上坂 良彦 日水製薬株式会社, 中央研究所, 研究員
可部野 久俊 日水製薬株式会社, 開発部長
NISHIBUCHI Mitsuaki Faculty of Medicine, Kyoto University, Associate Professor, 医学部, 助教授 (50189304)
UESAKA Yoshihiko Nissui Pharmaceutical Co., Senior Researcher
|Budget Amount *help
¥19,600,000 (Direct Cost : ¥19,600,000)
Fiscal Year 1990 : ¥9,100,000 (Direct Cost : ¥9,100,000)
Fiscal Year 1989 : ¥10,500,000 (Direct Cost : ¥10,500,000)
To diagnose bacterial infections, it is necessary to identify the causative agent. The methods routinely applied at clinical microbiology laboratory are based on isolation and examination of biochemical characters of the isolated causative bacteria. However, these methods usually require 20-30 hours, and in some instances more than 72 hours, thus the informations available are too late to be properly considered for a proper therapy of the infections. The present investigation is aimed to develop a rapid, sensitive method to identify pathogenic bacteria based on the bead-ELISA that has successfully been developed for detecting bacterial protein toxins.
In diarrheal stools, causative bacteria are usually included in the order of 10^6 bacteria per ml. Thus, even if it is necessary to dilute the specimen a thousand folds, an ELISA that can detect 10^3 bacteria per ml is sufficient for the detection of the objective bacteria.
Polyclonal antibodies against whole cell preparations of Shigella dysenteriae, S. flexneri, S. boydii, S. sonnei, Vibrio cholerae, V. parpahamemolyticus and Campylobacter jejuni were prepared and the specificities of each antibody to react only the homologous bacteria were confirmed by an agglutination test with appropriately treated antibodies. By using these specific antibodies, a bead-ELISA system was developed after examining various conditions of the reaction. A successful ELISA-kit was prepared with various bacteria listed above, and especially a kit to identify Campylobacter jejuni is to be tested in clinical microbiology laboratories.