrile, methanol and ethanol.

[0037] By adopting the above technical solution, the precipitate contains a majority of target analytes with lower polarity and a portion of target analytes with higher polarity. Reagent 4 configured in this application utilizes organic solvents of varying polarity in a specific ratio, enabling the extraction of a wide range of drugs with a strong protein removal and purification effect. Furthermore, the addition of citric acid not only improves extraction efficiency but also promotes drug dissociation, precipitates proteins, and further chelates divalent metal ions.

[0038] Furthermore, the ratio of the reagent 4 in S5 to the sample to be tested in S2 is: 1 g of the sample to be tested corresponds to 6-7 ml of the reagent 4; the centrifugal parameters are: centrifugal force of 8000 g, centrifugal temperature of 4° C., and centrifugal time of 10 min.

[0039] Furthermore, the ratio of the reagent 5 in S7 to the sample to be tested in S2 is: 1 g of the sample to be tested corresponds to 9-12 mg of the reagent 5; the centrifugal parameters are: centrifugal force of 4000 g, centrifugal temperature of 4° C., and centrifugal time of 5 min.

[0040] Furthermore, the nitrogen blowing concentration in S8 is carried out in a 40° C. water bath, and the volume after nitrogen blowing concentration is 6.5-7.5% of the volume before concentration; the volume after dilution and reconstitution is 2-2.5 times the volume before dilution.

[0041] By adopting the above technical solution, nitrogen blowing and concentration are performed to concentrate the volume of the mixed liquid, remove the organic solvent, and increase the concentration of the target analyte to meet the accuracy requirements.

[0042] Furthermore, the freezing parameters in S9 are: -20°C freezing for 20 minutes; the centrifugal parameters are: centrifugal force of 12000-15000g, centrifugal temperature of 4°C, and centrifugal time of 3 minutes.

[0043] By adopting the above technical solution, the freezing treatment causes the lipids to solidify, and then the lipids are removed by high-speed centrifugation to achieve a purification effect.

[0044] In summary, this application has the following beneficial effects:

[0045] In the present application, the sample to be tested is first treated with ammonium citrate, which chelates divalent metal ions to prevent target analytes such as tetracyclines, fluoroquinolones and macrolides from chelating with divalent metal particles to form precipitation; then β-glucuronidase and arylsulfatase are added to hydrolyze the conjugated bond, so that the target analyte is released from the conjugated form for extraction; the target analyte is then dissolved in a solvent with strong solubility and a wide solubility range; finally, N-propylethylenediamine is used to adsorb impurities, and lipids are removed by freeze centrifugation, thereby achieving pretreatment of multiple target analytes such as β2-receptor agonists, tetracyclines, anabolic steroids, macrolides, sulfonamides, amide alcohols, nitroimidazoles, fluoroquinolones, β-lactams, aminoglycosides, etc., greatly simplifying the detection steps, reducing the consumption of reagents and equipment, and improving work efficiency. DETAILED DESCRIPTION

[0046] The present application is further described in detail below with reference to the embodiments.

[0047] Preparation examples of raw materials and intermediates

[0048] raw material

[0049] The raw materials in the examples of this application can be obtained commercially:

[0050] Ammonium citrate, analytical grade;

[0051] β-Glucuronidase, activity 30 U / mL, purchased from Merck, Germany;

[0052] Arylsulfatase, activity 60 U / mL, purchased from Merck, Germany;

[0053] Acetonitrile, analytical grade;

[0054] Methanol, analytical grade;

[0055] Ethanol, analytical grade;

[0056] Citric acid, analytical grade.

[0057] Example

[0058] A universal pre-treatment method based on immunoaffinity rapid detection comprises the following steps:

[0059] S0. Reagent preparation:

[0060] Reagents 1. Ammonium citrate buffer: Add 1.22 g of ammonium citrate to 100 mL of water and stir to dissolve to obtain ammonium citrate buffer.

[0061] Reagent 2. β-glucuronidase;

[0062] Reagent 3. Arylsulfatase;

[0063] Reagent 4. Mix 200 mL of acetonitrile, 200 mL of methanol, and 100 mL of ethanol, then add 2.5 g of citric acid and stir to obtain Reagent 4.

