WO2014108016A1 - 检测肉制品中恩拉霉素残留的方法 - Google Patents
检测肉制品中恩拉霉素残留的方法 Download PDFInfo
- Publication number
- WO2014108016A1 WO2014108016A1 PCT/CN2013/089469 CN2013089469W WO2014108016A1 WO 2014108016 A1 WO2014108016 A1 WO 2014108016A1 CN 2013089469 W CN2013089469 W CN 2013089469W WO 2014108016 A1 WO2014108016 A1 WO 2014108016A1
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- WO
- WIPO (PCT)
- Prior art keywords
- enramycin
- add
- solution
- meat product
- microwell
- Prior art date
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- NJCUSQKMYNTYOW-MWUYRYRWSA-N enramicina Chemical group O.N1C(=O)NC(=O)C(C=2C=C(Cl)C(O)=C(Cl)C=2)NC(=O)C(CO)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(CC2N=C(N)NC2)NC(=O)C(CCCNC(N)=O)NC(=O)C(C(C)O)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C(C)O)NC(=O)N(CCCCN)C(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)/C=C/C=C/CCCCC(C)CC)C(C)OC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C)NC(=O)C1CC1CNC(N)=N1 NJCUSQKMYNTYOW-MWUYRYRWSA-N 0.000 title claims abstract description 87
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
Definitions
- the invention relates to a method for detecting residues of enramycin in meat products, and belongs to the technical field of food detection.
- Enramycin also known as lenidine, enmycin, amsandin, and oxytocin, is a peptide antibiotic produced by fermentation of the actinomycete Streptomyces fungi cidious NO. B5477 isolated from soil. .
- the drug was developed by Takeda Pharmaceutical Co., Ltd. in Japan in 1966, officially registered in Japan in 1974, and has been registered and widely used in many countries.
- the drug is against Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pyogenes, Pneumococcus, Bacillus subtilis, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, etc.
- the positive bacteria have strong bactericidal activity, which can change the balance of the flora in the intestine by inhibiting the growth and reproduction of harmful bacteria in the intestinal tract, and increase the absorption and utilization of nutrients in the feed. Long-term use of this drug is not easy to produce drug resistance, and it does not produce obvious cross-resistance with other antibiotics.
- Veterinary drug residue detection is an analytical technique for trace components in complex mixtures. Residues of antibiotics are generally in the microgram to nanogram range. Classical chemical analysis cannot detect or quantify is not accurate enough. Therefore, residual antibiotic detection method must be sensitive. Fast and efficient.
- the antibiotic residue detection method can be generally classified into three categories: microbial detection method, physical and chemical detection method and immunoassay technology. Microbiological detection is a widely used method. The principle of measurement is based on the antibacterial effect of antibiotics on the physiology and metabolism of microorganisms. The qualitative and quantitative determination of antibiotic microbial residues in samples is derived from the paper method (PD). The minimum detectable amount of chloramphenicol 0.
- TTC method Tripheye tetrazolium chloride
- STC method The minimum detectable amount of penicillin is 0. 004IU / mL
- tube disc method for penicillin Sensitivity is 0. 01U / mL
- Delvo detection method SP
- the sensitivity is: penicillin 3ng / mL, streptomycin 300ng / mL, gentamicin 400ng / mL.
- Microbiological assays can detect samples at levels close to the maximum residue limit, enabling high-volume, rapid, and sensitive assays, and can be performed in a non-laboratory environment.
- the microbiological detection method has the advantages of no special equipment, simple operation, easy promotion, high sensitivity, and suitable for simultaneous detection of large quantities of samples.
- the disadvantages are cumbersome operation, long detection period, high environmental requirements, and color judgment is judged by the naked eye.
- the results are subjective.
- Physical and chemical detection methods use special reactions or properties of groups in antibiotic molecules to determine their content, such as high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry, combined technology, etc.
- HPLC high performance liquid chromatography
- GC gas chromatography
- mass spectrometry combined technology, etc.
- Immunoassay technology is currently one of the most desirable methods for residue screening analysis.
- IAs immunoassays
- the second is the immunoassay technology and routine
- IAC immunoaffinity chromatography
- IAC-HPLC or IAC-GC are common combinations, such as chloramphenicol, avermectin and ivermectin.
- the future of immunoassay technology in veterinary drug residue analysis technology will be developed in the direction of standardization of reagents, the use of more sensitive and anti-interference markers and physical and chemical analysis techniques.
- the immunoassay method Compared with conventional physical and chemical analysis techniques, the most prominent advantages of immunoassay technology are simple operation, high speed and low cost of analysis.
- the immunoassay method has small sample amount, simple pretreatment, large capacity, low degree of instrumentation, similar detection to GC/MS or GC/ECD, and can reach ng/g-pg/g level, and the analytical efficiency is HPLC. Or dozens of times more than GC.
- most antibiotics have established immunoassays, such as sulfamethazine, chloramphenicol, sarafloxacin, streptomycin, tetracycline, monensin, and the like.
- the ELISA kit is currently the most widely used, rapid and sensitive method for detecting antibiotic residues, but most of the antibiotic kits are produced abroad, making the domestic testing cost very large, such as the antibiotic detection reagent produced by Bayer in Germany. Box, a 96 test kit costs 3,800 yuan, each sample costs 30 to 50 yuan, so it is necessary to develop and develop a domestic kit to reduce costs and improve economic efficiency.
- Japan has established a method for the determination of enramycin residues in livestock and aquatic foods. China has established the detection of enramycin premix and servo grade. Yuan Ersen (1999) has explored the detection of meat residue on this basis.
- the detection limit is 0.3 ppm, and the detection methods are all microbial detection methods. 2 ppm ⁇ Horie ( 1985) using a high performance liquid chromatography to analyze the residual porcine and chicken muscles, the detection limit was 0.2 ppm. Eramycin ELISA assays and kits have not been reported.
- the technical problem to be solved by the present invention is to provide a method for detecting residual enramycin in meat products with a lower detection limit.
- the method for detecting residues of enramycin in meat products of the present invention comprises the following steps:
- the meat product extract to be tested and a series of concentrations of the enramycin standard solution are added to the micropores pre-coated with the enramycin antigen; wherein the meat product extract is prepared by the following method 5 ⁇ 8000 ⁇ 12000rpm ⁇ 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8
- the preparation of the meat product extract by the method in the step a can improve the solubility and recovery rate of the enramycin in the meat product to be tested, and also improve the stability of the enramycin in the extract.
