Development of an ELISA system for tick-borne encephalitis virus infection in rodents

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.     

Adenosine induces dormancy in starfish blastulae

External application of 50 micrograms ml-1 adenosine inhibits development of the starfish Asterina pectinifera at the 256-cell stage when all the embryonic cells differentiate to epithelial cells. Intracellular concentration of adenosine in the adenosine-treated embryo is 2.7 times higher than those of the normal embryo whereas the contents of ATP, ADP, AMP and adenosine 3',5'-monophosphate are the same for both embryos. Adenosine causes more than 95% reduction in the rate of protein, DNA and RNA syntheses. By returning the embryo to normal sea water, macromolecular synthesis restarts and the embryo develops to the bipinnaria stage.     

Genetic polymorphism of coagulation factor XIII B subunit in the Japanese population: description of three new rare alleles

Polyacrylamide gel isoelectric focusing (PAGIEF) of neuraminidase-treated EDTA plasma samples followed by electroblotting with enzyme immunoassay was performed to further investigate coagulation factor XIII B subunit (FXIII B) polymorphism. In 435 Japanese subjects PAGIEF patterns of FXIII B were classified into five common and three rare allotypes. This suggested that the FXIII B*2 allele existed in the Japanese population in the same manner as in Caucasians. Three new rare allotypes were considered to be controlled by three rare alleles which were designated FXIII B*13, FXIII B*14, and FXIII B*15, respectively. The gene frequencies calculated from 435 Japanese subjects were FXIII B*1 = 0.2977, FXIII B*2 = 0.0184, FXIII B*3 = 0.6805, FXIII B*13 = 0.0011, FXIII B*14 = 0.0011, and FXIII B*15 = 0.0011, respectively.     

A novel antibody microarray format using non-covalent antibody immobilization with chemiluminescent detection

To date, protein and antibody microarrays have been used in reverse-phase and sandwich-based methods in order to detect known proteins such as biomarkers in samples. Our group developed "libraries" of antibodies against unknown proteins, referred to as mKIAA proteins, and we attempted to discover candidate novel biomarkers by protein expression profiling.To profile mKIAA protein expression using these antibodies, we established an antibody microarray system using chemiluminescent detection. A number of techniques for protein-antibody microarrays have been reported; however, no entirely suitable protocol for crude protein samples has been established. To address this issue, we immobilized purified antibodies on hydrophilic surface polymer slides (Maxisorp, Nunc). Although our system is based on the direct labeling of crude protein samples, we achieved sufficient sensitivity (detection limit: 50 pg mL(-1)) and low backgrounds. This sensitivity is on a level with the sandwich immunoassay-based antibody array system. Using our protocol, we developed an antibody microarray spotted with 960 anti-mKIAA antibodies (total: 3888 spots for quadruplicate assessments), and we carried out protein expression profiling of mKIAA proteins. In this study, we generated an expression profile of 960 mKIAA proteins and compared the present results with those obtained via cDNA microarray.     

TRICORE, a novel bead-based specimen concentration method for the culturing of Mycobacterium tuberculosis

Centrifugation is a necessary concentrating step for the detection of Mycobacterium tuberculosis in a liquid culture. However, centrifugation is biologically hazardous and presents an obstacle in the development of an automated culture system. A bead-based bacterial concentration method, TRICORE, was recently developed by Genetein Co., Ltd. We compared the efficacy of TRICORE and conventional centrifugation for concentrating M. tuberculosis in clinical sputum specimens by using liquid and solid culture systems. Among 90 pretreated clinical sputum specimens, 51 (57.3%) and 55 (61.8%) M. tuberculosis isolates were recovered by the MGIT culture system by using the centrifugation and TRICORE methods, respectively (chi-square test, p=0.5413). The detection time for the centrifugation method was 359.3±117.0 h, while that for the bead-based concentration method was 377.6±162.3 h (p=0.5637). However, the number of colonies recovered on solid media were significantly higher with the TRICORE method (p=0.003). In particular, among the smear-negative specimens, culture positivity of the TRICORE method was 39.6%, while that of the centrifugation method was 15.1%. The TRICORE bead-based concentration method was considered equivalent to centrifugation and enabled efficient collection of paucibacillary specimens in solution. Thus, the new noncentrifugation concentration method could yield more positive culture results.     

Identification of two isoforms of Dsk2-related protein XDRP1 in Xenopus eggs

The budding yeast UbL-UBA protein Dsk2 has a UbL domain at its N-terminus and a UBA domain at its C-terminus, and thus functions as a shuttle protein in the ubiquitin-proteasome pathway. In this report we describe two isoforms of Xenopus Dsk2-related protein, XDRP1L and XDRP1S. Difference of the two proteins in sequence was that the UbL domain of XDRP1S lacks 15 residues in the middle part of that of XDRP1L. Both XDRP1L and XDRP1S were expressed in Xenopus eggs. XDRP1L and XDRP1S bound to polyubiquitinated proteins via their UBA domains. XDRP1L also bound to the proteasome via its UbL domain, whereas the XDRP1S UbL domain was less likely to bind to the proteasome. Instead, XDRP1S not XDRP1L bound to monomeric cyclin A and prevented its degradation. The existence of such Dsk2-isoforms in Xenopus eggs suggests that the shuttling function via the UbL-UBA protein Dsk2 is evolutionally conserved across species.