喹乙醇单克隆抗体的制备及其免疫学快速检测方法的建立
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摘要
喹乙醇(Olaquindox,OLA)是化学合成的喹噁啉类广谱抗菌药物,具有蛋白同化作用,可提高饲料转化率与瘦肉率,促进动物生长,也可用于预防和治疗细菌感染性疾病。但是在生产实践中,喹乙醇的不规范使用不仅直接危害动物机体的健康,还可以通过食物链对人类健康造成极大的危害。随着科学技术的发展和国家的重视,其检测方法也不断发展。目前已有电化学分析法、光谱法、色谱法、液相色谱-质谱联用技术、免疫分析技术等检测方法。为了建立快速、灵敏的喹乙醇残留免疫学分析方法,本研究在分析喹乙醇免疫学特性的基础上,运用单克隆抗体技术制备了喹乙醇单克隆抗体,并组装了间接竞争ELISA试剂盒。主要研究结果如下:
1.喹乙醇人工抗原的制备与鉴定
琥珀酸酐法合成衍生物喹乙醇-半琥珀酸酯(OLA-HS);活化酯法合成全抗原OLA-BSA、OLA-OVA,紫外分光光度法(UV)、凝胶电泳(SDS-PAGE)鉴定;用OLA-BSA免疫BALB/c小鼠,间接ELISA测定多抗血清效价,间接竞争ELISA鉴定其敏感性、特异性。紫外扫描图谱显示合成的人工抗原的最大吸收峰发生偏移,证明偶联成功,并测得OLA-BSA、OLA-OVA中OLA-HS和BSA、OVA的分子结合比分别为分别为17.1:1和12.4:1;SDS-PAGE结果表明BSA的泳动速度大于OLA-BSA,说明OLA-BSA的分子量大于BSA,进一步说明OLA-HS与BSA偶联成功。3#小鼠抗血清的效价最高,对OLA的半数抑制浓度(IC50)为58.923 ng/mL,与卡巴氧的交叉反应率为1.841%,与其他药物无交叉反应。通过实验获得了高价、敏感、特异的多克隆抗体(pAb)。
2.喹乙醇单克隆抗体的制备及免疫学特性的鉴定
间接ELISA和间接竞争ELISA选择细胞融合备用鼠;杂交瘤技术制备喹乙醇单克隆抗体(OLAmAb),并对其效价、亲和力、敏感性、特异性、亚型进行鉴定;体内诱生腹水法大量制备单克隆抗体。筛选出了2B1、4F5、4E12、5B5、5E5五株敏感特异的杂交瘤细胞;鉴定单抗亚型均为IgG1;细胞培养上清间接ELISA效价在1:3.0×102~1:1.28×10~3之间,4E12细胞株制作的腹水效价为1:5.12×10~5,OLAmAb亲和常数Ka为3.75×10~(10)L/moL,对OLA的IC50为1.66ng/mL;与卡巴氧的交叉反应率为5.19%,与其它类药物无交叉反应。通过试验获得了高效价、敏感、特异的OLAmAb,可用于动物性食品中OLA的残留检测的免疫学试验。
3.喹乙醇残留免疫学快速检测方法的建立
根据酶联免疫吸附试验原理,应用OLAmAb研制出OLA残留快速检测间接竞争ELISA试剂盒(OLA-Kit),OLA-Kit的标准曲线呈S型,符合4参数logit曲线拟合,最低检测限为0.126μg/L,半数抑制浓度IC50为1.66ng/mL,平均批内和批间变异系数小于<15%。对猪肝、猪肉分别添加2、10、50、100ng/mL OLA标准品,平均回收率分别为77.6%和79.68%。OLA-Kit性能稳定,在4℃可保存6个月。
Olaquindox is a chemosynthetic broad-spectrum antibacterial drug. As a derivative of quinoxaline-1, 4-dioxide with protein assimilation, it can be used to improve feed conversion rate,lean meat percentage so as to promote the growth of animals and prevent and treat bacterial infection. Not standardized use of olaquindox is not only directly against the body of the animal health, but also on human health through the food chain causing tremendous harm in the production practic. Along with technological development and national attention, its detection method has also developed continuously. Diferent analytical methods are currently used: electrochemical analysis, Spectrometry, Chromatography, LC-MS, IAS, etc. In order to find a rapid and sensitive means of immunological analysis to detect the Olaquindox residue, in this study, based on the analysis of molecular structure and immunogenicity of Olaquindox, the technology of monoclonal antibodies production was applied to prepare the monoclone antibody against Olaquindox and then assemble rapid ELISA kit and strip for OLA residual detection. The main contents and resuLts of this study were as follows:
1. Synthesis and identification of the artificial antigen for Olaquindox
The derivative of olaquindox, called as OLA-SH, was synthesized by succinic anhydride method. Two complete antigens of olaquindox (OLA-BSA, OLA-OVA) were prepared by active ester method (AE). The characterization of hapten-protein conjugates were examined by two methods, ultraviolet spectrophotometry(UV), sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) respectively. BALB/c mice were immunized with OLA-BSA, the titre of polyclonal antibody(pAb)was detected by indirect ELISA, the sensitivity and specificity of pAb was identified by blocking ELISA. The result of UV spectrum show that the largest artificial antigen absorption peak migration occurred, the conjugation ratio of OLA-HS to BSA and OVA was about 17.1:1and 12.4:1. SDS-PAGE results show that the BSA's swimming faster than OLA-BSA, the OLA-BSA molecular weight greater than BSA, This further shows that OLA-BSA has been coupled with success. The titer of pAb for 3# mouse is the highest, its IC50 was 58.923ng/mL, cross reaction rate to carbadox was 1.841%, and no cross reaction to other antibiotics. The high-titer, sensitive and specific anti-OLA pAb had been produced.