- the method for detecting a residue of enramycin in a meat product according to the present invention wherein the enramycin antigen can be used as a method for synthesizing a complete antigen of enramycin by using the application number CN201010556432.
- the method disclosed in the patent application is prepared.
- the anti-enramycin antibody added in the step b of the method of the present invention may be replaced with serum containing an anti-enramycin antibody.
- the antigen-antibody concentration at an OD value of about 1.0 is an optimum working concentration, and the inventors of the present invention found that the pre-coated erazin antigen coating described in the step a of the method of the present invention The concentration was 2.30 ug/mL, and the dilution of the serum containing the anti-enramycin antibody added in the step b was 1:10000. At this time, the antigen-antibody concentration is the optimum working concentration.
- the terminating liquid described in the e step may be a stop solution commonly used in an ELISA enzyme-linked immunoassay, such as a sulfuric acid solution.
- the method for detecting residues of enramycin in a meat product of the present invention preferably comprises the following steps:
- lOOuL substrate solution can be a substrate solution commonly used in ELISA enzyme-linked immunoassay, and color development at 37 ° C lOmin;
- the method of the present invention is suitable for all common meat products, such as: at least one of pork, lamb, beef, chicken, duck, goose, and fish, preferably chicken.
- the detection limit of the method for the enramycin in the meat product is 8.9 ng/mL to 982 ng/mL, and the sensitivity is much higher than the existing method, and an unexpected technical effect is obtained.
- the method of the invention improves the detection limit of enramycin in the meat product, and the detection range is: 8. 9 ng/mL ⁇ 982 ng/mL, which is faster, simpler, more sensitive and flexible than the existing microbiological detection method, and also Compared with high-performance liquid chromatography, the detection cost is lower.
- the invention provides a feasible method for the inspection of the enramycin residue in meat products, and has broad application prospects.
- Figure 1 is a graph showing the determination of the anti-serum titer of enramycin
- Figure 2 is a square matrix titration curve of the working concentration of the enramycin antigen antibody
- Figure 3 is a graph of an indirect competitive ELISA standard curve for the detection of enramycin.
- the method for detecting residues of enramycin in meat products of the present invention comprises the following steps: a, respectively, the meat product extract to be tested and a series of concentrations of the enramycin standard solution are added to the micropores pre-coated with the enramycin antigen; wherein the meat product extract is prepared by the following method 5 ⁇ 8000 ⁇ 12000rpm ⁇ 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8
- the preparation of the meat product extract by the method in the step a can improve the solubility and recovery rate of the enramycin in the meat product to be tested, and also improve the stability of the enramycin in the extract.
- the method for detecting a residue of enramycin in a meat product according to the present invention wherein the enramycin antigen can be used as a method for synthesizing a complete antigen of enramycin by using the application number CN201010556432.
- the method disclosed in the patent application is prepared.
- the anti-enramycin antibody added in the step b of the method of the present invention may be replaced with serum containing an anti-enramycin antibody.
- the antigen-antibody concentration at an OD value of about 1.0 is an optimum working concentration
- the inventors of the present invention found that the pre-coated erazin antigen coating described in the step a of the method of the present invention
- the concentration was 2.30 ug/mL, and the dilution of the serum containing the anti-enramycin antibody added in the step b was 1:10000.
- the antigen-antibody concentration is the optimum working concentration.
- the terminating liquid described in the e step may be a stop solution commonly used in an ELISA enzyme-linked immunoassay, such as a sulfuric acid solution.
- the method for detecting residues of enramycin in a meat product of the present invention preferably comprises the following steps:
- lOOuL substrate solution can be a substrate solution commonly used in ELISA enzyme-linked immunoassay
- the value is B value, B/Bo is plotted on the ordinate, the logarithm of the concentration of enramycin is plotted on the abscissa, the standard curve is drawn, the regression equation is established, and the curve regression equation obtained from the standard solution of enramycin is calculated. The amount of enramycin in the meat product is measured.
- the method of the present invention is suitable for all common meat products, such as: at least one of pork, lamb, beef, chicken, duck, goose, and fish, preferably chicken.
- the method of the present invention is the detection limit of the range of meat products Enramycin is 8. 9n g / mL ⁇ 982n g / mL , the sensitivity is much higher than the conventional method, to obtain the unexpected technical effect.
- Example 1 Establishment of a method for detecting residues of enramycin in meat products of the present invention
- Enramycin artificial antigen (Er-BSA and Er_0VA, synthesized by the method disclosed in the patent application No. CN201010556432. 2, entitled “Synthesis method of a complete antigen of enramycin”);
- FCA Freund's complete adjuvant
- Enzyme-labeled secondary antibody (goat anti-rabbit I g G-HRP, imported by Huamei Bioengineering Co., Ltd.);
- Trimethylbenzidine (TMB, AMRESC0), the remaining chemical reagents were of analytical grade.
- Nafion reagent solution 11. 50gHgI and 8. 00gKI dissolved in 50mL of distilled water, stirred and dissolved, then added
- Saturated ammonium sulfate solution 425g ammonium sulfate was added to 500mL deionized water, and then stirred until heated to substantially dissolve, while hot filtered, after cooling to room temperature, adjust the pH to 7. 0 with ammonia water;
- Coating buffer (CBS, 0. 05M pH 9. 6): 0. 159g sodium carbonate and 0. 293g sodium bicarbonate dissolved in ionic water, to a volume of lOOmL: Dilution buffer (PBS, 0. 01M pH 7.4): 800gNaCL, 0. 20gKCL, 0. 20gKH 2 P04, 3. 58g, N3 ⁇ 4C03 ⁇ 12H 2 0, dissolved in 800mL deionized water, adjusted to pH7 with NaCL or HCL. 5 ⁇ ween-20; Adding PBS buffer to 0. 05 Tween-20;
- TMB substrate storage solution lOmgTMB is dissolved in 2mLDMS0 and stored in a 4°C refrigerator in the dark;
- Blocking solution gelatin 1. 0g added coating buffer (CBS) lOOmL;
- the artificial antigen is thawed, diluted with an appropriate amount of physiological saline under aseptic conditions, and added dropwise to an equal volume of adjuvant (FCA or
- the mixture was emulsified into a water-in-oil (W/0) state, and the final concentration of immunization was 1 mg/mL (calculated as carrier protein content).
