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地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值和溶解氧，因此不可能根据目标材料浓度的实时测量来控制发酵条件。在大多数情况下，生物制品的浓度由生物测定法或酶联免疫吸附测定法确定，这些方法需要半天或一整天才能得到结果。因此，一种快速测定生物制品的方法对于有效控制生物生产系统是必要的。本文建立了利用高效液相色谱柱和抗体的快速免疫分析方法，并将其应用于发酵的控制。研究了抗体偶联HPLC柱直接法和夹心法的性能。如果杂质的非特异性吸附影响较小，采用前一种方法可以在30min内测量生物制品的浓度。本方法应用于重组水稻细胞培养生产人α - 1抗胰蛋白酶，培养基采用简单成分。测定α - 1抗胰蛋白酶诱导后的产酶时间，确定适宜的产酶周期。提出了一种采用抗肽抗体和肽洗脱的夹心法，并将其应用于含杂质多的发酵体系。

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.