抗培氟沙星单克隆抗体杂交瘤细胞株的制备及免疫学鉴定
摘要
本试验旨在制备分泌培氟沙星抗体的杂交瘤细胞，并对其免疫学特性进行鉴定，为培氟沙星药物残留检测奠定基础。结果如下：
1.完全抗原的制备及鉴定通过EDC法将培氟沙星与牛血清白蛋白与鸡卵清白蛋白偶联，并通过SDS-PAGE及紫外分光光度法鉴定其偶联结果，通过获得的紫外吸收值及相关公式计算出其结合比。结果表明，成功偶联了PEF-BSA及PEF-OVA，并通过SDS-PAGE及紫外确定其偶联成功，通过相关公式计算免疫抗原PEF-BSA的偶联比为8:1，包被抗原PEF-OVA为6:1。
2.抗体血清的制备及检测应用制备的免疫抗原常规免疫小鼠，采血，利用Ci-ELISA检测其抗体效价，并对其包被抗原的选择进行可行性分析。结果显示，Ci-ELISA测定血清抗体效价，免疫5只小鼠的血清效价均到达4000以上，5号小鼠达到32000。通过Ci-ELISA确定用PEF-OVA为包被抗原是可行的。
3.分泌培氟沙星抗体的杂交瘤细胞的制备通过常规方法，制备滋养层细胞、脾细胞，复苏SP2/0细胞，进行计数后融合，并利用间接ELISA测定阳性率，筛选出阳性细胞，并采用有限稀释法进行克隆，筛选出全阳性细胞扩大培养；利用筛选出的阳性细胞制备腹水以获得大量抗体细胞。结果显示，筛选出2株分泌培氟沙星抗体的细胞，分别为5E2和4D8，融合率分别为31.18%和45.65%，三克后阳性率均达到100%。
4.杂交瘤细胞的检测采用秋水仙素法鉴定抗体细胞的染色体数，以鉴定融合是否成功；采用辛酸~硫酸铵法纯化腹水并以Ci-ELISA检测效价；采用蛋白试剂盒鉴定抗体亚型及分类；Ci-ELISA鉴定分泌抗体的稳定性。结果发现，杂交瘤细胞染色体数在90左右，确定免疫小鼠脾细胞与SP2/0融合成功；比较纯化前后腹水效价变化，纯化后腹水效价略有升高；试剂盒鉴定蛋白亚型为IgG1；Ci-ELISA鉴定冻存4个月后复苏的细胞，稳定性良好。
本研究成功筛选出分泌培氟沙星抗体的细胞株，并通过亚型、染色体、稳定性鉴定确定为免疫脾细胞与SP2/0融合的细胞，为培氟沙星残留检测提供依据。
The objective aimed at preparing the hybridoma of PEF and applied to explore itsimmunological characteristics for immunological rapid determination of PEF residues.Theresults showed that:
1. Basis on analysis of molecular structure and immunogenicity of PEF,immungen andcovering antigen were prepared by EDC coupling PEF to BSA or OVA.By the ultravioletabsorb spectral curve and SDS~PAGE electrophoresis,get the conjugate ratio. It turned outthat the PEF-BSA and PEF-OVA were synthesized successfully,and the molecule conjugateration of PEF to BSA and OVA were8:1and6:1respectively.
2. To immunize BALB/c mice using PEF-BSA,the antibody was identified byCi-ELISA,in order to prove that the PEF-OVA as a bag was antigen is feasible,we choose theCi-ELISA method.The result was the five mice immune serum antibody titer all reached1:4000by Ci-ELISA,the fifth mice was arived1:32000.Ci-ELISA test positive serum and resultsshowed that feasible.
3. BALB/c mice was chosen from five mice BALB/c immunized with PEF-BSA forcell fusion,the result of the positive by Ci-ELISA for the hybridoma.Scanning the positive cellwith the（limiting dilution analysis）LDA for the ascites.The result was that scanning twohybridoma lines of5E2and4D8,the cell fusion ration were31.18%and45.65%respectively，the ration of objective were100%.
4. Caryotype analysis of hybridoma of PEF mAb with the method of colchicine;comparison of ELISA titer of ascites and purified ascites;identify and classify the antibodysubtypes using the protein kit, assay the the stability of secretory antibodies in the indirectELISA.The results showed that the isotpe of them was IgG1, caryotype analysis ofhybridoma of PEF mAb was about90,comparison of ELISA titer of ascites and purifiedascites,the titer was higher;the anabiosis cells was indentified by Ci-ELISA,prove that hasgood stability.
In this study，we have successfully screened out of the cell line, which could secretePefloxacin antibody.Finally, we confirmed that the cell lines are fusioned by the cells ofimmune spleen and SP2/0through subtype analysis, chromosomal counts and identification ofstability, it has provided the basis for the detection of pefloxacin residue.
