Establishment and characterization of monoclonal antibodies against Chuzan virus K-47

We established sixteen mouse monoclonal antibodies reactive to Chuzan virus K-47 strain using P3-X63-Ag8-U1 cells as fusion partner cells. Among them, CG53/2/4 recognized a 100K structural protein of the virus. The 100K antigen lost it's antigenicity for CG53/2/4 after mild periodate oxidation treatment, suggesting that the 100K viral antigen is a glycoprotein. In addition, CG53/2/4 neutralized the viral infectivity. This indicates that the 100K glycoprotein is essential for the infection of the virus. The other monoclonal antibodies reacted with a 41K antigen of the virus. Especially CG1/1 showed the highest reactivity to the virus. Forward step sandwich assay using CG1/1 and biotinylated CG53/2/4 could detect the virus at 10TCID₅₀/ml. Therefore, these monoclonal antibodies can evantually predict the virus infection to the animals before their sideration.     

Epidemiological and serological survey of brucellosis in Mongolia by ELISA using sarcosine extracts

Brucellosis is an important zoonosis, and serological surveillance is essential to its control. However, cross-reactions of attenuated live cells of Brucella abortus strain S-19 and B. melitensis strain Rev-1 with Yersinia enterocolitica O9 or vaccinated animal sera interfere with accurate serological diagnosis by the Rose Bengal test (RBT). Therefore, we used ELISA with sarcosine extracts from the virulent B. abortus strain 544 to eliminate false-positives among RBT positive-sera. A total of 697 serum samples were collected in Mongolia from humans and animals in 23 nomadic herds. The herds were classified into three groups as brucellosis-endemic (BE), brucellosis-suspected (BS), or Brucella-vaccinated (BV). The number of 295 animals (43.0%) was positive by RBT, but 206 (69.8%) of these were positive according to ELISA; therefore, 30.2% of the RBT-positive sera were found to be false positives. The false positive samples for RTB represent 4.1%, 27.4%, and 68.2% of the animals from the BE, BS, and BV herds, respectively. In addition, 32% of RBT-positive human sera were also false positives. Thus, our ELISA would be more specific than RTB and useful for epidemiological surveillance for brucellosis.     

Fe(II)/Cu(I)-dependent P-type ATPase activity in the liver of Long-Evans cinnamon rats

This study examined Fe(II)-dependent ATPase activity in OTG (octylthioglucoside) -treated microsomes isolated from Wistar and LEC rats. The ATPase activity of the liver OTG-microsomes from Wistar rats increased sharply in the 5-150 microM range of Fe(II) with a K0.5 value of 23.9+/-3.6 microM, while the activity of LEC rat liver microsomes increased with increasing Fe(II) up to 500 microM with a K0.5 value of 64.4+/-8.1 microM. The K0.5 values for Fe(II)-dependent ATPase activity of spleen OTG-microsomes were nearly identical at 59.3 microM in the Wistar rat and 63.7 microM in the LEC rats with a similar level of activity at each Fe(II) concentration in both strains of animals. These results indicated that there are two types of Fe(II)-dependent ATPase with different Fe(II) sensitivity, a high sensitive (H) and a low sensitive (L) type, and that the H-type activity was specific to the liver. The H-type activity was, however, deficient in the liver of LEC rats that accumulate copper and iron in hepatocytes as a result of mutations in the Wilson's disease protein (WNDP). On the basis of these results, together with the similarity in optimal conditions required for full activity of the enzyme, we conclude that the Fe(II)-dependent ATPase (H-type) and WNDP may be identical.     

Epitope analysis of human monoclonal antibody specific for rice allergenic protein generated by in vitro immunization

We previously established an in vitro immunization protocol for generating antigen specific human monoclonal antibodies (mAbs). In vitro immunization was performed against the soluble protein of rice allergenic protein (RA), resulting in the generation of three B cell clones, AC7-1/F9, CB7-1/E2 and CB7-8/F5, all of which produce a RA-specific human monoclonal IgM antibody. We attempted to map the epitope regions recognized by thesem Abs to characterize their specificities. We performed two rounds of epitope mapping, rough mapping using 10-mer peptides covering the full-length RA with 5 amino acids overlapping, and fine mapping using 8-mer peptides covering the putative epitope regions from the rough mapping with 1amino acid overlapping. As a result of the fine mapping,we identified the epitope regions of these three mAbs as⁴⁵QVWQDCCRQ⁵⁴L, ⁵⁶AVDDGWCRCGA⁶⁷L and⁹¹FPGCRRG⁹⁸D on the RA molecule and found to be identical. Furthermore, we determined the putative core epitope regions, which are critical for mAb binding to each region, ⁴⁷WQDCC⁵²R and ⁶⁰GWC⁶³R. The information about the epitope region on the RA molecule,which might trigger the allergenic response, would be useful to establish a specific immunotherapy against rice allergy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced antibody production of human-human hybridomas by retinoic acid

The enhancement of human monoclonal antibody production by retinoic acid (RA) was evaluated usingthe human-human hybridoma cell line BD9 underserum-free culture condition. The amount of humanIgG secreted by BD9 hybriodmas was enhanced abouteight-fold by treatment with 10^-7 M of RA for 4days. Northern blot analysis showed that both mRNAlevels of the IgG light and heavy chains were markedlyincreased by RA when compared with control without RAtreatment. On the other hand, it was found thatcontinuous treatment of cells with RA was not alwaysrequired to exhibit the enhancing effect, suggestingthat RA may act as a trigger for IgG gene expression. The comparison between extra- and intracellular IgGamounts by immunoblot analysis suggests that thesecretion rate of IgG may be accelerated by RAtreatment. These results suggest that RA may be aneffective culture additive for efficient production ofhuman monoclonal antibody using human-humanhybridomas.     

Mechanism of the lifespan extension of Caenorhabditis elegans by electrolyzed reduced water--participation of Pt nanoparticles

Electrolyzed reduced water (ERW) contains a large amount of molecular hydrogen and a small amount of Pt nanoparticles (Pt NPs). We have found that ERW significantly extended the lifespan of Caenorhabditis elegans in a novel culture medium designated Water Medium. In this study, we found that synthetic Pt NPs at ppb levels significantly extended the nematode lifespan and scavenged reactive oxygen species (ROS) in the nematode induced by paraquat treatment. In contrast, a high concentration of dissolved molecular hydrogen had no significant effect on the lifespan of the nematode. These findings suggest that the Pt NPs in ERW, rather than the molecular hydrogen, extend the longevity of the nematode, at least partly by scavenging ROS.     

In vitro immunization of human peripheral blood lymphocytes: establishment of B cell lines secreting IgM specific for cholera toxin B subunit from lymphocytes stimulated with IL-2 and IL-4

In vitro immunization (IVI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. Although several cancer specific human MAbs were successfully generated by using this IVI method, it was not available for soluble antigens, which prompted us to improve the method for generation of human MAbs against soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 and interleukin-4. It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome by finding the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. B cells producing anti-CTB antibodies were directly cloned by a soft agar cloning method. This revised version was published online in August 2006 with corrections to the Cover Date.