19-Nortestosterone (NT) has been illegally used in horse racing to boost physical performance, and in animal husbandry to accelerate weight gain. To monitor the abuse of NT, our goal was to develop a commercial enzyme...19-Nortestosterone (NT) has been illegally used in horse racing to boost physical performance, and in animal husbandry to accelerate weight gain. To monitor the abuse of NT, our goal was to develop a commercial enzyme linked immunosorbent assay (ELISA) kit. For this purpose, hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mouse. Noncompetitive and competitive indirect ELISA were used to screen positive cell clones. To optimize the indirect competitive ELISA (icELISA) method, various methanol concentrations in assay buffer were evaluated. Matrix effects in urine and spiking test were also investigated. Finally, five hybridoma cell lines named NT-1, NT-2, NT-3, NT-4 and NT-5 were screened out. The corresponding monoclonal antibodies (mAbs) were of the IgG 1 isotype with a k light chain, and the antibody affinity of all mAbs were between 2.6×10 9 and 4.7×10 9 L/mol. The titer and IC 50 values of purified ascites were in the range of 0.64×10 5 2.56×10 5 and 0.55-1.0 ng/mL, respectively. Based on the NT-1 hybridoma, a heterologous icELISA method was developed for the quantitative detection of NT in cattle urine. The dynamic range was from 0.004 to 85.8 ng/mL, with a detection limit for the assay and IC 50 values of 0.002 and 0.55 ng/mL, respectively. Except for a high cross-reactivity (62%) to α-NT, negligible cross-reactivity to other compounds was observed. After optimization, 10% of methanol was used in the assay buffer, and a 20-fold dilution in cattle urine gave an inhibition curve almost the same as that in phosphate buffered saline. The correlation coefficient between the established icELISA and LC-MS/MS method was 0.9871. The results showed that the established heterologous icELISA method provides an excellent alternative for the detection of NT residues in food producing animals.展开更多
Objective To produce anti‐19‐Nortestosterone （NT） monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolate...Objective To produce anti‐19‐Nortestosterone （NT） monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate （CAAP） method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant （Kaff）. Results Five hybridoma cell lines, named NT‐1, NT‐2, NT‐3, NT‐4, and NT‐5, were identified and their corresponding mAbs were of the IgG 1 isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7×10 9 L/mol. The titers and IC 50 values of purified ascite fluids were in the range of （0.64–2.56）×10 5 and （0.55–1.0） ng/mL, respectively. Of all the cross‐reacting steroids, α ‐NT was the most reactive with the mAbs at 62% with NT‐1 mAb and 64% with NT‐2 mAb. Negligible cross‐reactivity （0.01%） with other steroids was observed. Conclusion The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.展开更多
目的制备人绒促性素(hCG)单克隆抗体。方法hCG抗原免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1融合,间接ELISA法筛选阳性孔,有限稀释法进行克隆化培养;制备腹水抗体;间接ELISA法测定抗体效价;采用HiTrap rProtein A FF亲和...目的制备人绒促性素(hCG)单克隆抗体。方法hCG抗原免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1融合,间接ELISA法筛选阳性孔,有限稀释法进行克隆化培养;制备腹水抗体;间接ELISA法测定抗体效价;采用HiTrap rProtein A FF亲和色谱柱纯化抗体。结果得到2株能稳定分泌单克隆抗体的杂交瘤细胞株,细胞培养上清中抗体效价达10-3以上,腹水抗体效价达10-7以上,纯化后的单抗纯度达98%以上,回收率达75%。结论成功获得两株能稳定分泌单克隆抗体的杂交瘤细胞,可用于早孕、肿瘤等诊断的研究。展开更多
To get the hybridoma cell lines secreting anti-thyrotropin monoclonal antibodies with high affinity and specificity. Methods: BALB/c mice were immunized with extract of human pituitaries. The spleen cells of one immun...To get the hybridoma cell lines secreting anti-thyrotropin monoclonal antibodies with high affinity and specificity. Methods: BALB/c mice were immunized with extract of human pituitaries. The spleen cells of one immunized mouse were fused with mouse myeloma cells in polyethylene glycol and the positive clones were subcloned 3 times. Results: Two hybridoma cell lines which secrete anti-thyrotropin monoclonal antibodies with high affinity and specificity have been collected. The antibodies were of the IgG1 subclass and their maximum binding with thyrotropin was 60% and 45. 1% respectively. Using competitive binding assay,the antibodies were found to direct against different epitopes of human thyrotropin. Conclusion: The extract of human pituitaries could be used to produce monoclonal anti-pituitary hormone antibodies. The two anti-thyrotropin monoclonal antibodies produced in this study could be used in the establishment of a sensitive measurement of human thyrotropin.展开更多
