Establishment Of ELISA For Trenbolone Residue And Preparation Of IAC For Three β Agonists

Recently, issues of food safty were reported frequently in China。The food safty has been regarded more and more。But those who were driven by benefit also use illegal additives which threat people'healthy。In order to guarantee the food safty, development of convenient methods to detect drug residue is very important。Trenbolone (TRE) is a kind of steroid hormone which has been widely used. TRE, belongs to synthetic derivative, is similar with male hormone in human body--testosterone on structure and activity. It can promote the synthesis of protein, increase appetite, grow muscle and accelerate deposition of calcium and phosphorus on bone. So TRE can be used in treating serious poor nourishment, osteoporosis and so on. Meanwhile, TRE is the most frequently used stimulant. In addition, TRE plays an important role in controlling and treating animal diseases. But it is also important to note that TRE has serious side effects, such as insomnia, hypertension, night sweat and so on. The using of TRE is stringently controlled in many countries and areas because of the reasons above. But those who were driven by benefit also use TRE illegally. Hence, to monitor TRE in animal feeds and tissues, a convenient, rapid and sensitive screening method is urgently required.The present methods to detect the TRE are instrumental methods and immunosorbent assays. Physical and chemical detection methods include high-performance liquid chromatography (HPLC), GC-MS, liquid chromatography (LC), LC-MS and so on. These methods are sensitive and steady and can be used as standard methods. But they require expensive and heavy instruments and time-consuming sample clean-up process so they are not suitable to be used as routing screen and high-capacity field detection methods for drugs. Enzyme linked immunosorbent assay (ELISA) is the most common method in immunosorbent assays. This method is a kind of simply manipulation, low cost, rapid and accurate testing method which overcomes the defect above. So it possesses very high application value and prospect.We aimed to develop a more sensitive ELISA method to detect TRE residues in animal feed, tissue and urine. The hapten of TRE was synthesized by succinic anhydride method. The TRE hapten was coupled to BSA and KLH via an EDC/NHS reaction to synthesize coating antigen and immunogen. The highly specific monoclonal antibody was prepared by immunizing mice. And enzyme linked immunosorbent assay was developed to detect TRE residue. And the results are as follows:IC50 value was 0.323 ng.mL-1 toward TRE and an average limit of detection (LOD) was 0.06 ng.mL-1. The results showed that this antibody was highly specific to detect TRE in six structually and functionally related compounds. Besides, the recovery rates in animal feed, animal tissue and urine system were 82.0-83.4%, 81.3-89.4% and 82.7-87.2%, respectively. The average CVs are 5.8-12.8%,9.3-16.2%, 9.1-11.2% for inter-assay and 10.1%,11.7%,10.1% for intra-assay, respectively.Clenbuterol, ractopamine and salbutamol belong toβ-adrenergic agonists. Beta-adrenergic agonists are a sort of nutrition redistribution. They are phenolethanolamine derivative which are similar with adrenalin and arterenol on structure and function. They can not only be used to treat animal diseases, but also can promote the growth of animal, reduce fat content and increase lean meat percentage. But research has shown thatβ-adrenergic agonists can be accumulated in animal bodies. Anyone who takes animal foodstuff that contains drugs residue for long time may get his liver, kidney hurt seriously. So lots of countries and areas have banned usage of manyβ-adrenergic agonists in livestock husbandry. But with the drive of benefit, many lawbreakers use these drugs illegally all the same. So it is urgent to develop detection technologies of drugs residues.So far, the methods to detect theβ-adrenergic agonists'residue are including immunoassays, HPLC, liquid chromatography-mass spectrometry, GC-MS other methods. Most of these studies used traditional solid phase extraction. But it is noteworthy that the sample pretreatment processes of traditional SPE technique are relatively complex and need abundant organic reagent. So this method is in dire need of improvement.We aimed to prepare an immunoaffinity column (IAC) that can separate and concentrate clenbuterol, ractopamine and salbutamol simultaneously. The anti-salbutamol antibody and anti-ractopamine antibody were covalently bonded to CNBr-activated Sepharose 4B gel. And then the complex substance was transferred into column to attain the IAC. Relative to the SPE technology, this IAC possesses specificity and efficiency and can reduce matrix effects obviously. Besides, IAC coupled with HPLC-UV-FLD carried out detection of clenbuterol, ractopamine and salbutamol simultaneously. And the results were as follows:coupling ratio of Abs and Sepharos 4B was 88.6%; the developed IAC had sufficient total column capacity for salbutamol, clenbuterol and ractopamine which were 275,232,514 ng, respectively. The IAC clean-up method has been successfully applied to the isolation and purification of clenbuterol, salbutamolvand ractopamine from swine muscle, liver and feed. The recovery rates of the test were in the range of 78.3-95.9%, while the coefficient variations were 1.3% to 5.0%.According to the study of this project, immunogen and coating antigen were synthesized successfully and monoclonal antibody of TRE was developed by immunobiologic methods. ELISA of TRE was established based on this antibody. In addition, an immunoaffinity column (IAC) coupled with HPLC-UV-FLD that can detect clenbuterol, ractopamine and salbutamol simultaneously was developed. These research provide new methods and thinking of detection for food security.