氧氟沙星免疫亲和色谱柱的研制
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摘要
本研究将氧氟沙星(ofloxacin,OFL)与两种载体蛋白偶联合成了两种人工抗原。用合成的免疫原免疫家兔,制备出高效价的抗OFL血清。经纯化后研制出针对氧氟沙星特异性吸附的免疫亲和色谱柱,并利用高效毛细管电泳(HPCE)对色谱柱进行了评价。
1.人工抗原的合成和鉴定
采用碳二亚胺法,将氧氟沙星分别与载体蛋白牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联,制备出两种人工抗原OFL-BSA和OFL-OVA。由FeCl3显色反应结果可知:彻底透析的人工抗原溶液与FeCl3反应后出现不同程度的橙黄色且略显浑浊,透析外液、BSA溶液和OVA溶液则呈现浅黄色,说明透析内液中存在大量连接在载体蛋白上的氧氟沙星,由此可初步推断氧氟沙星与载体蛋白BSA和OVA的交联成功。紫外扫描图谱显示人工抗原的最大吸收峰即不同于载体,也不同于半抗原,另外人工抗原的紫外扫描图谱还显示有半抗原的特征性吸收峰出现,进一步说明交联反应成功。
2.抗体制备和鉴定
用人工合成的免疫抗原OFL-BSA免疫家兔,制备出了抗OFL血清,抗OFL血清的效价为1:128×103。先后用饱和硫酸铵盐析沉淀法和DEAE-Sephadex A-50柱层析法对抗OFL血清进行了纯化。纯化后抗体的效价仍然保持在1:128×103。用抗OFL血清建立检测氧氟沙星的间接竞争ELISA法时,包被抗原OFL-OVA的工作浓度为2.5μg/mL,抗OFL血清的工作浓度为1:32×103,酶标二抗的稀释倍数为1:10000。实验对抗OFL抗体进行了交叉反应性的测定,测定结果表明,氧氟沙星与氟喹诺酮类药物都具有一定的交叉反应,交叉反应率在1.80%~4.52%之间,与磺胺嘧啶、氯霉素、土霉素和阿莫西林等非氟喹诺酮类抗菌药物的交叉反应率皆小于0.01%。测得的抗OFL抗体亲和常数为7.91×107 L /mol,从抗体性能这三个角度来看此纯化后的抗体符合氧氟沙星药物残留分析的要求,适合IAC的制备。
3.IAC制备和鉴定
建立检测氧氟沙星药物的毛细管电泳(HPCE)检测方法,绘制出标准曲线,线性方程为y=241.21x-158.03,R2=0.9981。最低检测限为2μg/mL。测得的免疫吸附剂的偶联率93%,动态柱容量为2204ng/mL,绝对柱容量为473.98ng/mL,分别添加5mL200ng/mL、300ng/mL和400ng/mL氧氟沙星标准工作液,5mL甲醇洗脱,高效毛细管电泳检测,连续测定6次,测得其回收率为94%、92.67%、95.5%,变异系数为5.32%、3.17%、3.84%。
In this study, ofloxacin(OFL) were linked respectively to two kinds of carrier proteins , by which the two kinds of artificial antigens were synthesized successfully.Using the artificial immunogens,rabbits were immunized to obtain the antiserum against ofloxacin. Then,using this purified antiserum,we produced the column of ofloxacin immunoaffinity chromatography which was evaluated by HPCE.
1.Synthesis and Identification of the Artifical Antigens for Ofloxacin.
Ofloxacin was coupled respectively to bovine serum albumin (BSA) by Carbodiimide method, Ofloxacin was also coupled respectively to ovalbumin(OVA) by Carbodiimide method.by which the Conjugates OFL-BSA and OFL-OVA were prepaired.The Conjugates were identified by colour reaction of FeCl3 and UV Spectrum.The results demonstrated that the four kinds of artificial antigens were synthesized successfully.
2.Preparation and dentification of Antisera.
OFL-BSA was used as immunogens to immunise rabbits, by which the antiserum against ofloxacin was obtained.The titer of the antiserum against ofloxacin was 1:128×103. The antiserum against ofloxacin whose titer still was 1:128×103, was purified from the antiserum by a series procedure of precipitation with saturated ammonium sulfate and chromatography with DEAE-Sephadex A-50. Using the antiserum against ofloxacin to establish Ci-ELISA methods for ofloxacin ,the working concentrations of the coating antigen (OFL-OVA) was 2.5μg/ml, the working concentrations of the antisera was 1:32×103, the working concentrations of Ab-HRP was 1:10000. By determinating the cross-reactivity of OFL, cross-reactivity rates were in the range from 1.80% to 4.52% between OFL and the fluoroquinolones. But OFL had no cross-reactivity with sulfadiazine, chloramphenicol, terramycin or penicillin. In this study the affinity constant was 7.91×107 L /mol.
3.The preparation for the column of ofloxacin immunoaffinity chromatography and the dynamic capacity of IAC was evaluated.
The regression equation of OFL in HPCE was y=241.21x-158.03, and the coefficient was 0.9981. the limits of detect was 2ug/ml. After evaluating the column,we have determined the coupling rate of 93%、the dynamic capacity of 2204ng/mL and the absolute capacity of 473.98ng/mL for ofloxacin. Using 5mL methanol as elution buffer,the recoveries were 94 %、92.6 %、95.5 %and C.V. was 5.32%、3.17%、3.84% at levels of 200ng/mL、300ng/mL、400ng/mL in 5mL standard working liquid detected by HPCE for six times.