1.完全抗原的制备及鉴定通过EDC法将培氟沙星与牛血清白蛋白与鸡卵清白蛋白偶联，并通过SDS-PAGE及紫外分光光度法鉴定其偶联结果，通过获得的紫外吸收值及相关公式计算出其结合比。结果表明，成功偶联了PEF-BSA及PEF-OVA，并通过SDS-PAGE及紫外确定其偶联成功，通过相关公式计算免疫抗原PEF-BSA的偶联比为8:1，包被抗原PEF-OVA为6:1。
2.抗体血清的制备及检测应用制备的免疫抗原常规免疫小鼠，采血，利用Ci-ELISA检测其抗体效价，并对其包被抗原的选择进行可行性分析。结果显示，Ci-ELISA测定血清抗体效价，免疫5只小鼠的血清效价均到达4000以上，5号小鼠达到32000。通过Ci-ELISA确定用PEF-OVA为包被抗原是可行的。
3.分泌培氟沙星抗体的杂交瘤细胞的制备通过常规方法，制备滋养层细胞、脾细胞，复苏SP2/0细胞，进行计数后融合，并利用间接ELISA测定阳性率，筛选出阳性细胞，并采用有限稀释法进行克隆，筛选出全阳性细胞扩大培养；利用筛选出的阳性细胞制备腹水以获得大量抗体细胞。结果显示，筛选出2株分泌培氟沙星抗体的细胞，分别为5E2和4D8，融合率分别为31.18%和45.65%，三克后阳性率均达到100%。
4.杂交瘤细胞的检测采用秋水仙素法鉴定抗体细胞的染色体数，以鉴定融合是否成功；采用辛酸~硫酸铵法纯化腹水并以Ci-ELISA检测效价；采用蛋白试剂盒鉴定抗体亚型及分类；Ci-ELISA鉴定分泌抗体的稳定性。结果发现，杂交瘤细胞染色体数在90左右，确定免疫小鼠脾细胞与SP2/0融合成功；比较纯化前后腹水效价变化，纯化后腹水效价略有升高；试剂盒鉴定蛋白亚型为IgG1；Ci-ELISA鉴定冻存4个月后复苏的细胞，稳定性良好。
本研究成功筛选出分泌培氟沙星抗体的细胞株，并通过亚型、染色体、稳定性鉴定确定为免疫脾细胞与SP2/0融合的细胞，为培氟沙星残留检测提供依据。
The objective aimed at preparing the hybridoma of PEF and applied to explore itsimmunological characteristics for immunological rapid determination of PEF residues.Theresults showed that:
1. Basis on analysis of molecular structure and immunogenicity of PEF,immungen andcovering antigen were prepared by EDC coupling PEF to BSA or OVA.By the ultravioletabsorb spectral curve and SDS~PAGE electrophoresis,get the conjugate ratio. It turned outthat the PEF-BSA and PEF-OVA were synthesized successfully,and the molecule conjugateration of PEF to BSA and OVA were8:1and6:1respectively.
2. To immunize BALB/c mice using PEF-BSA,the antibody was identified byCi-ELISA,in order to prove that the PEF-OVA as a bag was antigen is feasible,we choose theCi-ELISA method.The result was the five mice immune serum antibody titer all reached1:4000by Ci-ELISA,the fifth mice was arived1:32000.Ci-ELISA test positive serum and resultsshowed that feasible.
3. BALB/c mice was chosen from five mice BALB/c immunized with PEF-BSA forcell fusion,the result of the positive by Ci-ELISA for the hybridoma.Scanning the positive cellwith the（limiting dilution analysis）LDA for the ascites.The result was that scanning twohybridoma lines of5E2and4D8,the cell fusion ration were31.18%and45.65%respectively，the ration of objective were100%.
4. Caryotype analysis of hybridoma of PEF mAb with the method of colchicine;comparison of ELISA titer of ascites and purified ascites;identify and classify the antibodysubtypes using the protein kit, assay the the stability of secretory antibodies in the indirectELISA.The results showed that the isotpe of them was IgG1, caryotype analysis ofhybridoma of PEF mAb was about90,comparison of ELISA titer of ascites and purifiedascites,the titer was higher;the anabiosis cells was indentified by Ci-ELISA,prove that hasgood stability.
In this study，we have successfully screened out of the cell line, which could secretePefloxacin antibody.Finally, we confirmed that the cell lines are fusioned by the cells ofimmune spleen and SP2/0through subtype analysis, chromosomal counts and identification ofstability, it has provided the basis for the detection of pefloxacin residue.
引文
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