Development of rapid immmuno-assay methods utilizing HPLC and application to control of fermentation

为了通过基因工程微生物和细胞有效纯化有用的生物产品，发酵条件的控制和优化是最重要的。由于可测量的参数仅限于温度、pH和溶解氧，因此不可能根据目标物质浓度的实时测量来控制发酵条件。在大多数情况下，生物制品的浓度是通过生物测定或酶联免疫吸附测定方法测定的，这些方法需要半天或一整天才能得到结果。因此，一个快速的检测方法的生物产品将是必要的有效控制的生物生产system.In这项工作中，利用HPLC柱和抗体的快速免疫检测方法，并应用于控制发酵。研究了抗体偶联柱直接法和夹心法的性能。通过前一种方法，如果杂质的非特异性吸附的影响较小，则可以在30分钟内测量生物制品的浓度。将该方法应用于通过培养重组水稻细胞来生产人α 1-抗胰蛋白酶，其中使用简单组成的培养基。测定了α 1-抗胰蛋白酶诱导后的产酶时间，确定了适宜的产酶周期，并建立了一种新的利用抗肽抗体和肽洗脱的夹心法，并将其应用于含有多种杂质的发酵体系。

For effective purification of useful bioproducts by genetically engineered microorganisms and cells, control and optimization of fermentation conditions are most important. Because measurable parameters are limited to temperature, pH and dissolved oxygen, it was impossible to control fermentation conditions depending on real-time measurements of the concentrations of target materials. In most cases the concentrations of bioproducts are determined by bioassay or enzyme linked immunosorbent assay methods, and these methods require half or one full day to get results. Therefore, a rapid assay method of bioproducts will be necessary for effective control of bioproduction systems.In this work rapid immuno-assay methods utilizing HPLC columns and antibodies were developed and applied to control of fermentation. Performance of direct method and sandwich method using antibody-coupled HPLC columus were studied. By the former method it was possible to measure the concentration of bioproducts within 30min, if the effect of non-specific adsorption of impurities is minor. This method was applied to production of human alpha1-antitrypsin by culture of recombinant rice cells, in which a medium of a simple composition was used. The time-course of alpha1-antitrypsin production after induction was measured, and the suitable production period was determined.A new sandwich method using anti-peptide antibody and peptide elution was developed and applied to fermentation systems contaning many impurities.