多西环素多克隆抗体的制备及鉴定
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摘要
目的:采用人工偶联方法构建多西环素完全抗原,制备多克隆抗体。方法:分别采用直接偶联法、甲醛一步法、重氮法+混合酸酐法和重氮法+碳二亚胺法等4种方法将多西环素与牛血清白蛋白(bull serum albumin,BSA)或鸡卵清蛋白(ovalbumin,OVA)分别偶联,构建人工完全抗原。选择最好偶联效果制备的抗原免疫BALB/c小鼠,通过直接ELISA法检测多克隆抗体效价,竞争抑制ELISA法分析其灵敏性及特异性。结果:重氮法+碳二亚胺法偶联的多西环素人工完全抗原效果最好,其与BSA的偶联比为8.37∶1,与OVA的偶联比为4.92∶1。采用该方法偶联的抗原免疫小鼠获得的多克隆抗体效价在1∶8 000以上;该抗体对多西环素的半数抑制浓度(IC50)为98.89~120.32μg/L,与其他四环素类药物的交叉反应性较低。结论:重氮法+碳二亚胺法的偶联效率最高,获得的多西环素多克隆抗体效价较高,特异性和灵敏度均较理想。
Objective: To conjugate the doxycycline( Dox) with bull serum albumin( BSA) /ovalbumin( OVA) and generate polyclonal antibody against the Dox. Methods: The Dox was coupled with BSA / OVA via direct,one-step formaldehyde,diazo plus mixed anhydride and diazo plus carbodiimide conjugation method,respectively. The BALB / c mice were immunized with Dox-BSA which conjugated by most efficient method. The titer,sensitivity and specificity of antibody were detected and assayed by direct or indirect competitive inhibition ELISA. Results: It had been declared that the diazo plus carbodiimide conjugation method was most efficient,and the coupling ratio of Dox ∶ BSA was 8. 37 ∶ 1,and Dox∶ OVA was 4. 92 ∶ 1. The polyclone antibody( p Ab) titer was more than 1 ∶ 8 000 by indirect ELISA.The half maximal inhibitory concentration( IC50) was 98. 89- 120. 32 μg /L. There was less the cross reactivity with other tetracyclines. Conclusion: The conjugation method of diazo plus carbodiimide was the most efficient and the p Ab has high titer,better sensitivity and specificity.
Objective: To conjugate the doxycycline( Dox) with bull serum albumin( BSA) /ovalbumin( OVA) and generate polyclonal antibody against the Dox. Methods: The Dox was coupled with BSA / OVA via direct,one-step formaldehyde,diazo plus mixed anhydride and diazo plus carbodiimide conjugation method,respectively. The BALB / c mice were immunized with Dox-BSA which conjugated by most efficient method. The titer,sensitivity and specificity of antibody were detected and assayed by direct or indirect competitive inhibition ELISA. Results: It had been declared that the diazo plus carbodiimide conjugation method was most efficient,and the coupling ratio of Dox ∶ BSA was 8. 37 ∶ 1,and Dox∶ OVA was 4. 92 ∶ 1. The polyclone antibody( p Ab) titer was more than 1 ∶ 8 000 by indirect ELISA.The half maximal inhibitory concentration( IC50) was 98. 89- 120. 32 μg /L. There was less the cross reactivity with other tetracyclines. Conclusion: The conjugation method of diazo plus carbodiimide was the most efficient and the p Ab has high titer,better sensitivity and specificity.
引文
[1]Son K,Fujioka S,Iida T,et al.Doxycycline induces apoptosis in PANC-1 pancreatic cancer cells[J].Anticancer Res,2009,29(10):3995-4003.
[2]Tang H,Sampath P,Yan X,et al.Potential for enhanced therapeutic activity of biological cancer therapies with doxycycline combination[J].Gene Ther,2013,20(7):770-778.
[3]Berman B,Perez OA,Zell D.Update on rosacea and anti-inflammatory-dose doxycycline[J].Drugs Today(Barc),2007,43(1):27-34.
[4]Su W,Li Z,Li F,et al.Doxycycline-mediated inhibition of corneal angiogenesis:an MMP-independent mechanism[J].Invest Ophthalmol Vis Sci,2013,54(1):783-788.
