| Budget Amount *help |
¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 1996: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1995: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1994: ¥4,300,000 (Direct Cost: ¥4,300,000)
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| Research Abstract |
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,
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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
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