- the relevant immunization procedures and blood sampling were performed.
- lmL of ear vein blood was collected, and after 4 hours at 4 °C, the serum was centrifuged at 3000 rpm*10 mi n .
- the serum was mixed with glycerol (1:1) in equal volume and stored in a refrigerator at -20 °C for blank control.
- blood was collected by direct blood sampling, 2 (T30 mL/only; serum was separated as described above, dispensed and stored.
- a, coating Dilute the coated antigen with a coating solution to a series of concentrations, add to the microplate, lOOuL / well, incubate at 37 °C for 2h, then overnight at 4 °C;
- washing wash with washing liquid 5 times, 200uL / hole, each interval lmin, pat dry on absorbent paper;
- adding enzyme adding HRP - goat anti-rabbit lgG (1: 20000 dilution) 100uL / well, incubating at 37 °C for 1h; washing plate with (e); g, color development: adding lOOuL / hole TMB substrate use solution / hole, color development at 37 ° C lOmin;
- a, coating Dilute the coated antigen to a certain concentration with a coating solution to the enzyme plate lOOuL / well, incubate at 37 ° C for 2 h, then overnight at 4 ° C;
- Washing Wash 3 times with washing solution, 200uL/well, each time interval lmin, pat dry on absorbent paper;
- washing wash 3 times with washing solution, 250uL / hole, each interval lmin, pat dry on absorbent paper;
- Purification of the multi-antiserum was carried out by a saturated ammonium sulfate method. A part of the serum obtained after the last immunization of the rabbit was mixed with an equal amount of sterilized glycerin, and then stored in a 1.5 mL centrifuge tube; a part of the purified lgG was extracted. Mix and store with equal amount of sterilized glycerin.
- the specific steps of the law are as follows:
- the Naphthal reagent was used to measure the dialysis solution without yellowing, and the dialysis was completed. After purification, a portion of the serum was mixed with glycerol in an equal volume of 1:1, and the others were directly lyophilized and stored at -20 °C.
- . m has OD value f when Er is suppressed
- the crude product was processed according to the separation and purification method of the crude enramycin. 5 ⁇ The chicken muscle tissue was cut and homogenized for 2 min, placed in a 100 mL centrifuge tube, added with 20 ml of methanol-water solution, homogenized for 10 min, adjusted with a concentrated HCL pH 3. 5 ⁇ 4. 0, fully stirred for 10 min, Centrifuge at 1000 rpm for 10 min, take the supernatant, adjust the pH to 8.0 with NaOH, centrifuge for 10 min at 1000 rpm, and take the supernatant as a test solution.
- the highest titer of the anti-Er antiserum was measured to be greater than 1: 320000.
- the results showed that the immunization antigen was prepared by the glutaraldehyde method to immunize rabbits, and specific antibodies were produced. After four immunizations, the rabbits produced antibodies against Er.
- the antiserum titer of sample No. 4 was significantly higher than that of other rabbits. The results are shown in Fig. 1.
