微囊藻毒素-LR酶联免疫法的开发研究
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摘要
酶联免疫吸附分析(Enzyme-linked immunosorbent assay,ELISA)建立于1971年,并用于医学研究。微囊藻毒素的ELISA分析国外应用得早一些,国内也有应用,但不如国外成熟。本文以微囊藻毒素-LR的酶联免疫分析作为探讨对象,内容包括微囊藻的毒素-LR的提取与纯化、微囊藻毒素-LR的完全抗原化、以及微囊藻毒素-LR单克隆抗体细胞株的筛选培养。
(1)通过对从滇池捞取的新鲜藻样过筛选取、冷冻干燥制得蓝藻藻粉;用醋酸溶液破坏蓝藻细胞壁,使藻毒素溢出细胞;通过C18柱子的浓缩富集作用,用80%的甲醇溶液洗脱并收集藻毒素的提取液;对藻毒素提取液进行液相色谱分离分析,确定MC-LR保留时间为30min～31min,收集MC-LR分离液,浓缩、过C18柱纯化MC-LR,得到MC-LR粗提取液,在荧光硅胶薄层板的进一步精密分离下,得到MC-LR精制样品;通过液相色谱-质谱仪(LC-MS)鉴定,采用外标法计算,得到纯度为98.79%的MC-LR精制样品。本试验一共提取MC-LR精制样品6.4mg。
(2)MC-LR的分子量为995.2D,是典型的半抗原。在免疫反应过程中,只有反应性没有免疫原性,为使MC-LR具有免疫原性,我们利用蛋白质的偶联技术,使其在动物免疫的过程中产生有效的免疫应答。本文首先用二巯基乙胺活化MC-LR,在MC-LR的第七位氨基酸脱氢丙氨酸的双键位置引入一个氨基,然后选用分子量为67000D的牛血清白蛋白(BSA)为载体蛋白,通过戊二醛两步法使MC-LR与BSA得到耦联。通过生物质朴法对中间产物H2N-etMC-LR的分子量进行了鉴定,确定MC-LR的活化很成功;分别通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术、基质辅助激光解析电离飞行时间质朴(MALDI-TOF-MS)检测,对偶联物MC-LR-BSA进行定性、定量鉴定。结果显示,耦联比在3～18:1之间,平均耦联比为8:1。
(3)选用已鉴定的MC-LR-BSA免疫Balb/c实验小鼠,按100ug/只进行腹腔注射,加强免疫3～5次,每隔两周加强免疫一次,得到了效价为接近1:25600的血清。采用杂交瘤技术,取脾脏细胞与骨髓瘤细胞SP2/0进行细胞融合,用HAT选择性培养基进行选择培养,利用间接ELISA法筛选阳性细胞孔,将筛选出的阳性细胞进行细胞克隆欲获得稳定的细胞株,扩大培养。运用间接ELISA法测定MC-LR的最适包被浓度为10ng～20ng/孔。
Enzyme-linked immunosorbent assay (ELISA) was established in 1972 applied to Medical Research. ELISA analysis of microcystins was earlier applied abroad. It’s also applied at home, but it wasn’t as matuar as abroad. In this paper, We study mainly in ELISA analysis of microcystins. It include extracting and purification of MC-LR, making incomplete antigen to complete antigen of MC-LR, screening and cuiluring monoclonal antibody cell line of MC-LR.
(1)We got enough cyanobacteria from DianchiLake by sieving andlyophilization. Algae cell walls was damaged by acetic acid, and microcycstins overflowed from cell into acetic acid. Enrichment the acetic acid by Sep-pak C18 column, then elution the column by 80% methanol solution and gather the eluent. Using Liquid Chromatography Assay analyzed the Algae toxin extracts, discovering the retention time is 30th min to 31th min.Collect the separation liquid from LC within 30th min to 31th min. Enrichment the separation liquid by Sep-pak C18 column again, then elution the column by 80% methanol solution and gather the eluent. The eluent is crude extracts of MC-LR. Coating the crude extracts on Fluorescent silica gel thin layer plate, and by this further precise separation, we finally got the refined MC-LR. And its purity quotient is 96.88% by LC-MS identifiing and the external standard method. We totally got 4.6 mg refined MC-LR.
(2) Molecular weight of MC-LR is 995.2D. MC-LR is a kinds of typical incompl- -ete antigen. It has reactiveness but not immunogenicity during immunological response. So we adopted Protein-coupled technology to make MC-LR has immun- -ogenicity and have an effective immune response during animal immunization. In this paper, we activated MC-LR by cysteamine. The resault is an amino was linked in double bond of the seventh amino acid of MC-LR. Then,we choosed Bovine Serum Albumin(BSA) molecular weight is 67000D as vector. Made MC-LR and BSA connected by Two-step glutaraldehyde.We identified the molicular weight of H2N-etMC-LR by LC-MS, and it confirmed our activation was succesful. The qualitative identification and quantitative identification of MC-LR-BSA conjugate was separately by Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). The results showed that coupling ratio is 3 to 18:1, the average coupling ratio is 8:1.
(3) Using the identified MC-LR-BSA to immune Balb/c mice by intraperitoneal injection, and the dose is 100ug per each mice. It need 3 to 5 booster immunizations, and every two weeks it need one.We got serum titer was practically up to 1:256000. Fetch spleen cells of immuned mice and myeloma cells SP2 / 0 to fusion by Hybridoma technology. The fused cells was cultured by HAT selective medium. The results of indirect ELISA indicated that four McAbs could cross reacted with MC-LR. Cultured the cell lines under Large-scale cultivation. The results of indirect ELISA showed that the most appropriate concentration of coating antigen is between 10 and 20 nanogram per hole.
