Development of an ELISA-kit for a Rapid Identification of Pathogenic Bacteria

为了诊断细菌感染，有必要确定病原体。临床微生物学实验室常规应用的方法是对分离出的病原菌进行生化特性的分离和检测。然而，这些方法通常需要20-30小时，在某些情况下需要72小时以上，因此现有信息太晚，无法适当考虑感染的适当治疗。本研究旨在建立一种快速、灵敏的检测细菌蛋白毒素的方法。在腹泻粪便中，通常每毫升含有10^6个细菌。因此，即使需要将标本稀释一千倍，每毫升能检测到10^3个细菌的酶联免疫吸附试验(EL ISA)也足以检测出目的细菌。制备了副溶血弧菌和空肠弯曲菌，通过与适当处理的抗体的凝集试验，确认了每种抗体仅与同源细菌反应的特异性。通过使用这些特异性抗体，在考察了反应的各种条件后，建立了微珠-ELISA系统。用上述各种细菌制备了一种成功的ELISA试剂盒，特别是一种鉴定空肠弯曲菌的试剂盒即将在临床微生物学实验室进行测试。

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.