| Diethylstilbestrol (DES) and Dienestrol (DIEN) are non-steroidal, synthetic estrogen. They have been used in the area of animal husbandry as growth promoting agents for decades for its positive influences on economically important traits such as growth rate, lean meat rate and feed efficiency. Due to its adverse effects which cause a significant increase in carcinogenic response of progeny to human or animal, many countries have banned the use of DES and DIEN as feed additives. However, DES and DIEN abuse still occurs in livestock as result of its great economic interests. It is necessary to develop simple, quick, effective and sensitive detection method for them. So in this study, we have prepared monoclonal antibody (McAb) against DES and monoclonal antibody against DIEN. The aim of this project is to develop a sensitive ELISA based on McAb for detection of DES and DIEN residue in samples.1. DES and DIEN were activated by cross-linkers. Mono-o-carboxypropyl-diethylstilbestrol (DES-CP), hemi-succinate-diethylstilbestrol (DES-HS) and Mono-o-carboxymethyl-diethylstilbestrol (DES-CA) were synthesized, respectively. DES-CP was conjugated with covalently to bovine serum albumin (BSA) as immunogens. Then DES-CP, DES-HS and DES-CA were coupled to ovalbumin (OVA) as coating antigens, respectively. In addition, Mono-o-carboxypropyl-dienestrol (DIEN-CP) and hemi-succinate-dienestrol (DIEN-HS) were also synthesized. DIEN-CP was conjugated with covalently to BSA as immunogens. DIEN-HS was coupled to OVA as coating antigens. The absorption spectra of immunogens were scanned by UV–Vis spectrometer. The spectra exhibited that haptens were successfully coupled to carriers. The conjugate ratio of DES and DIEN to BSA were estimated by MALAD-TOF-MS according to the molecular weight alteration between BSA and hapten-BSA, which is 12:1 and 11:1, respectively.2. In order to evaluate the effect of spacer arm on the sensitivity of ELISA, different spacer arm of coating antigens (DES-CA-OVA, DES-CP-OVA and DES-HS-OVA) were synthesized. The female New Zealand white rabbits were immunized with DES-CP-BSA. The results showed that heterology in spacer structure of DES-HS-OVA with 4 carborchain presented the highest sensitivity of ELISA. The typical calibration curve of DES gave an IC50 value of 7.0 ng/ml and linear range of 1.0-30 ng/ml. Polyclonal antibody against DES was successfully prepared, which lays the foundation for preparation of McAb.3. To prepare McAb against DES and McAb DIEN, the BALB/c mice were immunized with immunogen.The best BALB/c mouse was selected as the spleen cell donor for fusion. The procedures include fusion, culture, hybridoma selection and clone. Finally, three established cell strains which excrete antibody against DES were obtained. They were named 3B9, 8F10 and 9C1. Similarly, two established cell strains which excrete antibody against DIEN were prepared. Five cell strains were injected into cavum abdominis of BALB/c mice. Ascites was purified by ammonia sulfate and identified by SDS-PAGE. The titres of two kinds of McAb all reach 1: 1.0×10~6. McAb which come from five cell lines belong to IgG1 and Kappa isotype.4. After the optimization of phycial and chemical parameters (concentration of McAb and coating antigen, coating condition, time of blocking, time of incubation, pH, and salt), the typical calibration curve of DES gave an IC50 value of 1.0 ng/ml, linear range of 0.3-3 ng/ml, a limit of detection of 0.065 ng/ml, a limit of quantitation of 0.15 ng/ml. The results showed cross-reactivity to hexestrol (7.7%), dienestrol (1.3%) and 17β-estradiol (<0.01%). The typical calibration curve of DIEN gave an IC50 value of 5.4 ng/ml, linear range of 1-30 ng/ml, a limit of detection of 0.14 ng/ml, a limit of quantitation of 0.24 ng/ml. The results showed no cross-reactivity to hexestrol, diethylstilbestrol and 17β-estradiol. It demonstrates that McAb against DES and McAb against DIEN process high specificity.5. The recoveries of DES and DIEN were obtained range between 71-107% and 62-64% when determining DES and DIEN residues in chicken, respectively. Sixteen chickens collected from Beijing city of local market were tested for residues of DES and DIEN. Data obtained showed all values not more than 1μg/kg. In order to estimate reliability of ELISA, DES kit of R-Biopharm was used to compare some parameters. The results presented recoveries and coefficient of variations do not occur different. Data of ELISA kit showed that sixteen chickens belong to negative samples. At present, DES and DIEN of limit of detection is 1μg/kg in GB. It indicates that established ELISA of DES and DIEN on can meet demand of resiudes determination.
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