Purification of treponema pallidum from rabbit testicular tissue

Lee, Min Geol; Cho, Sung Whan; Lee, Kwang Hoon; Lee, Jung Bock

Abstract

There are several different methods of purifying Treponema pallidum(TP) from rabbit testicular tissue. Among them, we compared the use of differential centrifugation, which has been most widely used, to Percoll density gradient centrifugatian, a newly applied method, in purifying TP from rabbit testicular tissue by checking the protein concentration of the TP suspension, hemagglutination assay using sheep erythrocytes sensitized by TP, IgM-TP-enzyme-linked immunosorhent, assay(IgM-TP-ELISA) and eJect,ron microscopic observation. The protein concent,ration af TP antigen suspension (2x10(8)TP/ml) purified by Percoll density gradient centrifugation (lower band) was the lowest (129.0pg/ml) when compared to those purified by differential centrifugation (324.0pg/ml) and Percoll density gradient centrifugatian (upper band) (560.2pg/ml). Sheep erythrocytes sensitized by TP purified by Percoll density gradient centrifugation(lower band) showed the same resiilts as those using a commercii1 TPHA kit when tested with positive and negative control sera. The sensitivity and specificity of the IgM-TP-ELISA were 88.5%(23/26') and 86.4%(19/2Z) respectively using TP as an antigen purified by differential centrifugation. The rates were 96.29% (25/26) and 95.5%(2l/22) using TP purified by Percoll density gradient centrifugation. As shown by the electron microscopy, T. pizllida purified by clifferential centritugation and Percoll density gradient centrifugatiori were structurally unaltered, and Percoll-purified TP contained much less tissue debris than TP prepared by differ ential centrifugation. Therefor e, Percoll density gradient centrifugation is considered to be a better method of purifying TP from rabbit testicular tissue when compared to differential centrifugatian, as a matter of fact, Perrol1 density gradient centrifugation has been applieci successfully in the study of the physiology, recombinant DNA techniques, and antigenic structure of TP and to the preparation of the antigen for the FTA-ARS and TP-ELISA