[0064] Reagent 5. N-propylethylenediamine;

[0065] S1. Take 10g of the animal tissue sample specified in the ELISA kit and homogenize it to obtain the sample to be tested;

[0066] S2. Place 1 g of the sample obtained in S1 into a 50 mL centrifuge tube and add 1 mL of Reagent 1. Vortex mix for 20 seconds. Then, add 20 μL each of Reagent 2 and Reagent 3. Vortex mix for 20 seconds to obtain a primary mixture.

[0067] S3. The initial mixture obtained in S2 3) was placed in a 45°C water bath shaker and shaken at 200 rpm for 120 min to obtain an enzymatic hydrolyzate;

[0068] S4. The enzymatic hydrolysate obtained in S3 was cooled to 25°C, and then centrifuged at 4000 g for 10 min at 4°C. The supernatant and precipitate were collected separately.

[0069] S5. Add 6 ml of Reagent 4 to the precipitate obtained in S4, vortex mix, shake at 500 rpm for 10 min, and then centrifuge at 8000 g and 4°C for 10 min, and collect the supernatant.

[0070] S6. The supernatant obtained in S4 is combined with the supernatant obtained in S5 to obtain a mixed solution;

[0071] S7. Add 10 mg of reagent 5 to the mixture obtained in S6, vortex mix for 1 min, and then centrifuge at a centrifugal force of 4000 g and a centrifugal temperature of 4°C for 5 min, and retain the supernatant;

[0072] S8. Concentrate the supernatant obtained in S7 by nitrogen purge in a 40°C water bath until the supernatant is concentrated to 0.5 mL. Then, dilute and reconstitute the supernatant with the sample diluent of the ELISA kit to a constant volume of 1 mL.

[0073] S9. Freeze the diluted and reconstituted solution obtained in S8 at -20°C for 20 minutes, then centrifuge at 15,000 g for 3 minutes, and collect the supernatant for testing.

[0074] Application Examples

[0075] Application Example 1

[0076] A method for detecting β2-receptor agonist drug residues, comprising the following steps:

[0077] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0078] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0079] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0080] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0081] The test values were compared with the precision and accuracy stated on commercial kits, as shown in Table 1. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunosorbent Assay Reagents (Kits)," the accuracy (recovery) requirement for ELISA testing is ≥40%, and the precision requirement is an intra-batch coefficient of variation of less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing as low as 40%. The lowest concentration added in the validation experiments was consistent with the lower limit of the kit.

[0082] Table 1 β2-receptor agonist drug residue detection results

[0083]

[0084] Application Example 2

[0085] A method for detecting tetracycline drug residues comprises the following steps:

[0086] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0087] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0088] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0089] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0090] The test values were compared with the precision and accuracy stated on commercial kits, as shown in Table 2. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)," the accuracy (recovery) requirement for ELISA testing is ≥40%, and the precision requirement is an intra-batch coefficient of variation of less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing as low as 40%. The lowest concentration added in the validation experiments was consistent with the lower limit of the kit.

[0091] Table 2 Tetracycline drug residue detection results

[0092]

[0093] Application Example 3

[0094] A method for detecting dexamethasone drug residues comprises the following steps:

[0095] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0096] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0097] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0098] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0099] The test values were compared with the precision and accuracy stated on commercial kits, as shown in Table 3. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)," the accuracy (recovery) requirement for ELISA testing is ≥40%, and the precision requirement is an intra-batch coefficient of variation of less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing as low as 40%. The lowest concentration added in the validation experiments was consistent with the lower limit of the kit.

[0100] Table 3 Dexamethasone drug residue test results

[0101]

[0102]

[0103] Application Example 4

[0104] A method for detecting erythromycin drug residues comprises the following steps:

[0105] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0106] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0107] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0108] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0109] The test values were compared with the precision and accuracy stated on commercial kits, as shown in Table 4. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)," the accuracy (recovery) requirement for ELISA testing is ≥40%, and the precision requirement is an intra-batch coefficient of variation of less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing as low as 40%. The lowest concentration added in the validation experiments was consistent with the lower limit of the kit.

