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

1991 Fiscal Year Final Research Report Summary

• Project/Area Number: 02650535
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: 工業分析化学
• Research Institution: The University of Tokyo, Faculty of Agriculture
• Principal Investigator: OKUBO Akira Univ. of Tokyo, Fac. of Agric., Assoc. Professor, 農学部, 助教授 (20111479)
• Co-Investigator(Kenkyū-buntansha): YOSHIMURA Etsuro Univ. of Tokyo, Dept. of Agric. Chem., Assist. Prof., 農学部, 助手 (10130303) ; YAMAZAKI Sunao Univ. of Tokyo, Dept. of Agric. Chem., Professor, 農学部, 教授 (00011982) ; TODA Shozo Univ. of Tokyo, Dept. of Agric. Che., Professor emeritus, 農学部, 名誉教授 (40011845)
• Project Period (FY): 1990 – 1991
• Keywords: Microscopic spectrometry / Enzyme immunostaining / FITC / Urease / Background fluorescence

Research Abstract

(1)Preparation of Anti-urease Antibody
Soy 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 Antigen
As 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 Staining
The antibodies obtained above were fixed on the section of soy bean cotyledon. Fluorescence(FIT 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 Spectrometry
FITC 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.