| Diethylstilbestrol (DES) is a non-steroidal estrogen and has been used in animal production as growth promoting additives for increasing animal lean meat percentage and feed conversion. However, DES was banned in ainimal production in many countries due to its teratogenicity, carcinogenicity and other side effects on humans. Anyhow, it is still illegally used by some farmers from time to time for increasing profits.In order to circumcent the disadvantages (e.g.labourious, time-consuming and costly etc.) of the existed methods for the detcction of DES. It is urgrntly need to develop a rapid, simple, sensitive,less labourious cost-effective method.for detection of DES, In present study, we developed an ELISA kit and a colloidal strip test for the rapid, specific and sensitive detection of DES, The developed ELISA Kit and the colloidal strip test were validated by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometer (GC-MS).To prepare the immunogen DES-bovine serum albumin (BSA) and the coating antigen DES-ovalbumin (OVA), an active caboxyl group (-COOH) was introduced to the hydroxy (-OH) locus of phenyl of DES by a series of chemical reactions to form DES-MCPE. Conjugation of DES-MCPE with BSA and OVA was confirmed by ultraviolet spectrophotometry (UV) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Compared with BSA alone, the absorption peak of DES-BSA shifted in UV spectrum, and DES-BSA migrated slower in SDS-PAGE, indicating that DES-BSA has been conjugated successfully. DES-OVA conjugate was characterized as DES-BSA. The conjugation ratio of DES-MCPE with BSA was calculated as 12.8:1, and DES-BSA was immunized against BALB/c mice. Indirect ELISA showed that the titers of the anti-DES polyclonal antibody (DES pAb) were all higher than 1:1.0×104. The pAb from one mouse gave the best IC50 (50% inhibitive concentration) of 18.221 μg/L by indirect competitive ELISA (ci-ELISA), and has 8.09% and3.63% cross-reactivity (CR) to Hexoestrolum and Dienestrol respectively, with no CR to other compounds. These data indicated that pAb against DES with high-titer, specificity and sensitivity was obtained.MAb to DES was produced by fusing spleen cells from the immunized mouse with SP2/0 cells in the presence of PEG4000. Four hybridoma cell lines of 1B8,2C4,4A1 and 4C7 were screened out using indirect and ci-ELISA. Indirect ELISA showed the titers of these four positive hybridoma cells were 1:2.56×102,1:4.0×102, 1:1.2×102.1:8.0×102 in the supernatant, and were 1:2.0×105, 1:2.56×105,1:1.28×103 and 1:5.12×105 in ascites, respectively. Their affinity constants (Ka) are 3.38x109,9.27×109,2.30×109, and 1.87×1010 L/moL, and their isotypes belong to IgG2a/κ、IgG1/κ、IgG2a/κ、IgG2a/κ, respectively.4C7 shows the best IC50 of 0.49 μg/L to DES and has 7.66% and 3.83% CR to Hexoestrolum and Dienestrol respectively, with no CR to other compounds. A ci-ELISA was established using the McAb 4C7, and based on the competitive binding of free DES in the sample and the coated DES-BSA with the 4C7. The optimal conditions for ci-ELISA were determined by chessboard ELISA as the followings:coating concentration of DES-OVA was 0.5μg/mL; the working concentration of 4C7was 1:6.4× 104; the diluted concentration of GoaMoIgG-HRP was 1:1×10O3; the plate was coated with 50 μL of DES-OVA at 37℃ for 120 min, blocked for 60 min with 5% porcine serum alubumin at room temperature (RT). TMB was used for color development at RT for 10 min. The calibration curve of ci-ELISA DES-K.it showed typical sigmoid and fitted to the four parameters logistic equation. The IC50 in ci-ELISA was 0.49 μg/L and the limit of detection was 0.5μg/L. The average recoveries of DES in negative carp meat and pork were 79.3% and 81.63%; the average coefficient variation were 9.28% and 8.70%, respectively; the average inter-assay coefficient variation were 79.3% and 81.63%, respectively; the average intra-assay coefficient variation were 7.6% and 7.0%, respectively; the average inter-assay coefficient variation was greater than intra-assay coefficient variation, and both were lower than 10%. The DES-Kit generally has 6.53% and 3.42%CR to Hexoestrolum and Dienestrol, respectively; and possesses no CR to other compounds. The DES-Kit was stable when stored at 4℃ for 6 months.A colloidal gold-based strip test was developed by conjugating the 4C7 with colloidal gold. The diameter of the gold particles was optimized as 25 nm. and the optimal labeling concentration of 4C7 was 7.2 μg/ml. The calibration curve of strip test read by a strip reader BioDot-TSR3000 was a typical sigmoid curve. The limit of detection of the strip test was 0.25 μg/L by BioDot-TSR3000, and 2.0 μg/L by naked eyes. The strip test possesses a little CR to Hexoestrolum and Dienestrol, and has no CR to other compounds. The DES-Strip is stable when stored at 4℃ for nine months.Validation withHPLC indicated that the differrences for the determination of DES between DES-Kit and HPLC was from 2.7% to 4.3%, and the differences between DES-strip and HPLC was from 1.5% to 4.9%.Validation with GC-MS indicated that the differences for the determination of DES between DES-Kit and GC-MS was from 1.4% to 4.1%, and the differences between DES-strip and HPLC was from 1.0% to 4.6%. These results demonstrated that both DES-Kit and DES-strip were comparable to either HPLC or GC-MS. |
PDF Full Text Request