[0110] Table 4 Erythromycin drug residue test results

[0111]

[0112] Application Example 5

[0113] A method for detecting sulfonamide drug residues comprises the following steps:

[0114] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0115] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0116] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0117] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0118] The test values were compared with the precision and accuracy stated on commercial kits, as shown in Table 5. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)," the accuracy (recovery) requirement for ELISA testing is ≥40%, and the precision requirement is an intra-batch coefficient of variation of less than or equal to 25%, or less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing as low as 40%. The lowest concentration added in the validation experiments was consistent with the lower limit of the kit.

[0119] Table 5 Erythromycin drug residue test results

[0120]

[0121] Application Example 6

[0122] A method for detecting chloramphenicol drug residues comprises the following steps:

[0123] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0124] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0125] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0126] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0127] The test values were compared with the precision and accuracy stated on commercial kits, as shown in Table 6. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunosorbent Assay Reagents (Kits)," the accuracy (recovery) of ELISA tests must be ≥40%, and the precision must be within the assay with a coefficient of variation of less than or equal to 25%, or less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing for accuracy as low as 40%. The lowest concentration used in validation experiments was consistent with the lower limit of the kit.

[0128] Table 6 Chloramphenicol drug residue test results

[0129]

[0130] Application Example 7

[0131] A method for detecting metronidazole drug residues comprises the following steps:

[0132] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0133] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0134] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0135] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0136] The test values were compared with the precision and accuracy indicated on commercial kits, as shown in Table 7. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)," the accuracy (recovery) requirement for ELISA testing is ≥40%, and the precision requirement is an intra-batch coefficient of variation of less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing as low as 40%. The lowest concentration added in the validation experiments was consistent with the lower limit of the kit.

[0137] Table 7 Metronidazole drug residue test results

[0138]

[0139] Application Example 8

[0140] A method for detecting fluoroquinolone drug residues comprises the following steps:

[0141] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0142] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0143] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0144] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0145] The test values were compared with the precision and accuracy indicated on commercial kits, as shown in Table 8. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)," the accuracy (recovery) requirement for ELISA testing is ≥40%, and the precision requirement is an intra-batch coefficient of variation of less than or equal to 25%, or less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing as low as 40%. The lowest concentration added in the validation experiments was consistent with the lower limit of the kit.

[0146] Table 8 Fluoroquinolone drug residue test results

[0147]

[0148] Application Example 9

[0149] A method for detecting ampicillin drug residues comprises the following steps:

[0150] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0151] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0152] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0153] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0154] The test values were compared with the precision and accuracy indicated on commercial kits, as shown in Table 9. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)," the accuracy (recovery) of ELISA tests must be ≥40%, and the precision must be within the batch coefficient of variation less than or equal to 25%, or less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing as low as 40%. The lowest concentration added in the validation experiments was consistent with the lower limit of the kit.

[0155] Table 9 Ampicillin drug residue test results

[0156]

[0157] Application Example 10

[0158] A method for detecting gentamicin drug residues comprises the following steps:

[0159] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0160] 2. Refer to the instructions of the commercial ELISA kit and set three spiked concentrations (detection limit, 2.5 times the detection limit, and 5 times the detection limit) of the test drug according to the nominal detection accuracy value of the kit. Add the drug to the blank sample. Repeat 6 replicates for each spiked concentration.

[0161] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0162] 4. Follow the instructions of each commercial kit to complete the ELISA test standard curve, blank sample and sample addition procedures, measure the data with a microplate reader, and calculate the drug value from the standard curve.

[0163] The test values were compared with the precision and accuracy indicated on commercial kits, as shown in Table 10. According to industry standards such as Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)," the accuracy (recovery) requirement for ELISA testing is ≥40%, and the precision requirement is an intra-batch coefficient of variation of less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, sometimes allowing as low as 40%. The lowest concentration added in the validation experiments was consistent with the lower limit of the kit.

[0164] Table 10 Gentamicin drug residue test results

[0165]

[0166]

[0167] As can be seen from Application Examples 1-10 and Tables 1-10, the pretreatment method of the present application is used to pretreat the sample to be tested, followed by drug residue determination. The test results for β2-receptor agonists, tetracyclines, anabolic steroids (dexamethasone), macrolides (erythromycin), sulfonamides, chloramphenicols (chloramphenicol), nitroimidazoles (metronidazole), fluoroquinolones, β-lactams (ampicillin), and aminoglycosides (gentamicin) all meet the industry standard requirements of Document No. 17 of the Agricultural and Medical Development

[2005] , "Technical Data Requirements for the Filing and Review of Veterinary Drug Residue Enzyme-Linked Immunosorbent Assay Reagents (Kits)." This indicates that the pretreatment method of the present application is suitable for the detection of multiple drug residues. By processing a single sample, multiple drug residues can be detected, greatly simplifying the detection steps, reducing the consumption of reagents and equipment, and improving work efficiency.

