| Ivermectin (IVM) is a kind of antibiotics that belongs to the family of marocyclic lactone. It has been widely used in livestock due to its broad spectrum of killing nemotodes and arthropods. However, IVM is taken as a poisonous compound, so its residue in animal tissue should not be ignored. The purpose of this study is to acquire and select the antibody to IVM, and further based on the antibody, develop a better method of ELISA to detect IVM residue in animal tissue.Haptens 4″-O-succinyl-IVM and 5-O-succinyl-IVM were prepaired by Using IVM. Mass spectrometry detection test showed that their molecular weight were 976 and 976.3, which was consistent to the anticipated product. Immunogen 4″-O-succinyl-IVM-BSA and 4″-O-succinyl-IVM-PLL were synthesized by coupling the hapten with carrier proteins BSA and PLL, respectively. Another immunogen, 5-O-succinyl-IVM-PLL was prepared by coupling the hapten 5-O-succinyl-IVM with PLL. And By linking hapten 5-O-succinyl-IVM with carrier protein OVA, coated antigen 5-O-succinyl-IVM-OVA was got. In order to select the better reaction conditions, an orthogonal experiment was designed, which showed that the best result was acquired at pH8.5, PLL35mg, 4℃, 8 hours recovery 52.6%, coupling ratio 30.2 during the preparation process of immunogen.The results of non-competition repression test of antiserum titers and Western blot showed that no significant difference existed between the antibodies of 4″-O-succinyl-IVM-PLL and 5-O-succinyl-IVM-PLL on their immunogen and specification to IVM. Due to the process of preparing 5-O-succinyl-IVM-PLL is much easier than 4″-O-succinyl-IVM-PLL, ELISA competition test were set up based on the antibodies of anti-4″-O-succinyl-IVM-BSA and anti- 5-O-succinyl-IVM-PLL, for the following ELISA detection.The appropriate concentrations of antigen to antibody were determined using square-matrix method. The results showed that the concentrations of coated antigen and enzyme-linked 2nd antigen were 30ng/ml and 1:1000, respectively, and the dilution times for the three antibodies were both 1∶10~5. According to the orthogonal test, all of pH (6.4~8.4), ionic strength (0.05~1.5MNaCl)and methanol concentration (5% ~ 15% (v/v) ) had no significant influence to the competition reaction. So, considering the convenience, the conditions of 0.50M NaCl, 10% methanol and pH 7.4 for the reaction system of two-antibody-competition reaction ELISA were chosed.For the three antibodie testing, the same coated antigen or different coated antigens were chosed. Four combination test were performed to analyze the standard curves, the detectable limits and sensitivity of the four combinations were also compared. The results indicated that all of the four experiments could meet the analysis requirement. And to the four experiments, the lowest detectable limit was 0.83 and the highest IC50 was 7.01ng/g. According to the analysis, PLL was better to be immuno-enhancing carrier.Using ELISA test, IVM in four tissues including muscle, liver, blood and milk were detected. Inthe study, different combinations of two antibodies and two coated antigens with the purpose of determining the recovery were designed. In the two experiments (experiment 5 in Chapter 4), the recovery rate of IVM varied between 93.9% and 101.2% and the variation coefficient was less than 5.1%;while in the other two experiments (experiment 6 in Chapter 4 ) the recovery rate of IVM varied between 92.6% and 101.7% and the variation coefficient was less than 6.3%. So the two methods were both reasonable and practicable.According to this research, it showed that using ELISA based on the antibodies of anti-4"-O-IVM-BSA antibody and anti-5-O-IVM-PLL to detect IVM, anti-5-O-succinyl-IVM-PLL antibody had higher affinity and specificity than anti-4"-O-succinyl-IVM-BSA. Therefor, the antigen 5-O-succinyl-IVM-PLL prepared in the study could induce animals to produce excellent antibody and it could be used to detect IVM and some AVMs by ELISA. |
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