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

(1)抗脲酶抗体的制备大豆脲酶是一种催化尿素分解为氨和二氧化碳的细胞质酶，通过层层析法制备大豆脲酶，得到纯化样品。免疫BALB/C小鼠3次，间隔2周。采用常规方法制备髓系融合细胞，ELISA法筛选高效价细胞。获得两种声效抗体，均属于IgG2a亚类。(2)抗原的固定由于对细胞溶性抗原如脲酶的固定报道较少，首次试验了细胞质抗原的固定方法，发现脲酶用多聚甲醛（PFA）法和周期性赖氨酸多聚甲醛（PLP）法都能很好地固定。(3)组织染色将上述抗体固定在大豆子叶切片上。一方面检测了荧光（FIT…More C）标记的抗小鼠IgG作为二抗，另一方面也检测了抗小鼠IgG生物素偶联碱性磷酸酶/链脲嘧啶的酶标记方法。对于FITC标记的标本，荧光显微镜证实了荧光被引入到组织中，但仍然存在一定的模糊性，无法将样品荧光与自然背景区分开来。(4)显微光谱法：对fitc标记的样品进行显微光谱监测。采用高压汞灯强化光源的傅里叶变换显微镜，配合图像强化光电二极管阵列探测器进行了高灵敏度的测量。滤光片法在一定程度上消除了激发光。然而，目前的方法不能完全消除核黄素等发出的荧光。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