- the antigen-antibody concentration when the 0D value is about 1.0 is selected as the optimal working concentration. Therefore, according to the results of the square array titration, the optimum coating concentration of the coated antigen Er-0VA was determined to be 2. 30 ug/mL, and the mixed serum working concentration was 1: 10000. The results are shown in Figure 2.
- the addition of a recovery test is a sign of determining the reliability of the analytical method of the present invention. ⁇ That is, the reliability of the method of the present invention meets the requirements.
- China's detection method with enramycin only uses microbial detection method to establish pre-mixture and feed-level detection means.
- the detection limits reported by Yuan Ersen et al. and Xie Mingquan are 0. 30ug/g and greater than 0. The limit of detection of 025mg/g;
- the detection limit was only 0. 20ug/ g .
- the invention adopts the enzyme-linked immunosorbent assay technology as a detection means, establishes an indirect competitive ELISA method, and tests Er, which can be applied in practice, and is compared with the domestic and foreign immunoassay methods.
- Table 3 compares Er detection method and detection limit
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Abstract
一种检测限更低的检测肉制品中恩拉霉素残留的方法，包括如下步骤：a 分别将待检测肉制品提取液和系列浓度的恩拉霉素标准液加入到预包被恩拉霉素抗原的微孔中；b 然后于各微孔中加入抗恩拉霉素抗体，混匀后孵育；c 于各微孔中加入辣根过氧化物酶HRP-羊抗兔IgG，混合后孵育；d 分别于各微孔中加入底物溶液，显色；e 分别于各微孔中加入终止液终止反应，用酶标仪读取各孔OD450值；f 对照恩拉霉素标准液所得的曲线回归方程，计算出待检测肉制品中恩拉霉素含量。
Description
检测肉制品中恩拉霉素残留的方法 技术领域
本发明涉及检测肉制品中恩拉霉素残留的方法， 属于食品检测技术领域。
背景技术
恩拉霉素 （Enramycin ) , 又名恩来霉素、 恩霉素、 安来霉素、 持久霉素， 是由土壤中 分离出来的放线菌 Streptomyces fungi cidious NO. B5477 发酵产生的一种多肽类抗生素。 该药于 1966年由日本武田药品工业株式会社研发， 1974年在日本正式注册， 其后在许多 国家被注册和广泛使用。 该药对金黄色葡萄球菌、 表皮葡萄球菌、 柠檬色葡萄球菌、 酿脓 链球菌、 肺炎球菌、 枯草杆菌、 炭疽杆菌、 破伤风梭菌、 肉毒梭菌、 产气荚膜梭菌等革兰 氏阳性菌有很强的杀菌活性， 可通过抑制肠道中有害细菌的生长和繁殖来改变肠道内的菌 群平衡， 增加伺料中营养物质的吸收和利用。 长期使用该药， 不易产生耐药性， 与其他抗 生素之间不产生明显交叉耐药， 同时有化学性能稳定， 无致突、 致畸、 致癌作用， 不易被 吸收， 残留少， 适口性好等优势， 因此被世界上许多国家推荐作为抗生素促生长剂， 常作 为伺料添加剂用于促进动物增重和提高伺料利用效率。 1988年我国农业部批准进口该药， 现在已有恩拉霉素预混料销售， 并且呈现良好的市场发展前景。
随着全球贸易自由化进程的加快， 以及 "二噁英" 、 药物残留等有毒有害物质造成的重 大动物源性食品污染事件的频发，世界各国都构筑起各具特色的动物产品质量安全管理体系， 从法律法规体系、 检验检疫监管体系、 标准体系、 认证体系等方面来保证动物源性产品的质 量安全。 由于对动物产品药残检测研究起步较晚， 我国的药残检测标准和检测水平落后于发 达国家。 发达国家凭借在科技、 管理、 环保等方面的优势， 从技术法规、 标准、 合格评定程 序等方面对我国动物产品出口不断设置新的 "绿色贸易壁垒" 门槛， 致使在我国国内合格的 动物产品在出口后被进口国检出抗生素、农药、兽药残超标而被退回或销毁的事件屡见不鲜， 造成的直接和间接经济损失在百亿美元以上。 因此， 提高我国动物产品药残的检测水平势在 必行。
兽药残留检测是复杂混合物中痕量组分的分析技术， 抗生素的残留量一般都在微克到纳 克级，经典的化学分析法无法检测或定量不够准确，因此要求残留抗生素检测法必须达到灵 敏、 快速、 高效。 抗生素残留检测法总体可以归为 3大类:微生物检测法、 理化检测法和免疫 分析技术。
微生物检测法是应用较广泛的方法， 其测定原理是根据抗生素对微生物的生理机能、 代 谢的抑制作用， 来定性或定量确定样品中抗生素微生物药物残留， 其衍生出的纸片法 （PD) 对氯霉素最低检出量 0. 01mg/L; 氯化三苯基四氮唑法 ( tripheye tetrazolium chloride, TTC法） 对青霉素的最低检出量为 0. 004IU/mL; 管碟法对青霉素的敏感度为 0. 01U/mL; 戴尔 沃检测法 （SP) 其灵敏度为： 青霉素 3ng/mL， 链霉素 300ng/mL, 庆大霉素 400ng/mL。 随着 研究的深入， 还出现了拭子法、牛的抗生素和磺胺实验法（CAST)、快速抗生素筛选法（FAST) 等更为灵敏、 准确、 简便、 快速的微生物检测方法。 微生物检测方法能检测出接近最高残留 限量浓度水平的样品， 能够进行大批量、 快速、 灵敏的测定， 且可以非实验室环境下进行。 微生物检测法具有不需要特殊设备、 操作简单、 易推广、 灵敏度高、 适用于大批量样品的同 时检测等优点， 缺点是操作繁琐， 检测周期长， 环境要求高， 显色状态判断通过肉眼判断， 其结果的主观性强。
