【作者】 侯春喜；

【导师】 赵寿经；

【作者基本信息】 吉林大学 ， 农产品加工及贮藏工程， 2006， 硕士

【摘要】 人参皂苷Rb1是人参和西洋参的主要成分之一,具有广泛的生理活性和药用价值,目前对人参皂苷Rb1的分离和检测方法有很多种,但存在分离成本高、破坏生理活性等缺点。常用的HPLC法检测人参皂苷最低限一般可达到数微克至数十微克每毫升,而ELISA法可检测到纳克甚至是皮克级的人参皂苷。本课题研究了人参皂苷Rb1的多克隆和单克隆抗体的制备方法,为ELISA法检测和亲和层析分离提供了物质基础。人参皂苷Rb1分子量小,不具备免疫原性,需交联成大分子才可能免疫成功。本实验合成了交联抗原GRb1-BSA(GRb1与BSA的结合比为10:1)和GRb1-OVA (GRb1与OVA结合比为8:1)。免疫小鼠获得多抗,其中一只小鼠分泌抗体效价达1:16000。取免疫脾细胞与小鼠骨髓瘤细胞进行融合,HAT筛选,有限稀释法克隆,建立分泌单克隆抗体的杂交瘤细胞系。获得两株分泌GRb1的单克隆抗体的细胞系,分别命名为1F7和1G3,腹水中单抗浓度分别为3.8 mg/ml、3.1 mg/ml。单抗的检测显示在0.01-1μg/ml呈线性关系,GRb1的检测范围达50ng/ml-350ng/ml,两株单抗针对相同的抗原决定簇。腹水型单抗耐热性较差,抗体耐冻融性好,-20℃反复冻融5次效价不变,可以反复冻融,-20℃保存一个月效价才下降一个稀释度。抗体在酸或者碱性条件下也比较容易失效,而在中性条件下则保存良好。1F7和1G3杂交瘤细胞株在体外连续传15代,反复5次液氮冻存复苏和液氮冻存1、2个月后复苏,杂交瘤细胞1F7和1G3能够稳定分泌抗GRb1的抗体,制备的单克隆抗体可用于GRb1含量的检测和分离。更多还原

【Abstract】 Ginseng is one of the most important Chinese medicines used in the world in tonics to combat stress and cancer, disturbances of the central nervous system, and so on. It contains many dammarane and oleanane saponins,polyacetylene derivertives and polysaccharides of which the biological activity has been studied widely.Molecular weight being low, GRb1 is a half antigen. Bovine serum albumin (BSA) has complicated constructer, multiple available site, satisfactoryantigenicity, strong resistance, well dissolvability, and easy to get. All thesereveal BSA an optimal carrier. Ovalbumin is chosed in the same time for controlled trial and detection. Ginsenoside Rb1 reacted with serum albumin(BSA) and ovalbumin (OVA) respectively to produce cross-linking antigens,GRb1-BSA(the ratio was about 10) and GRb1-OVA(ratio was about 8). BALB/c female mice were injected with GRb1-BSA dissolved in phosphate buffer saline (PBS) five times. The first immunization (50μg protein) was injected as a 1:1 emulsion in Freund’s complete adjuvant. The following three times (50μg protein in each injection) were injected as a 1:1 emulsion in Freund’s incomplete adjuvant..On the third day after the final immunization, splenocytes were isolated and fused with a HAT-sensitive mouse myeloma cell line, SP2/0, by the polyethylene glycol (PEG) method.Immune time of the protein antigen is 10-20 days betweens intervals generally. Intervals of hapten immunization is generally longer, for 3-4 weeks,sometimes there are 50 days, but this is not a conclusion. The research discovers that immunization interval is 15 days for haptena as well as thesmall amount of injection also can produce high particular antibody.Discovers in our experiment, the appropriate amount of injecting with the artificialantigen is 50 μg. Antibody acquired has a high titer value two months later after immunization, and has a low titer value one month later after immunization. Considering the reason, we may have something to do with injecting measure, also may have something to do with immunization interval.If the titer value of is low, it will lead to failures of producing hybridomas, so we increase immunization times to improve the titer value of serumantibody. After immunizations 5 times, the positive cell bore that secreted large quantity monoclonal antibodies had been obtained after the cell fusion.It increases the successful rate of cell fusion consumedly.Monoclonal antibodies are produced by cell fusion technology. In contrast to polyclonal antibodies, monoclonal antibodies have following virtues: identical structures, uniform component, high specificity, animate biotic-activityand fewer cross reacts.Antibody titers were determined using indirect ELISAwith OVA-GRb1 in coating the micro-wells.The specific anti-GRb1 sera were detected in all mice and were suficient for succedent experiment.SP2/0myelomas cell were chosen for cell fusion. Keep it in the bloom of proliferation before cell fusion.Feeder cells should be prepared before cell fusion. Kill one or more Balb/C mouse and draw its celiac macrophage and lymphocyte, then pave themin the cell culture wells. These should be operated one day before cell fusion.Electro-fusion, laser-fusion, chem-fusion and bio-fusion are optional. Chem.-fusion is chosen. Highly purified Polyethylene 1000(PEG-1000) was usedfor cell fusion.Cell fusion was proceeded in a 50-ml centrifuge tube. Minglespleen cells and myeloma cells (5:1) into the centrifuge tube, and then instill PEG-1000 scrupulously at a certain dose. Put the mixed cells into the cellculture wells that had been paved with feeder cells, and selected-culture with HAT and HT. Avoiding contamination is crucial to the cell fusion. Inspect the cells next day.When cells have paved the whole 96-well plates,screen positive cells using indirectELISA. And then proliferate the positive cells. Subclone the positive cells by limiting dilution for three times to get identical cells.And then inject them into mouse abdominal cavity. Retrieve ascites in the fifteenth day.After repetitious efforts, we got two positive hybridomas named 1F7 and1G3with indirect ELISA. The fusion conditions of the hybridoma cells were studied. The results showed that PEG concentration, the quality of new-born calf serum and the time for replacing HAT selective media affected the fusionrates. The fusion rates were higher with 50% PEG of MW 1000 and replacement of HAT medium on the fifth day after fusion.The 96-well micro titerplates needn’t to be pretreated. Hybridoma clones appeared on the third dayafter fusion.It was usually appropriate to begin to detect antibodies on the fifteenth day. There were very few new hybridoma clonies to appear after thirteen days. Two fusions were conducted and two hybridoma cell lines secreting high level of monoclonal antibody were screened out.The linear detection ranged well from 0.01μg/ml to 0.05μg/ml. The fullmeasuring range extends from 50ng/ml to 350ng/ml of GRb1 by competitiveELISA. The two McAbs for ginsenoside Rb1 aim at the same determiningcluster and they made it possible to establish ELISA for GRb1 detection and GRb1 isolation.The McAb in ascites from the mice injected with 1F7 was examined by the heat-resistant and freezing-thawing tests. Kept at 4℃ for one week, the McAb activity of the ascites was not changed. The McAb activity of theascites was decreased from 1:16000 to 1:8000, While Kept at 37℃ , the McAb activity of the ascites was decreased from 1:16000 to 1:8000 at the 2ndday, to 1:4000 at the 4th day, to 1:2000 at the 6th day. The titer of the ascites McAb by freezing-thawing treatment was equal to that stored at -20℃.更多还原