Danofloxacin, Gatifloxacin, Terbutaline Establishment Of His Forest Residues In The Enzyme-linked Immunoassay

With the development of the livestock husbandry, veterinary drug, including veterinary drug additives, plays a vital role in reducing incidence of diseases and death rate, raising feed using rate, and promoting animals' growth. At the same time, however, because of lacking of related knowledge and wanting to make more money, some farmers use veterinary drug illegally. The illegal use of these drugs in animal husbandry is inevitable to result in the presence of residues in edible tissues and can have potential health risks to humans, such as allergy, deformity, mutation, cancer and so on. Therefore, the safety issue of food especially the safety issue of drug residues in livestock products has been highly concerned in the world. It becomes important to control drug residues level in animal products.There are a lot of problems in livestock husbandry in China such as in scale, not proficient in management, no standard in regular usage of drugs, short in withdrawal time and etc. They are harmful to our health. In recent years, Chinese animal food exports were blocked repeatedly because of the problem of drug residues. It caused huge economic losses, and damaged the image of the quality of Chinese animal food seriously. Chinese government laws to impose restrictions on drug residues as well as withdrawal time. In order to control drug residues in animal products, protect import and export trade, guarantee people's health, Chinese government is strengthening in fiancial support in research to develop analytical techniques to detect drug residues.At present the analytical techniques to detect drug residues mainly include physical and chemical detection methods (such as HPLC,LC-MS,LC-MS/MS), microbial detection method, immunosorbent assay (including ELISA, RIA, EIA, FIA, MAIA). Physical and chemical detection methods can be considered as the standard method to detect drug residues, because it is accurate, stable and reliable, but the equipment used in these methods is expensive, plus complex, time-consuming and laborious sample treatment procedure. So it is difficult to popularize. The results measured by the microbial detection methods are usually higher than the Maximum Residue Limits (MRLs). A rapid, sensitive and effective detection method must be established. Enzyme-linked immunoassay (including ELISA, EICA, CLEIA) overcomes the shortcomings of the methods mentioned above and is a rapid, sensitive, convenient, easy universal detection method. This paper aims at establishing enzyme-linked immunosorbent assay (ELISA) to detect danofloxacin, electrochemical immunoassay (ECIA) to detect gatifloxacin, and chemiluminescence enzyme-linked immunoassay (CLEIA) to detect terbutaline.For danofloxacin, using the indirect ELISA, we obtained the results: The anti-danofloxacin shows high sensitivity, with an IC50 value of 2.0 ppb toward danofloxacin; The antibody has a detective limit of 0.8 ppb and a linear range of 0.8-6.0 ppb; For this antibody, there is only lower cross-reactivity with fleroxacin (20.6 %) and pefloxacin(21.7 %); Recovery rates from the danofloxacin-fortified chicken liver samples were in the range of 84.0-109.6 %, while the intra-assay and inter-assay coefficients of variation were <15.3 % and <19.0 % respectively.For gatifloxacin, using the indirect ECIA, we obtained the results: The anti-gatifloxacin shows high sensitivity, with an IC50 value of 10 ppb toward gatifloxacin; The antibody has a detective limit of 1.0 ppb and a linear range of1.0-100 ppb; For this antibody, there is only lower cross-reactivity with lomefloxacin (3.0 %) , ofloxacin (3.0 %) and ciprofloxacin (21.7 %); Recovery rates from the gatifloxacin -fortified urine samples were in the range of 93.0-117.0 %, while the inter-assay coefficients of variation was <12.5 % respectively.For terbutaline and furaltadone, using the indirect CLEIA, we obtained the results: The anti-terbutaline and anti-furaltadone show high sensitivity, with IC50 value of 1.0 ppb toward terbutaline and 5.0 ppb toward furaltadone. The anti-terbutaline has a detective limit of 0.1 ppb and a linear range of 0.1-10 ppb; For this antibody, there is only lower cross-reactivity with Clenbuterol (62.9 %) and salbutamol (62.9 %); Recovery rates from the terbutaline-fortified urine samples were in the range of 83.8-112.9 %, while the intra-assay and inter-assay coefficients of variation were <19.9 % and <7.2 % respectively. The anti- furaltadone has a linear range of 0.5-50 ppb; With high specificity, there is only lower cross-reactivity with NPAMOZ (45.5 %), AMOZ (41.7 %) and furazolidone (4.1 %); Recovery rates from the terbutaline in water samples were in the range of 90.3-111.8%, while the intra-assay and inter-assay coefficients of variation were <11.8 % and <16 % respectively.Through this study, we prepared specific polyclonal antibody for danofloxacin , gatifloxacin, terbutaline and furaltadone. Using these antibodies, we established rapid sensitive methods to detect the residues of for danofloxacin , gatifloxacin, terbutaline and furaltadone. There researches can be used to develop various immunoassay kits danofloxacin, terbutaline and furaltadone CLEIA kits and gatifloxacin.