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

为了最优地控制哺乳动物细胞培养过程，监测底物、代谢物和产物（如葡萄糖、谷氨酰胺、乳酸、氨、抗体和细胞因子）的浓度是重要的。本研究的目的是开发一种利用傅里叶变换红外光谱仪作为检测器对哺乳动物细胞培养过程进行在线监测的方法。由于培养液中葡萄糖、谷氨酰胺、乳酸等物质的浓度相对较低，无法用FT-IR光谱仪检测，我们尝试用反渗透膜对培养液进行浓缩。使用聚酰胺/聚乙烯醇膜，在2MPa的操作压力下，可以将谷氨酰胺或葡萄糖溶液浓缩到比初始浓度高约50倍的浓度。但由于反渗透膜法对乳酸的排斥系数低，不适合用于提高乳酸浓度。抗体或细胞因子的浓度也极低，每窝小于几十毫克，培养液中含有几窝血清白蛋白，有必要对产生的蛋白进行选择性和敏感的检测。本文采用酶联免疫吸附法（ELISA），对酶联免疫吸附法中酶和底物的最佳组合进行了研究。结果表明，以碱性磷酸酶和磷酸酚分别为酶和底物，检测苯酚形成的ELISA方法在灵敏度上最适合FT-IR测定。传统的酶联免疫吸附试验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，因此很难控制偶联反应的位点和与抗体偶联的酶分子数。因此，靶蛋白浓度与酶活性之间的标准曲线随酶标记二抗的数量而变化。来克服这个问题。我们设计并构建了一种细菌表达载体，可以在大肠杆菌中产生抗体单链可变区与碱性磷酸酶的融合蛋白。该嵌合蛋白保持了与亲本酶和抗体几乎相同的酶活性和抗原结合亲和力。基于抗体可变区分离的VH链和VL链的链间相互作用，开发了一种检测单表位抗原的新型ELISA系统，命名为“Open Sandwich 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