[5]Dalvi PS,Singh A,Trivedi HR,et al.Effect of doxycycline in patients of moderate to severe chronic obstructive pulmonary disease with stable symptoms[J].Ann Thorac Med,2011,6(4):221-226.
[6]Su W,Li Z,Li Y,et al.Doxycycline enhances the inhibitory effects of bevacizumab on corneal neovascularization and prevents its side effects[J].Invest Ophthalmol Vis Sci,2011,52(12):9108-9115.
[7]Chen X,Xia S,Li R,et al.Doxycycline enhances the Ras-MAPK signaling and proliferation of mouse thymic epithelial cells[J].J Cell Biochem,2009,107(3):494-503.
[8]Huang Y,Li R,Chen X,et al.Doxycycline up-regulates the expression of IL-6 and GM-CSF via MAPK/ERK and NF-κB pathways in mouse thymic epithelial cells[J].Int Immunopharmacol,2011,11(9):1143-1149.
[9]甘金华,邓薇,李进平,等.强力霉素人工抗原的合成与抗体制备[J].食品与生物技术学报,2011,30(2):316-320.
[10]万亮.三种真菌毒素蛋白质微阵列检测方法的初步研究以及抗四环素多克隆抗体的制备[D].南昌:南昌大学,2008.
[11]乐涛,严佩峰,王喜亮,等.多西环素人工抗原的合成与鉴定[J].中国兽药杂志,2010,44(1):30-34,55.
[12]Zhang Y,Lu S,Liu W,et al.Preparation of anti-tetracycline antibodies and development of an indirect heterologous competitive enzyme-linked immunosorbent assay to detect residues of tetracycline in milk[J].J Agric Food Chem,2007,55(2):211-218.
[2]Tang H,Sampath P,Yan X,et al.Potential for enhanced therapeutic activity of biological cancer therapies with doxycycline combination[J].Gene Ther,2013,20(7):770-778.
[3]Berman B,Perez OA,Zell D.Update on rosacea and anti-inflammatory-dose doxycycline[J].Drugs Today(Barc),2007,43(1):27-34.
[4]Su W,Li Z,Li F,et al.Doxycycline-mediated inhibition of corneal angiogenesis:an MMP-independent mechanism[J].Invest Ophthalmol Vis Sci,2013,54(1):783-788.
[5]Dalvi PS,Singh A,Trivedi HR,et al.Effect of doxycycline in patients of moderate to severe chronic obstructive pulmonary disease with stable symptoms[J].Ann Thorac Med,2011,6(4):221-226.
[6]Su W,Li Z,Li Y,et al.Doxycycline enhances the inhibitory effects of bevacizumab on corneal neovascularization and prevents its side effects[J].Invest Ophthalmol Vis Sci,2011,52(12):9108-9115.
[7]Chen X,Xia S,Li R,et al.Doxycycline enhances the Ras-MAPK signaling and proliferation of mouse thymic epithelial cells[J].J Cell Biochem,2009,107(3):494-503.
[8]Huang Y,Li R,Chen X,et al.Doxycycline up-regulates the expression of IL-6 and GM-CSF via MAPK/ERK and NF-κB pathways in mouse thymic epithelial cells[J].Int Immunopharmacol,2011,11(9):1143-1149.
[9]甘金华,邓薇,李进平,等.强力霉素人工抗原的合成与抗体制备[J].食品与生物技术学报,2011,30(2):316-320.
[10]万亮.三种真菌毒素蛋白质微阵列检测方法的初步研究以及抗四环素多克隆抗体的制备[D].南昌:南昌大学,2008.
[11]乐涛,严佩峰,王喜亮,等.多西环素人工抗原的合成与鉴定[J].中国兽药杂志,2010,44(1):30-34,55.
[12]Zhang Y,Lu S,Liu W,et al.Preparation of anti-tetracycline antibodies and development of an indirect heterologous competitive enzyme-linked immunosorbent assay to detect residues of tetracycline in milk[J].J Agric Food Chem,2007,55(2):211-218.