(1)通过对从滇池捞取的新鲜藻样过筛选取、冷冻干燥制得蓝藻藻粉;用醋酸溶液破坏蓝藻细胞壁,使藻毒素溢出细胞;通过C18柱子的浓缩富集作用,用80%的甲醇溶液洗脱并收集藻毒素的提取液;对藻毒素提取液进行液相色谱分离分析,确定MC-LR保留时间为30min～31min,收集MC-LR分离液,浓缩、过C18柱纯化MC-LR,得到MC-LR粗提取液,在荧光硅胶薄层板的进一步精密分离下,得到MC-LR精制样品;通过液相色谱-质谱仪(LC-MS)鉴定,采用外标法计算,得到纯度为98.79%的MC-LR精制样品。本试验一共提取MC-LR精制样品6.4mg。
(2)MC-LR的分子量为995.2D,是典型的半抗原。在免疫反应过程中,只有反应性没有免疫原性,为使MC-LR具有免疫原性,我们利用蛋白质的偶联技术,使其在动物免疫的过程中产生有效的免疫应答。本文首先用二巯基乙胺活化MC-LR,在MC-LR的第七位氨基酸脱氢丙氨酸的双键位置引入一个氨基,然后选用分子量为67000D的牛血清白蛋白(BSA)为载体蛋白,通过戊二醛两步法使MC-LR与BSA得到耦联。通过生物质朴法对中间产物H2N-etMC-LR的分子量进行了鉴定,确定MC-LR的活化很成功;分别通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术、基质辅助激光解析电离飞行时间质朴(MALDI-TOF-MS)检测,对偶联物MC-LR-BSA进行定性、定量鉴定。结果显示,耦联比在3～18:1之间,平均耦联比为8:1。
(3)选用已鉴定的MC-LR-BSA免疫Balb/c实验小鼠,按100ug/只进行腹腔注射,加强免疫3～5次,每隔两周加强免疫一次,得到了效价为接近1:25600的血清。采用杂交瘤技术,取脾脏细胞与骨髓瘤细胞SP2/0进行细胞融合,用HAT选择性培养基进行选择培养,利用间接ELISA法筛选阳性细胞孔,将筛选出的阳性细胞进行细胞克隆欲获得稳定的细胞株,扩大培养。运用间接ELISA法测定MC-LR的最适包被浓度为10ng～20ng/孔。
Enzyme-linked immunosorbent assay (ELISA) was established in 1972 applied to Medical Research. ELISA analysis of microcystins was earlier applied abroad. It’s also applied at home, but it wasn’t as matuar as abroad. In this paper, We study mainly in ELISA analysis of microcystins. It include extracting and purification of MC-LR, making incomplete antigen to complete antigen of MC-LR, screening and cuiluring monoclonal antibody cell line of MC-LR.
(1)We got enough cyanobacteria from DianchiLake by sieving andlyophilization. Algae cell walls was damaged by acetic acid, and microcycstins overflowed from cell into acetic acid. Enrichment the acetic acid by Sep-pak C18 column, then elution the column by 80% methanol solution and gather the eluent. Using Liquid Chromatography Assay analyzed the Algae toxin extracts, discovering the retention time is 30th min to 31th min.Collect the separation liquid from LC within 30th min to 31th min. Enrichment the separation liquid by Sep-pak C18 column again, then elution the column by 80% methanol solution and gather the eluent. The eluent is crude extracts of MC-LR. Coating the crude extracts on Fluorescent silica gel thin layer plate, and by this further precise separation, we finally got the refined MC-LR. And its purity quotient is 96.88% by LC-MS identifiing and the external standard method. We totally got 4.6 mg refined MC-LR.
(2) Molecular weight of MC-LR is 995.2D. MC-LR is a kinds of typical incompl- -ete antigen. It has reactiveness but not immunogenicity during immunological response. So we adopted Protein-coupled technology to make MC-LR has immun- -ogenicity and have an effective immune response during animal immunization. In this paper, we activated MC-LR by cysteamine. The resault is an amino was linked in double bond of the seventh amino acid of MC-LR. Then,we choosed Bovine Serum Albumin(BSA) molecular weight is 67000D as vector. Made MC-LR and BSA connected by Two-step glutaraldehyde.We identified the molicular weight of H2N-etMC-LR by LC-MS, and it confirmed our activation was succesful. The qualitative identification and quantitative identification of MC-LR-BSA conjugate was separately by Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). The results showed that coupling ratio is 3 to 18:1, the average coupling ratio is 8:1.
(3) Using the identified MC-LR-BSA to immune Balb/c mice by intraperitoneal injection, and the dose is 100ug per each mice. It need 3 to 5 booster immunizations, and every two weeks it need one.We got serum titer was practically up to 1:256000. Fetch spleen cells of immuned mice and myeloma cells SP2 / 0 to fusion by Hybridoma technology. The fused cells was cultured by HAT selective medium. The results of indirect ELISA indicated that four McAbs could cross reacted with MC-LR. Cultured the cell lines under Large-scale cultivation. The results of indirect ELISA showed that the most appropriate concentration of coating antigen is between 10 and 20 nanogram per hole.
引文
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