理化检测法是利用抗生素分子中的基团所具有的特殊反应或性质来测定其含量， 如高效 液相色谱法 (HPLC)、气相色谱法（GC)、质谱法、联用技术等， 能进行定性、定量和药物鉴定， 敏感性较高， 但有的检测程序较复杂， 检测成本高， 仪器设备要求高， 操作规范严格。
免疫分析技术是目前最理想的残留筛选性分析方法之一。 在目前药物残留分析中主要分 为两大类： 一为相对独立的分析方法， 即免疫测定法 ( Immunoassays, IAs )， 如 RIA、 ELESA、 固相免疫传感器等； 二是将免疫分析技术与常规理化分析技术联用， 如免疫亲合色谱（IAC)， 它可使兽药残留分析将免疫技术的高选择性和理化技术的快速分离和灵敏性融为一体， 避免 了 IA 直接测定样本信息量太少、 假阳性或理化分析技术选择性低等不足， 分析过程简化。 IAC-HPLC或 IAC-GC是常见的联用方式， 如氯霉素、 阿维菌素和伊维菌素等残留分析。 未来 兽药残留分析技术中的免疫分析技术将向试剂标准化、 使用更灵敏和抗干扰性强的标记物和 理化分析技术联用方向发展。
免疫分析技术与常规的理化分析技术相比， 最突出的优点是操作简单， 速度快、 分析成 本低。 如 ELISA法， 免疫测定方法取样量小， 前处理简单， 容量大， 仪器化程度低， 检测与 GC/MS或 GC/ECD相似， 可达到 ng/g-pg/g级， 分析效率则为 HPLC或 GC的几十倍以上。 目前 大部分抗生素已经建立了免疫测定法， 如磺胺二甲嘧啶、 氯霉素、 沙拉沙星、 链霉素、 四环 素、 莫能菌素等。
ELISA试剂盒是目前使用最广泛、 快速、 灵敏的检测抗生素残留的方法， 但是各类抗生 素试剂盒多为国外生产， 使得国内检测花费非常大， 如德国拜耳公司生产的抗生素检测试剂
盒， 一个 96次检测试剂盒要花 3800元， 每个样品的检测要花 30~50元， 因此很有必要研制 和开发出国产的试剂盒， 以降低成本， 提高经济效益。
日本已建立畜、 水产性食品中恩拉霉素残留检测方法， 我国建立了恩拉霉素预混料和伺 料级的检测， 袁而森 （1999) 在此基础上探索过对肉制品残留检测， 检测限量为 0. 3ppm， 其 检测方法都是微生物检测法。 Horie ( 1985)利用高效液相色谱分析猪、鸡肌肉恩拉霉素残留， 检测限量为 0. 2ppm。 恩拉霉素的 ELISA检测方法和试剂盒都未曾见报道。 由于目前世界各国 的食品安全卫生控制指标限量逐步降低，食源性危害关键检测技术趋向高科技化、系列化（多 残留检测）、 速测化、 器具便携化， 建立新的高敏感度、 便携化的、 低成本的恩拉霉素药残检 测方法以适应现在的动物产品检验检疫要求是非常有必要的。 发明内容 本发明所要解决的技术问题是提供一种检测限更低的检测肉制品中恩拉霉素残留的方 法。
本发明检测肉制品中恩拉霉素残留的方法包括如下步骤：
a、分别将待检测肉制品提取液和系列浓度的恩拉霉素标准液加入到预包被恩拉霉素抗原 的微孔中； 其中， 所述的肉制品提取液采用下述方法制备得到： 取待检测肉制品剪碎， 匀浆， 加浓度为 50%v/v的甲醇-水溶液，匀浆，用浓 HC1调整 pH值 3. 5〜4. 0，混匀， 8000〜12000rpm 离心 8〜12min， 取上清液， 用 NaOH调节 pH值至 8. 0， 8000〜 12000离心 8〜 12min， 取上 清液， 即得待检测肉制品提取液； 所述的恩拉霉素抗原采用申请号为 CN201010556432. 2 的 方法制备得到；
b、 然后于各微孔中加入抗恩拉霉素抗体， 混匀后孵育；
c、 于各微孔中加入辣根过氧化物酶 HRP—羊抗兔 IgG， 混合后孵育；
d、 分别于各微孔中加入底物溶液， 显色；
e、 分别于各微孔中加入终止液终止反应， 用酶标仪读取各孔 0D45。值；
f、 对照恩拉霉素标准液所得的曲线回归方程， 计算出待检测肉制品中的恩拉霉素含量。 其中， 采用 a步骤中的方法制备肉制品提取液可以提高待检测肉制品中的恩拉霉素的溶 解性和回收率， 同时还提高了提取液中的恩拉霉素的稳定性。
其中， 本发明检测肉制品中恩拉霉素残留的方法， 所述的恩拉霉素抗原可以采用申请号 为 CN201010556432. 2， 发明名称为 "一种恩拉霉素完全抗原的合成方法" 的专利申请公开的 方法制备得到。
其中， 为了节约成本， 本发明方法的 b步骤中加入的抗恩拉霉素抗体可以采用含有抗恩 拉霉素抗体的血清代替。
其中， 0D值为 1. 0左右时的抗原抗体浓度为最适工作浓度， 经本发明的发明人研究发 现， 本发明方法的 a步骤中所述的预包被恩拉霉素抗原的包被浓度为 2. 30ug/mL， b步骤中 加入的含有抗恩拉霉素抗体的血清的稀释度为 1 : 10000。 此时抗原抗体浓度为最适工作浓 度。
其中， 上述检测肉制品中恩拉霉素残留的方法， 其 e步骤中所述的终止液可以为 ELISA 法酶联免疫实验常用的终止液， 如硫酸溶液。
进一步的， 本发明检测肉制品中恩拉霉素残留的方法， 优选包括如下步骤：
a、分别将待检测肉制品提取液和系列浓度的恩拉霉素标准液加入到预包被恩拉霉素抗原 的微孔中； 待检测肉制品提取液和系列浓度的恩拉霉素标准液的加入量为 50uL/孔；
b、 然后于各微孔中加入含有抗恩拉霉素抗体的血清 50uL/孔， 血清稀释度为 1 : 10000，
37°C孵育 lh， 洗涤， 拍干；
c、 于各微孔中加入辣根过氧化物酶 HRP—羊抗兔 IgG 1 : 20000倍稀释， lOOuL/孔， 混 合后于 37°C孵育 lh， 洗涤， 拍干；
d、分别于各微孔中加入 lOOuL底物溶液（可以为 ELISA酶联免疫实验常用的底物溶液）， 37°C显色 lOmin;
e、 分别于各微孔中加入终止液 2moL/mLH2S04终止反应， 用酶标仪读取各孔 0D45。值； f、 以无恩拉霉素抑制时 0D值为 Bo，各相应浓度恩拉霉素抑制时的 0D45。值为 B值， 以 B/Bo为纵坐标， 以恩拉霉素浓度的对数为横坐标， 绘制标准曲线， 建立回归方程， 对照恩 拉霉素标准液所得的曲线回归方程， 计算出待检测肉制品中的恩拉霉素含量。
其中， 本发明方法适合于所有常见的肉制品， 比如： 为猪肉、 羊肉、 牛肉、 鸡肉、 鸭肉、 鹅肉、 鱼肉中至少一种， 优选为鸡肉。
本发明方法对肉制品中的恩拉霉素的检测限范围为 8. 9ng/mL〜982ng/mL， 其灵敏度远高 于现有方法， 取得了意料不到的技术效果。
本发明方法提高了肉制品中的恩拉霉素的检测限， 其检测范围为： 8. 9ng/mL〜982ng/mL， 比已有的微生物检测法更快捷、 简便、 灵敏和灵活， 同时也比高效液相色谱法的检测成本更 低， 本发明为肉制品中的恩拉霉素残留的检验提供了一种可行的方法， 具有广阔的应用前景。 附图说明
图 1 为恩拉霉素抗血清效价测定曲线图；
图 2 为恩拉霉素抗原抗体工作浓度的方阵滴定曲线图；