[0168] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A universal pretreatment method based on rapid immunoaffinity detection, characterized in that: The following steps are involved: S1. Take the animal product to be tested, homogenize it, and obtain the test sample; S2. Add reagent 1 to the sample obtained in S1 and mix well, then add reagent 2 and reagent 3 in sequence and mix well to obtain an initial mixed solution; S3. The initial mixed solution is enzymatically hydrolyzed to obtain an enzymatic hydrolyzate; S4. Centrifuge the enzymatic hydrolyzate obtained in S3 and collect the supernatant and precipitate respectively; S5. Add reagent 4 to the precipitate obtained in S4, mix, centrifuge, and collect the supernatant; S6. The supernatant obtained in S4 is combined with the supernatant obtained in S5 to obtain a mixed solution; S7. Add reagent 5 to the mixture obtained in S6, mix well, and centrifuge to retain the supernatant; S8. The supernatant obtained in S7 was concentrated by nitrogen blowing and then diluted and reconstituted with the sample diluent of the kit; S9. Freeze and centrifuge the diluted and reconstituted solution obtained in S8, and remove the supernatant for testing; The reagent 1 is ammonium citrate buffer; the ammonium citrate buffer is an aqueous solution of ammonium citrate with a concentration of 1.20-1.25 g / ml; The reagent 2 is β-glucuronidase; The reagent 3 is arylsulfatase; The reagent 4 is a mixture of acetonitrile, methanol, ethanol and an organic acid; the organic acid is citric acid or formic acid; the volume ratio of acetonitrile, methanol and ethanol is 2:2:1; the organic acid is citric acid, and the weight of the organic acid is 0.5-0.6% of the total weight of acetonitrile, methanol and ethanol; The reagent 5 is N-propylethylenediamine.

2. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The ratio of the sample to be tested to reagent 1 in S2 is 1 g of the sample to be tested corresponding to 1-1.5 ml of reagent 1, the ratio of reagent 1 to reagent 2 is (45-55):1, and the ratio of reagent 1 to reagent 3 is (45-55):

1.

3. A universal pretreatment method based on rapid immunoaffinity detection according to claim 1, characterized in that: The enzymatic hydrolysis in S3 was carried out under shaking in a 45°C water bath shaker.

4. A universal pretreatment method based on rapid immunoaffinity detection according to claim 1, characterized in that: In the S4, the enzymatic hydrolysate is cooled to 25-27° C. before centrifugation. The centrifugal parameters are as follows: centrifugal force of 4000 g, centrifugal temperature of 4° C., and centrifugal time of 10 min.

5. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The ratio of the reagent 4 in S5 to the sample to be tested in S2 is: 1 g of the sample to be tested corresponds to 6-7 ml of the reagent 4; the centrifugal parameters are: centrifugal force of 8000 g, centrifugal temperature of 4° C., and centrifugal time of 10 min.

6. A universal pretreatment method based on rapid immunoaffinity detection according to claim 1, characterized in that: The ratio of the reagent 5 in S7 to the sample to be tested in S2 is: 1 g of the sample to be tested corresponds to 9-12 mg of the reagent 5; the centrifugal parameters are: centrifugal force of 4000 g, centrifugal temperature of 4° C., and centrifugal time of 5 min.

7. A universal pretreatment method based on rapid immunoaffinity detection according to claim 1, characterized in that: The nitrogen blowing concentration in S8 is carried out in a 40° C. water bath, and the volume after nitrogen blowing concentration is 6.5-7.5% of the volume before concentration; the volume after dilution and reconstitution is 2-2.5 times the volume before dilution.

8. A universal pretreatment method based on rapid immunoaffinity detection according to claim 1, characterized in that: The freezing parameters in S9 are: freezing at -20°C for 20 min; the centrifugal parameters are: centrifugal force of 12000-15000g, centrifugal temperature of 4°C, and centrifugal time of 3 min.

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