Plant wilt bacteria (P. solanacearum)have a wide host range and cause wilt diseases of more than 200 species from 33 families. Because of no suitable resistant materials to wilt diseases, Fan Yun-liu et al. first prop...Plant wilt bacteria (P. solanacearum)have a wide host range and cause wilt diseases of more than 200 species from 33 families. Because of no suitable resistant materials to wilt diseases, Fan Yun-liu et al. first proposed that this disease would be controlled with the genetic engineering of bacteriocin. Since bacteriocin M2 from strain M2 of P.展开更多
基金supported by the Eleventh Five-Year Plan for National Science and Technology of China (2006BAK02A21/1)Henan Innovation Project for University Prominent Research Talents (2010HASTIT026)
文摘19-Nortestosterone (NT) has been illegally used in horse racing to boost physical performance, and in animal husbandry to accelerate weight gain. To monitor the abuse of NT, our goal was to develop a commercial enzyme linked immunosorbent assay (ELISA) kit. For this purpose, hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mouse. Noncompetitive and competitive indirect ELISA were used to screen positive cell clones. To optimize the indirect competitive ELISA (icELISA) method, various methanol concentrations in assay buffer were evaluated. Matrix effects in urine and spiking test were also investigated. Finally, five hybridoma cell lines named NT-1, NT-2, NT-3, NT-4 and NT-5 were screened out. The corresponding monoclonal antibodies (mAbs) were of the IgG 1 isotype with a k light chain, and the antibody affinity of all mAbs were between 2.6×10 9 and 4.7×10 9 L/mol. The titer and IC 50 values of purified ascites were in the range of 0.64×10 5 2.56×10 5 and 0.55-1.0 ng/mL, respectively. Based on the NT-1 hybridoma, a heterologous icELISA method was developed for the quantitative detection of NT in cattle urine. The dynamic range was from 0.004 to 85.8 ng/mL, with a detection limit for the assay and IC 50 values of 0.002 and 0.55 ng/mL, respectively. Except for a high cross-reactivity (62%) to α-NT, negligible cross-reactivity to other compounds was observed. After optimization, 10% of methanol was used in the assay buffer, and a 20-fold dilution in cattle urine gave an inhibition curve almost the same as that in phosphate buffered saline. The correlation coefficient between the established icELISA and LC-MS/MS method was 0.9871. The results showed that the established heterologous icELISA method provides an excellent alternative for the detection of NT residues in food producing animals.
基金supported by the Henan Innovation Project for University Prominent Research Talents, 2010HASTIT026
文摘Objective To produce anti‐19‐Nortestosterone （NT） monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate （CAAP） method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant （Kaff）. Results Five hybridoma cell lines, named NT‐1, NT‐2, NT‐3, NT‐4, and NT‐5, were identified and their corresponding mAbs were of the IgG 1 isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7×10 9 L/mol. The titers and IC 50 values of purified ascite fluids were in the range of （0.64–2.56）×10 5 and （0.55–1.0） ng/mL, respectively. Of all the cross‐reacting steroids, α ‐NT was the most reactive with the mAbs at 62% with NT‐1 mAb and 64% with NT‐2 mAb. Negligible cross‐reactivity （0.01%） with other steroids was observed. Conclusion The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.
文摘目的制备人绒促性素(hCG)单克隆抗体。方法hCG抗原免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1融合,间接ELISA法筛选阳性孔,有限稀释法进行克隆化培养;制备腹水抗体;间接ELISA法测定抗体效价;采用HiTrap rProtein A FF亲和色谱柱纯化抗体。结果得到2株能稳定分泌单克隆抗体的杂交瘤细胞株,细胞培养上清中抗体效价达10-3以上,腹水抗体效价达10-7以上,纯化后的单抗纯度达98%以上,回收率达75%。结论成功获得两株能稳定分泌单克隆抗体的杂交瘤细胞,可用于早孕、肿瘤等诊断的研究。
文摘To get the hybridoma cell lines secreting anti-thyrotropin monoclonal antibodies with high affinity and specificity. Methods: BALB/c mice were immunized with extract of human pituitaries. The spleen cells of one immunized mouse were fused with mouse myeloma cells in polyethylene glycol and the positive clones were subcloned 3 times. Results: Two hybridoma cell lines which secrete anti-thyrotropin monoclonal antibodies with high affinity and specificity have been collected. The antibodies were of the IgG1 subclass and their maximum binding with thyrotropin was 60% and 45. 1% respectively. Using competitive binding assay,the antibodies were found to direct against different epitopes of human thyrotropin. Conclusion: The extract of human pituitaries could be used to produce monoclonal anti-pituitary hormone antibodies. The two anti-thyrotropin monoclonal antibodies produced in this study could be used in the establishment of a sensitive measurement of human thyrotropin.
文摘Plant wilt bacteria (P. solanacearum)have a wide host range and cause wilt diseases of more than 200 species from 33 families. Because of no suitable resistant materials to wilt diseases, Fan Yun-liu et al. first proposed that this disease would be controlled with the genetic engineering of bacteriocin. Since bacteriocin M2 from strain M2 of P.