图 3 为检测恩拉霉素的间接竞争性 ELISA标准曲线图。
具体实施方式
本发明检测肉制品中恩拉霉素残留的方法包括如下步骤：
a、分别将待检测肉制品提取液和系列浓度的恩拉霉素标准液加入到预包被恩拉霉素抗原 的微孔中； 其中， 所述的肉制品提取液采用下述方法制备得到： 取待检测肉制品剪碎， 匀浆， 加浓度为 50%v/v的甲醇-水溶液，匀浆，用浓 HC1调整 pH值 3. 5〜4. 0，混匀， 8000〜12000rpm 离心 8〜12min， 取上清液， 用 NaOH调节 pH值至 8. 0， 8000〜 12000离心 8〜 12min， 取上 清液， 即得待检测肉制品提取液； 所述的恩拉霉素抗原采用申请号为 CN201010556432. 2 的 方法制备得到；
b、 然后于各微孔中加入抗恩拉霉素抗体， 混匀后孵育；
c、 于各微孔中加入辣根过氧化物酶 HRP—羊抗兔 IgG， 混合后孵育；
d、 分别于各微孔中加入底物溶液， 显色；
e、 分别于各微孔中加入终止液终止反应， 用酶标仪读取各孔 0D45。值；
f、 对照恩拉霉素标准液所得的曲线回归方程， 计算出待检测肉制品中的恩拉霉素含量。 其中， 采用 a步骤中的方法制备肉制品提取液可以提高待检测肉制品中的恩拉霉素的溶 解性和回收率， 同时还提高了提取液中的恩拉霉素的稳定性。
其中， 本发明检测肉制品中恩拉霉素残留的方法， 所述的恩拉霉素抗原可以采用申请号 为 CN201010556432. 2， 发明名称为 "一种恩拉霉素完全抗原的合成方法" 的专利申请公开的 方法制备得到。
其中， 为了节约成本， 本发明方法的 b步骤中加入的抗恩拉霉素抗体可以采用含有抗恩 拉霉素抗体的血清代替。
其中， 0D值为 1. 0左右时的抗原抗体浓度为最适工作浓度， 经本发明的发明人研究发 现， 本发明方法的 a步骤中所述的预包被恩拉霉素抗原的包被浓度为 2. 30ug/mL， b步骤中 加入的含有抗恩拉霉素抗体的血清的稀释度为 1 : 10000。 此时抗原抗体浓度为最适工作浓 度。
其中， 上述检测肉制品中恩拉霉素残留的方法， 其 e步骤中所述的终止液可以为 ELISA 法酶联免疫实验常用的终止液， 如硫酸溶液。
进一步的， 本发明检测肉制品中恩拉霉素残留的方法， 优选包括如下步骤：
a、分别将待检测肉制品提取液和系列浓度的恩拉霉素标准液加入到预包被恩拉霉素抗原 的微孔中； 待检测肉制品提取液和系列浓度的恩拉霉素标准液的加入量为 50uL/孔；
b、 然后于各微孔中加入含有抗恩拉霉素抗体的血清 50uL/孔， 血清稀释度为 1 : 10000， 37°C孵育 lh， 洗涤， 拍干；
c、 于各微孔中加入辣根过氧化物酶 HRP—羊抗兔 IgG 1 : 20000倍稀释， lOOuL/孔， 混 合后于 37°C孵育 lh， 洗涤， 拍干；
d、分别于各微孔中加入 lOOuL底物溶液（可以为 ELISA酶联免疫实验常用的底物溶液）， 37°C显色 lOmin;
e、 分别于各微孔中加入终止液 2mOL/mLH2S04终止反应， 用酶标仪读取各孔 0D45。值； f、 以无恩拉霉素抑制时 0D值为 Bo，各相应浓度恩拉霉素抑制时的 0D45。值为 B值， 以 B/Bo为纵坐标， 以恩拉霉素浓度的对数为横坐标， 绘制标准曲线， 建立回归方程， 对照恩 拉霉素标准液所得的曲线回归方程， 计算出待检测肉制品中的恩拉霉素含量。
其中， 本发明方法适合于所有常见的肉制品， 比如： 为猪肉、 羊肉、 牛肉、 鸡肉、 鸭肉、 鹅肉、 鱼肉中至少一种， 优选为鸡肉。
本发明方法对肉制品中的恩拉霉素的检测限范围为 8. 9ng/mL〜982ng/mL， 其灵敏度远高 于现有方法， 取得了意料不到的技术效果。
下面结合实施例对本发明的具体实施方式做进一步的描述， 并不因此将本发明限制在所 述的实施例范围之中。
实施例 1 本发明检测肉制品中恩拉霉素残留的方法的建立
1、 试验材料与试验方法
1. 1 试验材料
新西兰大白兔；
恩拉霉素人工抗原（Er-BSA和 Er_0VA，采用申请号为 CN201010556432. 2,发明名称为"一 种恩拉霉素完全抗原的合成方法" 的专利申请公开的方法合成）；
弗氏完全佐剂 （FCA， Sigma公司）；
弗氏不完全佐剂 (FICA, Sigma公司）；
酶标二抗（山羊抗兔 IgG-HRP， 华美生物工程公司进口分装）；
96孔酶标板（C0STAR)；
三甲基联苯胺 (TMB， AMRESC0公司）， 其余化学试剂均为分析纯。
1. 2试剂的配制与用品准备
1. 2. 1 配制试剂
0. 85 %生理盐水溶液： 0. 85gNaCL溶于 lOOmL去离子水；
萘氏试剂溶液： 11. 50gHgI和 8. 00gKI溶于 50mL蒸馏水中，搅拌溶解后，再加入
50mL20%Na0H;
饱和硫酸铵溶液： 425g硫酸铵加入 500mL去离子水中， 然后边加热边搅拌至基本溶解， 趁热过滤， 待其冷却到室温后用氨水调 pH到 7. 0；
包被缓冲液 （CBS, 0. 05M pH9. 6)： 0. 159g碳酸钠和 0. 293g碳酸氢钠溶解于离子水中， 定容到 lOOmL:
稀释缓冲液 （PBS, 0. 01M pH7. 4)： 800gNaCL, 0. 20gKCL, 0. 20gKH2P04, 3. 58g, N¾C03 · 12H20， 溶于 800mL去离子水中， 用 NaCL或 HCL调 pH7. 2〜7. 4， 定容到 lOOOmL; 磷酸盐洗涤缓冲液： 将 PBS缓冲液中添加 0. 05 Tween-20;
TMB底物储存液： lOmgTMB溶于 2mLDMS0中， 在 4°C冰箱中避光保存；
柠檬酸缓冲液 （PH5. 0)： 2. 3328g柠檬酸， 9. 2006gN¾C03 · 12¾0, 定容到 500mL; 底物缓冲液： 将柠檬酸缓冲液 （pH5. 0) 500mL中加入 0. 5mLTWeen-20;
样品稀释液； 1. 0LPBS加 1. OmLTween-20和 lg明胶；
终止液： 2mol/LH2S04;
封闭液： 明胶 1. 0g加入包被缓冲液 （CBS) lOOmL;
1. 2. 2 主要仪器
酶标仪 (Bio-Tek ELX800型) ；
各种规格移液枪 (Eppendorf)；
BS100S型分析天平 (北京赛多利斯公司）；
DF-101S型恒温加热磁力搅拌器 (河南予华仪器公司）；
漩涡振荡仪；
恒温水浴锅；
ULT1386-3-V38型低温冰箱（美国 REVC0公司）；
DCD-172K型常规冰箱 (美国伊莱克斯公司）；
培养箱 （浙江科通仪器公司） ；
RE-52AA旋转蒸发器 （上海亚荣生化仪器厂） ；
KQ-600型超声波清洗器；
1. 3 试验方法
1. 3. 1 免疫方案
选 10只健康的 2月龄雄性实验用清洁级新西兰大白兔， 体重为 2. (Γ2. 50kg/只， 用兔子分 笼伺养， 自由饮食； 在免疫前采集阴性血清备用，免疫程序见表 1。
实验动物免疫方案程序
注： 70天免疫结束后采血。
将人工抗原解冻， 在无菌状态下以适量生理盐水稀释后， 滴加到等体积的佐剂 （FCA 或
