Tetracycline, Salbutamol, Terbutaline Enzyme-linked Immunosorbent Assay To Establish

With the development of society and the improvement of life level, people increasingly consume animal food. Therefore, modern animal husbandry is developed very rapidly in recent years. To make more money, in animal husbandry many farmers use some drugs, such as tetracycline, salbutamol, and terbutaline to treat diseases or promote growth as feed additives. The illegal use of these drugs in animal husbandry, however, is inevitable to result in the presence of residues in edible tissues and can have potential health risks to humans. Therefore, the safety issue of food has been highly concerned in the world. To ensure the safety of food for consumers, more and more countries had established analytical techniques to detect residues of drugs. The traditionally instrumental analyses such as LC-MS, GC-MS, and HPLC are the most widely used methods to detect residues of drugs in food and food products. These methods are sensitive, accurate and highly specific, but require expensive equipment, large volume of solvents, derivative treatment, and time-consuming sample clean-up process. Therefore, they are not suitable to be used as routing screen and field detection methods for drags. The ELISA technique has long been known as a rapid, sensitive, specific, and cost-effective analytical method and has been used for diagnostic and residue detection purpose for many years. The aim of this research is to establish the ELISA techniques for tetracycline, salbutamol, and terbutaline.In this study, a simple and convenient indirect heterologous competitive ELISA method for tetracycline was developed using polyclonal antibody prepared. Three new immunogens, TC-Tolidine-BSA, TC-ABA-cBSA, and TC-CDI-cBSA, were synthesized in this research to develop anti-tetracycline antibodies. All antibodies raised in rabbits and coating antigens synthesized were screened and characterized using homologous and heterologous ELISA formats to select the best combination. An optimized ELISA gave an IC₅₀ value of 3.92 μg/mL towards tetracycline in PBST, and the limit of detection for tetracycline was 0.01 μg/mL. This method is sensitive and has a linear range from 0.1 to 100 μg/mL. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally closely related compounds of chlortetracycline (112%) and oxytetracycline (<2%). The recovery rates from the tetracycline-fortified raw milk samples were in the range of 74-116%, while the intra- and inter-assay coefficients of variation were <14.5 and <25.0, respectively.For salbutamol, two new immunogens, SAL-ABA-cBSA and SAL-CDI-cBSA, were synthesized in this research to develop anti-salbutamol antibodies. The antibody prepared with SAL-CDI-cBSA shows high sensitivity in the heterologous assay using SAL-BDE-OVA as a coating antigen, with an IC50 value of 9.86 ng/mL toward salbutamol. This antibody has a detective limit of 0.8 ng/mL and a linear range of 2.6-50 ng/mL. The antibody prepared with SAL-ABA-cBSA shows high sensitivity in the heterologous assay using CL-OVA as a coating antigen, with an IC50 value of 8.97 ng/mL toward salbutamol. This antibody has a detective limit of 0.1 ng/mL and a linear range of 0.8-1000 ng/mL. The antibody prepared with SAL-ABA-cBSA was assessed in an indirect competitive enzyme immunoassay to exploit the specificity obtained. The antibody prepared with SAL-ABA-cBSA shows high cross-reactivity with clenbuterol (107%) and is promising for the simultaneous determination of salbutamol and clenbuterol residues in food and food products. Recovery rates from the salbutamol-fortified swine liver samples were in the range of 70-99%, while the intra-assay and inter-assay coefficients of variation were <13.3 and <14.3, respectively.In our study to terbutaline, two new immunogens, TER-ABA-cBSA and TER-BDE-cBSA were synthesized to develop anti-terbutaline antibodies. The antibody prepared with TER-ABA-cBSA shows high sensitivity in the heterologous assay using TER-BDE-OVA as a coating antigen, with an IC₅₀ value of 21.4 ng/mL toward terbutaline. This antibody has a detective limit of 1 ng/mL and a linear range from 4 to 200 ng/mL. The antibody prepared with TER-BDE-cBSA shows high sensitivity in the heterologous assay using TER-ABA-OVA as a coating antigen, with an IC₅₀ value of 9.25 ng/mL toward terbutaline. This antibody has a detective limit of 0.3 ng/mL and a linear range from 0.8 to 100 ng/mL. The antibody prepared with TER-BDE-cBSA shows high cross-reactivity with clenbuterol (93.9%) and salbutamol (94.9%) and is promising for the simultaneous determination of terbutaline, salbutamol and clenbuterol residues in food and food products. Recovery rates from the terbutaline-fortified swine urine samples were in the range of 80-99%, while the intra-assay and inter-assay coefficients of variation were <12.5 and <12.2, respectively.In conclusion, we have successfully prepared polyclonal antibodies of tetracycline, salbutamol, and terbutaline. The competitive ELISA methods developed in this study provide alternative, sensitive, specific, and stable analysis for the detection of drug residues and can be used in preparation of tetracycline, salbutamol, and terbutaline ELISA kits.