BASIC RESEARCH AIMING CONSTRUCTION OF ON-LINE MONITORING SYSTEM FOR MAMMALIAN CELL CULTURE SYSTEM

为了最佳地控制哺乳动物细胞培养过程，重要的是监测底物、代谢物和产物如葡萄糖、谷氨酰胺、乳酸盐、氨、抗体和细胞因子的浓度。本研究的目的是建立一种以FT-IR光谱仪为检测器的哺乳动物细胞培养过程的在线监测方法，由于培养液中葡萄糖、谷氨酰胺和乳酸等物质的浓度较低，难以用FT-IR光谱仪检测，因此尝试用反渗透膜对培养液进行浓缩。在2 MPa的操作压力下，使用聚酰胺/聚乙烯醇膜可以将谷氨酰胺或葡萄糖溶液浓缩至初始浓度的约50倍。但反渗透膜法对乳酸的截留率较低，不适合提高乳酸浓度，抗体或细胞因子的浓度也极低， ...更多信息 小于几十mg/升，培养液中含有几g/升的血清白蛋白，有必要选择性和灵敏地检测产生的蛋白。本文采用酶联免疫吸附试验（ELISA）方法，通过红外光谱检测，探讨了酶联免疫吸附试验中酶与底物的最佳组合。结果表明，以碱性磷酸酶为酶，以苯酚磷酸盐为底物，检测苯酚生成的酶联免疫吸附法（ELISA）灵敏度高，最适合于傅立叶变换红外光谱法（FT-IR）检测;而常规ELISA则采用化学偶联法标记酶的二抗。由于化学偶联反应不是位点特异性反应，因此难以控制偶联反应位点和与抗体偶联的酶分子的数量。因此，靶蛋白浓度和酶活性之间的标准曲线根据酶标记二抗的批次而变化。为了克服这个问题，我们设计并构建了一个细菌表达载体，它允许在大肠杆菌中产生抗体的单链可变区和碱性磷酸酶的融合蛋白。该嵌合蛋白保留了亲本酶和抗体几乎相同的酶活性和抗原结合亲和力。基于单个抗体可变区分离的VH和VL链的链间相互作用，还开发了用于检测具有单个表位的抗原的新型ELISA系统，并命名为“开放夹心ELISA”。少

In order to control mammalian cell culture process optimally it is important to monitor the concentrations of substrates, metabolites and products such as glucose, glutamine, lactate, ammonia, antibody and cytokine. The aim of this study is to develop an on-line monitoring method for mammalian cell culture process using FT-IR spectrometer as a detector.Since the concentrations of substances such as glucose, glutamine and lactate in culture broth are relatively low for detection by FT-IR spectrometer, we tried to concentrate culture broth by using reverse osmosis membrane. It was possible to concentrate glutamine or glucose solution up to about fifty times higher concentration than initial concentration by use of membrane made of poly-amide/poly-vinyl-alcohol at operating pressure of 2MPa. However reverse osmosis membrane method is not suitable for increasing lactate concentration because of low rejection factor of lactate.The concentration of antibody or cytokine is also extremely low, … More less than several ten mg per litter and culture broth contains several gper litter of serum albumin, it is necessary to detect produced protein selectively and sensitively. Here we employed Enzyme-Linked-Immuno-Sorbent-Assay (ELISA) method and investigated the optimal combination of enzyme and substrate for ELISA method detected by FT-IR spectrometer. It was shown thatt the ELISA method using alkaline-phosphatase and phenol phosphate as enzyme and substrate, respectively, and detecting the formation of phenol is most suitable for FT-IR measurement in its sensitivity.For conventional ELISA,the secondary antibody labeled with enzyme by chemical conjugation method has been used. Since chemical conjugation reaction is not site specific reaction, it is difficult to control the conjugation reaction site and the number of enzyme molecules conjugated with antibody. Consequently, standard curve between the concentration of target protein and enzymatic activity changes depending on the lot of enzyme-labeled secondary antibodies. To overcome this problem.we have designed and constructed a bacterial expression vector, which allows to produce a fusion protein of single-chain variable region of antibody and alkaline-phosphatase in Escherichia coli. This chimeric protein retained almost the same enzymatic activity and antigen binding affinity of parent enzyme and antibody. Novel ELISA system for detecting antigen with a single epitope was also developed based on interchain interaction of separated VH and VL chains from single antibody variable region and was named "Open Sandwich ELISA". Less