FICA) 中， 混合乳化后成油包水 (W/0)状态， 使免疫的最终浓度为 lmg/mL (以载体蛋白含量 计算）， 据表 1设计方案进行相关免疫程序和采血检测。 免疫前采耳缘静脉血 lmL， 4°C放置 4h后 3000rpm*10min离心取血清， 将血清与甘油（1 : 1)等体积混和， 保存于 _20°C冰箱， 用于 空白对照， 在 70d 免疫结束后采用心脏直接采血法采血， 2(T30mL/只； 按上述方法分离血 清， 分装和保存。
1. 3. 2 EL1SA方法的建立
1. 3. 2. 1 抗原抗体最佳工作浓度的测定
分别从每只家兔的耳缘静脉采血 lmL， 室温静置 l h， 4°C冰箱 2~3h， 3000g/minl5min 离心分离血清。 采用方阵滴定法确定抗原、 抗体的最适工作浓度， 具体步骤如下：
a、 包被： 用包被液将包被抗原稀释成一系列浓度加至酶标板， lOOuL/孔， 37 °C孵育 2h， 然后 4°C过夜；
b、 洗涤： 用洗涤液洗 3遍， 200uL/孔， 每次间隔 lmin， 在吸水纸上拍干； c、 封闭： 加入封闭液 150uL/孔， 37 °C孵育 lh后， 4°C过夜， 洗涤同 b ;
d、 加样： 加入事先系列稀释好的抗体， lOOuL/孔， 37 °C孵育 lh;
e、 洗涤： 用洗液洗 5遍， 200uL/孔， 每次间隔 lmin， 在吸水纸上拍干；
f、加酶：加入 HRP—羊抗兔 lgG (1 : 20000倍稀释）100uL/孔， 37 °C孵育 lh;洗板同（e) ; g、 显色： 加入 lOOuL/孔 TMB底物使用液 /孔， 37 °C显色 lOmin;
h、 终止： 加入 2mOL/mlH2S0450uL/孔， 用酶标仪读取各孔 0D45。；
判定标准： 阳性血清 0D45。在 1. 0左右抗原包被浓度和抗体稀释度为最佳工作浓度。 阳性血清与阴性血清的 P/N比值 0倍的血清稀释倍数为血清效价。
1. 3. 2. 2间接竞争 ELISA方法的建立
a、 包被： 用包被液将包被抗原稀释成一定浓度加至酶标板 lOOuL/孔， 37°C孵育 2 h， 然后 4°C过夜；
b、 洗涤： 用洗涤液洗 3遍， 200uL/孔， 每次间隔 lmin， 在吸水纸上拍干；
c、 封闭： 加入封闭液 150uL/孔， 37°C孵育 lh后拍干；
d、 加样： 加入系列稀释的 Er标准品 50uL/孔， 再加入适当稀释的抗体 50uL/孔， 37 °C孵育 lh;
e、 洗涤： 用洗涤液洗 3遍， 250uL/孔， 每次间隔 lmin， 在吸水纸上拍干；
f、 加二抗：加入 HRP—羊抗兔 IgG (l : 20000倍稀释）100uL/孔， 37°C孵育 lh; 洗板同 (e)；
g、 显色： 分别加入 lOOuL底物溶液 /孔， 37°C显色 lOmin;
h、 终止： 加入 2moL/mLH2S0450uL/孔， 用酶标仪读取各孔 0D45。；
1. 3. 2. 3 饱和硫酸铵法纯化多抗血清
多抗血清的纯化采用饱和硫酸铵法， 将最后一次免疫后的兔子取血后获得的血清一部 分与等量灭菌甘油混合后于 1. 5mL离心管中分装保存； 一部分纯化提取 lgG后再与等量灭 菌甘油混合分装保存。 该法具体操作步骤如下：
取血清加等量生理盐水， 于搅拌下逐滴加入与稀释血清等量的饱和硫酸铵 [终浓度为 50
%饱和（NH4) 2S04]， 然后于 4°C， 3 小时以上， 使其充分沉淀， 再 离心（3000rpm) 20 分钟， 弃上清， 以生理盐水溶解沉淀至 Xml。 再逐滴加饱和硫 酸铵 X / 2 ml， 置 4°C， 3小时 以上 [此时 (NH4) 2S04的饱和度为 33 %) ]。 重复上述第二步过程 2次。 将末次离心后所得沉 淀物以 0. 02M PH7. 4 PBS溶解至 Xml装入透析袋。 对 PBS充分透析、 除盐换液 3次， 至萘氏 试剂测透析外液为无黄色， 透析结束。 纯化后血清一部分与甘油 1 : 1等体积混合， 其余直接 冷冻干燥后保存于 -20°C。
1. 3. 2. 4 标准曲线的制作
将 1. 3. 2. 2中的 0D45。nm以无 Er抑制时 0D值为 Bo,各相应浓度 Er抑制时的 0D值为 B值， 以 B/Bo为纵坐标， 以 Er浓度的对数为横坐标， 绘制标准曲线， 建立回归方程。
。 m 有 Er抑制时的 OD值 f
B Bo = x 100%
无&竞争时的 OD值
1. 3. 2. 5样品提取方法
按照恩拉霉素粗品的分离、 纯化的方法进行粗品处理。 取 5. 0g 鸡肌肉组织剪碎， 匀浆 广 2min， 置于 100mL离心管中， 加 20毫升甲醇-水溶液 中， 匀浆 10min， 用浓 HCL 调整 pH3. 5~4. 0， 充分搅拌 10min， lOOOOrpm离心 10min， 取上清液， 用 NaOH调节 pH至 8. 0， lOOOOrpm离心 10min， 取上清液， 作为检测液备用。
1. 3. 2. 6 添加回收率的测定
取空白的鸡肌肉组织， 按照 500、 250、 50n/g的浓度添加 Er, 且使其在肌肉组织中混 匀， 并室温静置 20min， 按照 1. 3. 2. 5中的提取方法进行提取， 所得提取液进行 ELISA检 测。 每个添加浓度做 5份样品， ELISA检测时每份样品的提取液做 2个平行重复孔。 各样 品经上述方法处理后，按 1. 3. 2. 2 中的方法进行操作，检测 0D45。nm的吸收值，根据 1. 3. 2. 4 的方法计算抑制率， 代入标准回归方程， 计算 Er 的含量， 并根据以下公式计算添加回收 w , .、 实测值
添加回收率 (％) = 100%
添加值（ng)
2. 结果分析
2. 1 Er抗血清效价的测定
采用间接 ELISA方法， 测得抗 Er抗血清的最高效价大于 1 : 320000， 结果表明用戊二 醛法制备免疫抗原来免疫家兔， 能够产生特异性抗体。 在进行了 4次免疫后， 试验家兔均 产生了针对 Er的抗体， 其中 4号样品的抗血清效价明显高于其他家兔的抗血清效价， 结 果见图 1。
2. 2 ELISA检测方法的建立
2. 2. 1 包被抗原 Er-0VA与纯化后抗 Er抗血清工作浓度的确定
根据 ELISA检测的基本要求，选择 0D值为 1. 0左右时的抗原抗体浓度为最适工作浓度。 因此根据方阵滴定的结果确定包被抗原 Er-0VA的最适包被浓度为 2. 30ug/mL, 混合血清工 作浓度为 1 : 10000。 结果见图 2。
2. 3. 2标准曲线的建立
用间接竞争 ELISA法建立检测 Er的标准曲线， 呈良好的线性相关。 抑制率 (B/B。)与浓度 的对数 (LogC，母液为 lmg/mL)之间的线性回归方程为： y=-0. 2985x+l. 0148； y:抑制率 (B/B。)， x: 浓度的对数， 相关系数 r2=0. 9924; 其抑制中点即抑制率为 50%时对应的浓度是 53. Ong/mL; 检出范围在 8. 9ng/mL~982ng/mL之间， 其标准曲线见图 3.
