| 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℃.
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