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Objective:Two anti-human cardiac troponin I(cTnI)monoclonal antibodies were prepared to establish Luminol chemiluminescence enzyme immunoassay.Methods: Obtaining ascites monoclonal antibody was purified by ammonium sulfate fractionation and protein G affinity chromatography column, and their specificity was identified by SDS-PAGE and ELISA methods.Luminol chemiluminescence enzyme immunoassay was successfully established,which was used to detect 30 clinical serum samples.Results:Two antibodies named Ab1 and Ab3 could recognize different forms of cTnI including cTn I monomers,cTn I-C complexes and cTn I-T-C complexes.Both Ab1 and Ab3 are IgG1.The titers of the Ab1 and the Ab3 were up to 1:800 000 and 1:400 000,respectively.The affinity constants of Ab1 and Ab3 were 1.62×109L/mol and 2.60×108L/mol,respectively.The detection range of Lumino chemiluminescence enzyme immunoassay established by two monoclonal antibodies was 6.25 ng/ml to 400 ng/ml. Detecting 30 clinical samples attained the positive detection rate of 77.3% and the negative detection rate of 100%.Conclusion:The chemilu minescence enzyme immunoassay has been established to detect different forms of cTn I including cTn I monomer,cTn I-C complexes and cTn I-T-C complexes,which has better practicality.
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<p><b>OBJECTIVE</b>To study the synergistic inhibitory effects of basic fibroblast growth factor (bFGF) monoclonal antibody (bFGF mAb) and irinotecan on the proliferation of small cell lung cancer H223 cells.</p><p><b>METHODS</b>CCK-8 assay and flow cytometry were used to assess the effects of bFGF mAb combined with irinotecan on the proliferation and apoptosis of H223 cells, respectively. Western blotting was performed to analyze the effect of bFGF-mAb combined with irinotecan on AKT and ERK1/2 phosphorylation in the cells.</p><p><b>RESULTS</b>Both bFGF mAb and irinotecan alone inhibited H223 cell proliferation in a dose-dependent manner (P<0.05). The inhibitory rate was significantly higher in H223 cells treated with bFGF mAb + irinotecan (54.30%) than in cell treated with bFGF mAb (18.73%) or irinotecan (21.96%) alone (P<0.05). Both bFGF mAb and irinotecan induced H223 cell apoptosis in a dose-dependent manner (P<0.05), and the combined treatment resulted in a significantly higher early apoptosis rates (6.5%) than treatment with bFGF mAb (2.7%) or irinotecan (4.3%) alone (P<0.05). bFGF mAb and irinotecan, either alone or in combination, significantly inhibited the levels of p-AKT protein and p-ERK1/2 protein without obviously affecting AKT and ERK1/2 protein levels.</p><p><b>CONCLUSION</b>bFGF mAb and irinotecan produce synergistic inhibitory effects on small cell lung cancer H223 cells by suppressing proliferation and promoting apoptosis of the cells, and can effectively block the MAPK/ERK and PI3K/AKT signaling pathways associated with bFGF.</p>
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Objective:To study the enhancing effect of bFGF-targeted antisense phosphorothioate oligonucleotide (APO)on the chemosensitivity of human laryngeal squamous carcinoma cell line Hep2 to Doxorubicin,5-Fluorouracil, and Cisplatin.Methods:bFGF-specific APO was designed,constructed and transfected into Hep2 cells with jetPEI (polyethyleneimine).Expression of bFGF mRNA was evaluated by semi-quantitative RT-PCR after transfection;immuno- cytochemical method was used to examine the expression of bEGF expression before and after transfection of Hep2;the in- duction of cell apoptosis was analyzed by flow cytometry;cell proliferation was then analYzed by MTT assay after treatment with bFGF-specific APO or chemotherapeutic drugs,or a combination of both.Results:bFGF-specific APO inhibited the growth of Hep2 cells in a dose-and time-dependent manner,with the peak inhibitory rate being 25.5%.The expression of bFGF mRNA and protein decreased by 52.0% and 41.1%,respectively.The apoptosis rate of Hep2 cells was 20.5% after transfection,bFGF-specifie APO reduced the 50% inhibitory concentration of Doxorubicin,5-Fluorouracil,and Cis- platin in Hep2 cells by 75.5%,83.5% and 65.4%,respectively.Conclusion:bFGF-specific APO can enhance the chemosensitivity of Hep2 cells,which paves a new way for potential biologic chemotherapy of laryngeal squamous carcino- ma.
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In order to produce recombinant human anti-HBsAg Fab antibody in Pichia pastoris, the recombinant yeast was fermented using fed-batch system in a 30 L bioreactor. The fermentation temperature was 30 degrees C, the pH was 5.0 approximately 5.3, and the DO was 20% approximately 30%. The recombinant Fab antibody was purified from crude culture supernatant by ion exchange and analyzed by SDS-PAGE and western blot and ELISA. When the absorbance (OD600) of broth reach 300 at the end of fed-batch phase, the induced phase was initiated. The results showed that recombinant human anti-HBsAg Fab antibody was high-level expressed in recombinant Pichia pastoris using a fed-batch fermentation system. Both chains of the Fab were successfully expressed upon methanol induction. After 192 h of induction, the expression level of recombinant Fab (soluble) reached 412 mg/L. The recombinant Fab antibody was purified effectively by ion-exchange chromatography from the fermentation supernatant to a purity of 95%. And the affinity activities of the purified recombinant Fab antibdy and fermentation supernatant were detected, and both of them showed high affinity activities. The results demonstrated that recombinant human anti-HBsAg Fab antibody could be high level produced by fed-batch fermentations in Pichia pastoris. Which can be efficiently used in industrial production.