Development of the screening method for the active tubercular patients by urine

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 14570365
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Public health/Health science
• Research Institution: Kansai Medical University
• Principal Investigator: NISHIYANIA Toshimasa Kansai Medical University, Faculty of Medicine, Professor, 医学部, 教授 (10192254)
• Co-Investigator(Kenkyū-buntansha): KANDA Seiji Kansai Medical University, Faculty of Medicine, Associate Professor, 医学部, 講師 (70295799)
• Project Period (FY): 2002 – 2004
• Keywords: active tuberculosis / screening method / MPB64 / Dot-blot assay

Research Abstract

According to the results of the report of last year for the reactivity with patients' sera compared with MPB64 antigen and other MPB70 or MPB83 antigen, MPB64 specifically reacted with the sera of active tuberculosis patients. We reported that the possibility of the antigen for the diagnosis of tuberculosis. Moreover we examined this antigen with Dot-blot assay. MPB64 protein was expressed as a fusion protein with (His)6 tag, and we obtained as a recombinant protein after affinity purified using with (His)6 tag. For the assay, a series of the antigen dilutions that diluted by 4 times from 300 μg/ml (one spot, 300ng) was developed on a nitrocellulose membrane. After blocking with skim milk, the reactivity was examined with the tuberculosis patients' sera (1000 times). The results were showed as the lowest concentration of the antigen detected by the limiting dilutions and were examined the correlation of the antigen reactivity with the date for the hospitalization, Gaffky data, and colo ny numerical data of expectoration culture culture.
As a result of the active tuberculosis patients' sera, the reactivity was detected at the low protein concentration, and it gradually increased from a start of a treatment just after hospitalization. Furthermore, the curve of it was showed the correlation with Gaffky data and the number of the colonies of expectoration culture.
A clone (BCG1) was cloned by screening from Mycobacterium Bovis cDNA library last year. BCG1 also examined the reactivity with patients' sera. As a result of Western blotting, BCG 1 showed specific reaction with patients' sera, but the difference was not detected between patients' sera and healthy volunteer's.
In this report, we examined a search of an antigen to use for screening of an active tuberculosis patients and the effectiveness with patients' sera. It was very difficult to detect the difference with healthy volunteer and tuberculosis patient so that BCG was used as a vaccination for the prevention of the tuberculosis. From this result, a MPB64 antigen specifically reacted with patients' sera, and the reactivity was decreased by a treatment and showed correlation with clinical symptoms such as Gaffky data. We were able to get the knowledge that MPB64 was useful for screening of active tuberculosis patients. We are planning to develop the screening method that can detect the active tuberculosis patients using by patients' urine in future.