1.人工抗原的合成和鉴定
采用碳二亚胺法,将氧氟沙星分别与载体蛋白牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联,制备出两种人工抗原OFL-BSA和OFL-OVA。由FeCl3显色反应结果可知:彻底透析的人工抗原溶液与FeCl3反应后出现不同程度的橙黄色且略显浑浊,透析外液、BSA溶液和OVA溶液则呈现浅黄色,说明透析内液中存在大量连接在载体蛋白上的氧氟沙星,由此可初步推断氧氟沙星与载体蛋白BSA和OVA的交联成功。紫外扫描图谱显示人工抗原的最大吸收峰即不同于载体,也不同于半抗原,另外人工抗原的紫外扫描图谱还显示有半抗原的特征性吸收峰出现,进一步说明交联反应成功。
2.抗体制备和鉴定
用人工合成的免疫抗原OFL-BSA免疫家兔,制备出了抗OFL血清,抗OFL血清的效价为1:128×103。先后用饱和硫酸铵盐析沉淀法和DEAE-Sephadex A-50柱层析法对抗OFL血清进行了纯化。纯化后抗体的效价仍然保持在1:128×103。用抗OFL血清建立检测氧氟沙星的间接竞争ELISA法时,包被抗原OFL-OVA的工作浓度为2.5μg/mL,抗OFL血清的工作浓度为1:32×103,酶标二抗的稀释倍数为1:10000。实验对抗OFL抗体进行了交叉反应性的测定,测定结果表明,氧氟沙星与氟喹诺酮类药物都具有一定的交叉反应,交叉反应率在1.80%~4.52%之间,与磺胺嘧啶、氯霉素、土霉素和阿莫西林等非氟喹诺酮类抗菌药物的交叉反应率皆小于0.01%。测得的抗OFL抗体亲和常数为7.91×107 L /mol,从抗体性能这三个角度来看此纯化后的抗体符合氧氟沙星药物残留分析的要求,适合IAC的制备。
3.IAC制备和鉴定
建立检测氧氟沙星药物的毛细管电泳(HPCE)检测方法,绘制出标准曲线,线性方程为y=241.21x-158.03,R2=0.9981。最低检测限为2μg/mL。测得的免疫吸附剂的偶联率93%,动态柱容量为2204ng/mL,绝对柱容量为473.98ng/mL,分别添加5mL200ng/mL、300ng/mL和400ng/mL氧氟沙星标准工作液,5mL甲醇洗脱,高效毛细管电泳检测,连续测定6次,测得其回收率为94%、92.67%、95.5%,变异系数为5.32%、3.17%、3.84%。
In this study, ofloxacin(OFL) were linked respectively to two kinds of carrier proteins , by which the two kinds of artificial antigens were synthesized successfully.Using the artificial immunogens,rabbits were immunized to obtain the antiserum against ofloxacin. Then,using this purified antiserum,we produced the column of ofloxacin immunoaffinity chromatography which was evaluated by HPCE.
1.Synthesis and Identification of the Artifical Antigens for Ofloxacin.
Ofloxacin was coupled respectively to bovine serum albumin (BSA) by Carbodiimide method, Ofloxacin was also coupled respectively to ovalbumin(OVA) by Carbodiimide method.by which the Conjugates OFL-BSA and OFL-OVA were prepaired.The Conjugates were identified by colour reaction of FeCl3 and UV Spectrum.The results demonstrated that the four kinds of artificial antigens were synthesized successfully.
2.Preparation and dentification of Antisera.
OFL-BSA was used as immunogens to immunise rabbits, by which the antiserum against ofloxacin was obtained.The titer of the antiserum against ofloxacin was 1:128×103. The antiserum against ofloxacin whose titer still was 1:128×103, was purified from the antiserum by a series procedure of precipitation with saturated ammonium sulfate and chromatography with DEAE-Sephadex A-50. Using the antiserum against ofloxacin to establish Ci-ELISA methods for ofloxacin ,the working concentrations of the coating antigen (OFL-OVA) was 2.5μg/ml, the working concentrations of the antisera was 1:32×103, the working concentrations of Ab-HRP was 1:10000. By determinating the cross-reactivity of OFL, cross-reactivity rates were in the range from 1.80% to 4.52% between OFL and the fluoroquinolones. But OFL had no cross-reactivity with sulfadiazine, chloramphenicol, terramycin or penicillin. In this study the affinity constant was 7.91×107 L /mol.
3.The preparation for the column of ofloxacin immunoaffinity chromatography and the dynamic capacity of IAC was evaluated.
The regression equation of OFL in HPCE was y=241.21x-158.03, and the coefficient was 0.9981. the limits of detect was 2ug/ml. After evaluating the column,we have determined the coupling rate of 93%、the dynamic capacity of 2204ng/mL and the absolute capacity of 473.98ng/mL for ofloxacin. Using 5mL methanol as elution buffer,the recoveries were 94 %、92.6 %、95.5 %and C.V. was 5.32%、3.17%、3.84% at levels of 200ng/mL、300ng/mL、400ng/mL in 5mL standard working liquid detected by HPCE for six times.
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[2] Gootz,T.D.,McGuirk,P.R.,Moynihon,M.S&Haskell,S.L..Placement of alkyl substituents on the C7 piperazine ring of fluoroquinolones:Dramatic differential effects on mammalian top-oisomeraseⅡand DNA gyrase [J].Antimicrobial Agents and Chemotherapy,1994,38,130~ 131
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[4] HoriiT ,M onjiA ,U emuraK ,N agura0 .J. Microbiol.Methods.2006,65:557~561
[5]聂青和,张开瑞.氟喹诺酮类抗菌药物不良反应的临床观察[J].中级医刊,1993,5:45
[6]杨晓,杨烨.喹诺酮类药物致过敏性休克165例文献分析[J].中国药房,2006,17(5):372
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