2. Preparation of monoclonal antibodies and immunological characterization of Olaquindox
The titre and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA, so as to select the mouse used in cell fusion. OLAmAb was prepared by hybridoma technology. The titer, affinity, sensitivity, specificity and subtype of the mAb were characterized. Massive OLAmAb were induced from in vivo method. There hybridoma cell lines of 2B1、4F5、4E12、5B5 and 5E5 were screened for specificity to OLA, all the isotypes of the mAb were IgG1. The indirect ELISA titer of the mAb were 1:3.0×10~2 ~1:1.28×10~3 in supernatant, 1:5.12×10~5 of 4E12 in ascites, and the affinity constant(Ka) was 3.75×1010L/moL, the mAb of 4E12 showed good sensitivity with IC50 of 1.66ng/mL to OLA. The rate of cross reaction of OLAmAb with carbadox was 5.19%, and there was no cross-reactivity to other compounds. OLAmAb of high-titer, sensitivity and specificity had been generated, it is possible to establish immunoassay of OLA residues in animal food.
3. Development of rapid determination of Difloxacin residue method
Based on the enzyme-linked immunosorbent assay principle, a competitive ELISA kit for determination of OLA (OLA-Kit) was developed with OLAmAb. The calibration curve of OLA-Kit with standard OLA inhibitor was typical sigmoid curve fitted to the four parameters logistic equation, the detection limit for OLA was 1ng/mL and IC50 was1.66ng/mL. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. The recoveries from pig liver and pork were 77.6% and 79.68%, respectively, when 2, 10, 50, 100ng/mL OLA were spiked. The validity of OLA-Kit in 4℃was above six months.
1.喹乙醇人工抗原的制备与鉴定
琥珀酸酐法合成衍生物喹乙醇-半琥珀酸酯(OLA-HS);活化酯法合成全抗原OLA-BSA、OLA-OVA,紫外分光光度法(UV)、凝胶电泳(SDS-PAGE)鉴定;用OLA-BSA免疫BALB/c小鼠,间接ELISA测定多抗血清效价,间接竞争ELISA鉴定其敏感性、特异性。紫外扫描图谱显示合成的人工抗原的最大吸收峰发生偏移,证明偶联成功,并测得OLA-BSA、OLA-OVA中OLA-HS和BSA、OVA的分子结合比分别为分别为17.1:1和12.4:1;SDS-PAGE结果表明BSA的泳动速度大于OLA-BSA,说明OLA-BSA的分子量大于BSA,进一步说明OLA-HS与BSA偶联成功。3#小鼠抗血清的效价最高,对OLA的半数抑制浓度(IC50)为58.923 ng/mL,与卡巴氧的交叉反应率为1.841%,与其他药物无交叉反应。通过实验获得了高价、敏感、特异的多克隆抗体(pAb)。
2.喹乙醇单克隆抗体的制备及免疫学特性的鉴定
间接ELISA和间接竞争ELISA选择细胞融合备用鼠;杂交瘤技术制备喹乙醇单克隆抗体(OLAmAb),并对其效价、亲和力、敏感性、特异性、亚型进行鉴定;体内诱生腹水法大量制备单克隆抗体。筛选出了2B1、4F5、4E12、5B5、5E5五株敏感特异的杂交瘤细胞;鉴定单抗亚型均为IgG1;细胞培养上清间接ELISA效价在1:3.0×102~1:1.28×10~3之间,4E12细胞株制作的腹水效价为1:5.12×10~5,OLAmAb亲和常数Ka为3.75×10~(10)L/moL,对OLA的IC50为1.66ng/mL;与卡巴氧的交叉反应率为5.19%,与其它类药物无交叉反应。通过试验获得了高效价、敏感、特异的OLAmAb,可用于动物性食品中OLA的残留检测的免疫学试验。
3.喹乙醇残留免疫学快速检测方法的建立
根据酶联免疫吸附试验原理,应用OLAmAb研制出OLA残留快速检测间接竞争ELISA试剂盒(OLA-Kit),OLA-Kit的标准曲线呈S型,符合4参数logit曲线拟合,最低检测限为0.126μg/L,半数抑制浓度IC50为1.66ng/mL,平均批内和批间变异系数小于<15%。对猪肝、猪肉分别添加2、10、50、100ng/mL OLA标准品,平均回收率分别为77.6%和79.68%。OLA-Kit性能稳定,在4℃可保存6个月。
Olaquindox is a chemosynthetic broad-spectrum antibacterial drug. As a derivative of quinoxaline-1, 4-dioxide with protein assimilation, it can be used to improve feed conversion rate,lean meat percentage so as to promote the growth of animals and prevent and treat bacterial infection. Not standardized use of olaquindox is not only directly against the body of the animal health, but also on human health through the food chain causing tremendous harm in the production practic. Along with technological development and national attention, its detection method has also developed continuously. Diferent analytical methods are currently used: electrochemical analysis, Spectrometry, Chromatography, LC-MS, IAS, etc. In order to find a rapid and sensitive means of immunological analysis to detect the Olaquindox residue, in this study, based on the analysis of molecular structure and immunogenicity of Olaquindox, the technology of monoclonal antibodies production was applied to prepare the monoclone antibody against Olaquindox and then assemble rapid ELISA kit and strip for OLA residual detection. The main contents and resuLts of this study were as follows:
1. Synthesis and identification of the artificial antigen for Olaquindox
The derivative of olaquindox, called as OLA-SH, was synthesized by succinic anhydride method. Two complete antigens of olaquindox (OLA-BSA, OLA-OVA) were prepared by active ester method (AE). The characterization of hapten-protein conjugates were examined by two methods, ultraviolet spectrophotometry(UV), sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) respectively. BALB/c mice were immunized with OLA-BSA, the titre of polyclonal antibody(pAb)was detected by indirect ELISA, the sensitivity and specificity of pAb was identified by blocking ELISA. The result of UV spectrum show that the largest artificial antigen absorption peak migration occurred, the conjugation ratio of OLA-HS to BSA and OVA was about 17.1:1and 12.4:1. SDS-PAGE results show that the BSA's swimming faster than OLA-BSA, the OLA-BSA molecular weight greater than BSA, This further shows that OLA-BSA has been coupled with success. The titer of pAb for 3# mouse is the highest, its IC50 was 58.923ng/mL, cross reaction rate to carbadox was 1.841%, and no cross reaction to other antibiotics. The high-titer, sensitive and specific anti-OLA pAb had been produced.
2. Preparation of monoclonal antibodies and immunological characterization of Olaquindox
The titre and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA, so as to select the mouse used in cell fusion. OLAmAb was prepared by hybridoma technology. The titer, affinity, sensitivity, specificity and subtype of the mAb were characterized. Massive OLAmAb were induced from in vivo method. There hybridoma cell lines of 2B1、4F5、4E12、5B5 and 5E5 were screened for specificity to OLA, all the isotypes of the mAb were IgG1. The indirect ELISA titer of the mAb were 1:3.0×10~2 ~1:1.28×10~3 in supernatant, 1:5.12×10~5 of 4E12 in ascites, and the affinity constant(Ka) was 3.75×1010L/moL, the mAb of 4E12 showed good sensitivity with IC50 of 1.66ng/mL to OLA. The rate of cross reaction of OLAmAb with carbadox was 5.19%, and there was no cross-reactivity to other compounds. OLAmAb of high-titer, sensitivity and specificity had been generated, it is possible to establish immunoassay of OLA residues in animal food.
3. Development of rapid determination of Difloxacin residue method
Based on the enzyme-linked immunosorbent assay principle, a competitive ELISA kit for determination of OLA (OLA-Kit) was developed with OLAmAb. The calibration curve of OLA-Kit with standard OLA inhibitor was typical sigmoid curve fitted to the four parameters logistic equation, the detection limit for OLA was 1ng/mL and IC50 was1.66ng/mL. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. The recoveries from pig liver and pork were 77.6% and 79.68%, respectively, when 2, 10, 50, 100ng/mL OLA were spiked. The validity of OLA-Kit in 4℃was above six months.
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