Development of Highly Sensitive and Selective Analytical Method Combining Microscopic Spectrometry and Enzyme Immunostaining Method

（1）抗尿素酶抗体的制备大豆尿素酶是一种催化尿素分解为氨和二氧化碳的细胞质酶，通过连续层析从新鲜大豆中制备并获得纯化的样品。免疫BALB/C小鼠，间隔2周，分3次免疫。以常规方式制备髓细胞融合细胞，并通过ELISA选择高滴度细胞。获得了两种声克隆抗体，均属于IgG 2a亚类。它们与其它脲酶如刀豆脲酶、细菌脲酶等有交叉反应。（2）抗原的固定由于对尿素酶等细胞可溶性抗原的固定报道较少，本文首先对细胞质抗原的固定方法进行了试验，发现多聚甲醛（PFA）法和过氧化赖氨酸多聚甲醛（PLP）法都能较好地固定尿素酶。（3）组织培养将上述抗体固定在大豆子叶切片上。荧光（FIT 关于我们 C）一方面检测标记的抗小鼠IgG作为第二抗体，另一方面还测试了使用偶联有碱性磷酸酶/链霉亲和素的抗小鼠IgG-生物素的酶标记方法。在FITC标记的样本的情况下，通过荧光显微镜证实荧光被引入组织中，但仍然存在一些模糊性以区分样品荧光与自然背景。（4）显微分光光度法用显微分光光度法监测FITC标记的样品。高灵敏度的测量是使用傅里叶变换显微镜与增强光源的高压汞灯，连同图像增强的光电二极管阵列检测器。用滤光片法在一定程度上消除了激发光。然而，完全消除了核黄素等发出的荧光，用目前的方法是不成功的。为了克服这一困难，正在考虑另一种具有不同激发波长的荧光化合物。少

(1)Preparation of Anti-urease AntibodySoy bean urease, a cytoplasnic enzyme catalyzing decomposition of urea to ammonia and carbon dioxide, was prepared from fresh soy bean by successive chromatography and obtained purified specimen. It was immunized to BALB/C mouse three tines at intervals of two weeks. Myelona fused cells were prepared in the usual manner and high titer cells were selected by ELISA. Two kinds of sonocional antibody were obtained, which belonged both to the subclass IgG2a. They were crossreacted with other ureases, such as Jack bean urease, bacterial urease, etc.(2)Fixation of AntigenAs there has been few report- on the fixation of cyto-soluble antigen like urease, fixation method for cytoplasmic antigen was first tested and urease was found to be fixed rather well both by the paraformaldehyde(PFA)method and the periodafe lysine paraformaldehyde(PLP)method.(3)Tissue StainingThe antibodies obtained above were fixed on the section of soy bean cotyledon. Fluorescence(FIT … More C)labeled anti-mouse IgG was examined in one hand as a secondary antibody, and on the other hand, the enzyme labeling method using anti-mouse IgG-biotin coupled with alkaline phosphatase/streptoavidine was also tested. In case of FITC labeled specimen, fluorescence was confirmed to be introduced into the tissue by fluorescence microscopy, but still remained some ambiguousness to distinguish sample fluorescence from natural background.(4)Microscopic SpectrometryFITC labeled specimen was monitored by microscopic spectrometry. Highly sensitive measurement was done using Fourier-transform microscope with intensified light source by high pressure Hg lamp, together with the image-intensified photo-diode array detector. Excitation light was eliminated to some extent by the filter method. However complete elimination of fluorescence emitted from riboflavin, etc., was not successful by the present methods. To overcome this difficulty another fluorescence compound with different excitation wavelength is under consideration. Less