2. 3. 4添加回收率测定
在鸡肌肉组织中以 500、 250、 50n/g的浓度添加 Er， 提取液用 ELISA检测， 计算出相应 Er的含量， 并根据添加的量计算回收率， 结果见表 2。
表 2 样品中添加 Er的回收结果 (n=5)
目前， 我国对与恩拉霉素的检测方法仅仅利用微生物检测法建立了预混料和伺料级的检 测手段， 袁而森等和谢明权等报道的检测极限分别为 0. 30ug/g和大于 0. 025mg/g的检测限; 另外， 据文献报道， 日本厚生省利用高效液相色谱法建立了畜、 水产食品恩拉霉素残留检测 法， 检测限量也仅为 0. 20ug/g。 本发明采用酶联免疫分析技术作为检测手段， 建立了间接竞 争 ELISA法， 对 Er进行了检测， 在实际中可以应用， 与国内外免疫检测方法的比较见表 3。
表 3对 Er检测法及检测限的比较
Claims
1、 检测肉制品中恩拉霉素残留的方法， 其特征在于包括如下步骤：
a、分别将待检测肉制品提取液和系列浓度的恩拉霉素标准液加入到预包被恩拉霉素抗原 的微孔中； 其中， 所述的肉制品提取液采用下述方法制备得到： 取待检测肉制品剪碎， 匀浆， 加浓度为 50%v/v的甲醇-水溶液，匀浆，用浓 HC1调整 pH值 3. 5〜4. 0，混匀， 8000〜12000rpm 离心 8〜12min， 取上清液， 用 NaOH调节 pH值至 8. 0， 8000〜 12000离心 8〜 12min， 取上 清液， 即得待检测肉制品提取液； 所述的恩拉霉素抗原采用申请号为 CN201010556432. 2 的 方法制备得到；
b、 然后于各微孔中加入抗恩拉霉素抗体， 混匀后孵育；
c、 于各微孔中加入辣根过氧化物酶 HRP—羊抗兔 IgG， 混合后孵育；
d、 分别于各微孔中加入底物溶液， 显色；
e、 分别于各微孔中加入终止液终止反应， 用酶标仪读取各孔 0D45。值；
f、 对照恩拉霉素标准液所得的曲线回归方程， 计算出待检测肉制品中的恩拉霉素含量。
2、 根据权利要求 1所述的检测肉制品中恩拉霉素残留的方法， 其特征在于： b步骤中加 入抗恩拉霉素抗体为含有抗恩拉霉素抗体的血清。
3、 根据权利要求 2所述的检测肉制品中恩拉霉素残留的方法， 其特征在于： a步骤中所 述的预包被恩拉霉素抗原的包被浓度为 2. 30ug/mL， b步骤中加入的含有抗恩拉霉素抗体的 血清的稀释度为 1 : 10000。
4、 根据权利要求 1所述的检测肉制品中恩拉霉素残留的方法， 其特征在于： e步骤中所 述的终止液为硫酸溶液。
5、根据权利要求 1所述的检测肉制品中恩拉霉素残留的方法，其特征在于包括如下步骤: a、分别将待检测肉制品提取液和系列浓度的恩拉霉素标准液加入到预包被恩拉霉素抗原 的微孔中； 待检测肉制品提取液和系列浓度的恩拉霉素标准液的加入量为 50uL/孔；
b、 然后于各微孔中加入含有抗恩拉霉素抗体的血清 50uL/孔， 血清稀释度为 1 : 10000， 37°C孵育 lh， 洗涤， 拍干；
c、 于各微孔中加入辣根过氧化物酶 HRP—羊抗兔 IgG 1 : 20000倍稀释， lOOuL/孔， 混 合后于 37°C孵育 lh， 洗涤， 拍干；
d、 分别于各微孔中加入 lOOuL底物溶液， 37°C显色 lOmin;
e、 分别于各微孔中加入终止液 2mOL/mLH2S04终止反应， 用酶标仪读取各孔 0D45。值；
f、 以无恩拉霉素抑制时 0D值为 Bo，各相应浓度恩拉霉素抑制时的 0D45。值为 B值， 以 B/Bo为纵坐标， 以恩拉霉素浓度的对数为横坐标， 绘制标准曲线， 建立回归方程， 对照恩 拉霉素标准液所得的曲线回归方程， 计算出待检测肉制品中的恩拉霉素含量。
6、 根据权利要求 1〜5任一项所述的检测肉制品中恩拉霉素残留的方法， 其特征在于： 所述的待检测肉制品为猪肉、 羊肉、 牛肉、 鸡肉、 鸭肉、 鹅肉、 鱼肉中至少一种。
7、根据权利要求 6所述的检测肉制品中恩拉霉素残留的方法， 其特征在于： 所述的待检 测肉制品为鸡肉。
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