titer(titre)

1. The ELISA titre was 1:2000.By cell fusion, 46 hybridoma cell lines were screened, and 10 lines were cloned with limited dilution method.16 lines secreting anti-bFGF monoclonal antibody were been developed, and 2 lines targeted fusion protein. Sensitive ELISA and dot-ELISA for bFGF was developed with this mAb. The detection limit of them were 0.1 ng/well and 0.5 ng/well. The expression level of anti-bFGF mAb by different rebuilt engineering cells were identified by western blot and to direct rebuild recombiment engineering cell. The dose and character of anti-bFGF mAb inhibiting bFGF biology activity were searched by 3T3 cell line. Searching 20 tissue of liver cancer, liver cancer cell lines and general tissue of liver, finding bFGF were highly expressed in tissues of liver cancer and liver cancer cell lines. Affinity chromatography purifying bFGF was set up by mAb binging CNBr-pepharose 4B, and the purification was 95%. We found that the titer of anti-bFGF antibody was very high in serum of neuropathic amyotrophia.
应用细胞融合制备46株杂交瘤，对其中10株进行克隆化，获得bFGF特异单抗16株，2株针对融合蛋白；应用该单抗建立了0.1ng/孔灵敏度的ELISA，0.5ng/孔敏度的斑点ELISA；用Western-blotting鉴别了经改造不同工程菌蛋白表达，指导重组工程菌改造；用3T3细胞培养研究了单抗抑制bFGF生物学活性的剂量和特点；合作研究了20例肝癌、肝癌细胞株和正常肝组织，发现前者bFGF高表达；应用单抗偶联CNBr-sepharose 4B建立了小量免疫亲和层析纯化bFGF，纯度达到95%；发现神经性肌萎缩患者血清中含有高滴度的bFGF抗体，已有10多家单位引用单抗或进行合作。

2. Diazotization was used to conjugate sulfadimethoxine to carrier protein BSA and obtained immunizing antigen. The coating antigen OVA-SDM was obtained in the same way, ultravioletand SDS-PAGE were used to identify SDM artificial antigen. BALB/C mice were immunized with BSA-SDM, the titre of polyclonal antibody was detemined by indirect ELISA and blocking ELISA. The hybridoma lines that secrete SDM mAb were established with using monoclonal antibody hybridoma technology. The immunological traits such as titer, affinity, sensitivity and specificity of the mAb were characterized.
用重氮化法将SDM偶联于载体蛋白BSA和OVA，合成免疫原BSA-SDM和包被原OVA-SDM，并用紫外扫描、SDS-PAGE进行鉴定；用BSA-SDM免疫BALB/C小鼠，间接ELISA和阻断ELISA选择细胞融合备用鼠；应用杂交瘤技术建立分泌SDM mAb细胞株，用体内诱生腹水法制备SDM mAb；对SDM mAb的效价、亲和性、敏感性和特异性等免疫学特性进行鉴定。

3. The recombinant adenovirus of Ad-Cp-CDglyTK was packaged, amplified and purified in 293 cells, and the virus titre was determined by TCID50 method. The CDglyTK gene expression in CNE1 and NP69 were examined by reverse transcription-polymerase chain reaction after in vitro transfection in CNE1 and NP69 cells. The killing effect of Ad-Cp-CDglyTK/GCV+5-FC on CNE1 cells wasdetected by MTT method. RESULTS:The results of restriction enzyme digestion and DNA sequencing showed that the tk, cd, and Cp gene were inserted into the pDC316 plasmid in correct orientations. The titer of the recombinant adenovirus was 5.6×1012 TCID 50/L. The Cp fragment was amplified from the total RNA of the transfected CNE1 cells by RT-PCR.
结果：经DNA测序、限制性酶切法分析显示pDC316-Cp-CDglyTK含完整正确的tk、cd、Cp基因序列，在293细胞中包装扩增后病毒滴度为5.6×1012 TCID50/L，体外转染鼻咽癌CNE1细胞株与正常鼻咽NP69细胞株后，采用RT-PCR法从CNE1细胞株总RNA中扩出Cp片段，而NP69细胞株未检测到相应基因mRNA表达，MTT结果显示经前体药物处理转染后CNE1细胞株与NP69细胞株，5-FC+GCV联合用药较单一前体药物对CNE1细胞具有更强的抑制作用（P＜0.05），联合用药对NP69细胞株未见明显杀伤作用。