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胞质酶，催化尿素分解为氨和二氧化碳。每隔两周免疫BALB/C小鼠3次。按常规方法制备Myelona融合细胞，用ELISA法筛选高滴度细胞。获得了两种声学抗体，均属于IgG2a亚类。(2)抗原的固定由于对胞溶抗原如尿素酶的固定报道较少，因此首次尝试了胞质抗原的固定方法，发现用多聚甲醛(PFA)法和多聚赖氨酸多甲醛(PLP)法都能较好地固定尿素酶。(3)组织染色荧光(Fit…一方面检测了更多C~(2+)标记的抗鼠免疫球蛋白作为二抗，另一方面也检测了抗鼠免疫球蛋白-生物素结合碱性磷酸酶/链霉亲和素的酶标记法。对于FITC标记的样品，荧光显微镜证实了荧光进入组织，但在区分样品和自然背景的荧光方面仍存在一些模糊之处。(4)显微光谱对FITC标记的样品进行显微光谱分析。采用高压汞灯光源增强型傅里叶变换显微镜和像增强型光电二极管阵列探测器进行高灵敏度测量。滤光法在一定程度上消除了激发光。然而，用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，正在考虑另一种不同激发波长的荧光化合物。较少

(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