Masahisa Okada

Reactivity of recombinant treponema pallidum (r-Tp) antigens with anti-Tp antibodies in human syphilitic sera evaluated by ELISA

We evaluated the immunoreactivity of recombinant Treponema pallidum (r‐Tp) antigens with human sera by indirect enzyme‐linked immunoabsorbant assay (ELISA). We expressed antigens with a molecular weight (MW) of 17KDa, 15KDa, 47KDa, and 42KDa, which are believed to be major immunoreactive membrane proteins of Tp cells. The expressed proteins were described by adding the prefix M, S, and G to the corresponding Tp antigens, namely, mature antigens, signal sequence containing antigens, and glutathione s‐transferase (GST)‐fused antigen in this report. A rather high expression occurred for M47 and S42 proteins in the Escherichia coli system, whereas for M15 and M17 proteins, a poor expression was observed. However, a fairly high expression occurred for G15 and G17. Thus expressed proteins were purified by means of chromatographies to a level of > 95%, and the purified proteins were found to be reactive with TPHA positive serum by Western blotting (WB). An ELISA performed with a serum of 1/1000 dilution using these purified antigens for coating on the solid phase showed that G17 antigen was more effective in detecting syphilis antibodies in human serum than M47, S42, and G15. There was a good consistency between ELISA and TPHA, whereby the cutoff indexes (CI) on ELISA showed a correlation coefficient of 0.7276 in logarithmic TPHA titers. J. Clin. Lab. Anal. 11:315–322, 1997.

Novel rapid immunochromatographic test based on an enzyme immunoassay for detecting nucleocapsid antigen in SARS-associated coronavirus.

A novel severe acute respiratory syndrome‐associated coronavirus (SARS‐CoV) has been discovered. The detection of both antigens and antibodies in SARS‐CoV from human specimens with suspected SARS plays an important role in preventing infection. We developed a novel rapid immunochromatographic test (RICT) based on the sandwich format enzyme immunoassay (EIA) with an all‐in‐one device for detecting the native nucleocapsid antigen (N‐Ag) of SARS‐CoV using monoclonal antibodies (MoAbs), which we produced by immunizing recombinant N‐Ag to mice. RICT is a qualitative assay for respiratory aspirates and serum specimens. With this assay, a positive result can be judged subjectively by the appearance of a blue line on the device 15 min after the sample is applied. RICT with several pairs of MoAbs showed a high sensitivity for the detection of recombinant N‐Ag as well as viral N‐Ag of SARS‐CoV. rSN122 and rSN21‐2 were the best MoAbs for immobilized antibody and enzyme labeling, respectively. With regard to analytical sensitivity, RICT detected N‐Ag at 31 pg/mL for recombinant N‐Ag, and at 1.99×102 TCID50/mL for SARS‐CoV. The specificity of RICT was 100% when 150 human sera and 50 nasopharyngeal aspirates (NSPs) were used. RICT based on an EIA using the rSN122/rSN21‐2 pair is a sensitive, specific, and reliable rapid assay for detecting N‐Ag in SARS‐CoV treated with either heat or Triton X‐100. J. Clin. Lab. Anal. 19:150–159, 2005.