Hitoshi Takeuchi

An application of tetrazolium (MTT) colorimetric assay for the screening of anti-herpes simplex virus compounds

A rapid and sensitive procedure was developed for in vitro evaluation of anti-herpes simplex virus (HSV) agents. The procedure is based on spectrophotometrical assessment for viability of virus- and mock-infected cells via in situ reduction of a tetrazolium dye MTT, which has already been used for the detection of anti-human immunodeficiency virus (HIV) agents (Pauwels et al., 1988). Monolayer cells such as human embryonic fibroblast, VERO, or HeLa cells were not suitable for this purpose. Among the non-adherent cell lines examined for susceptibility to HSV type 1 (HSV-1), a B-lymphoblastoid cell line NC-37 was found to be the most sensitive. The cell line was found to have a good correlation between the viable cell number and the reduction of MTT. In addition, centrifugation of the virus-infected cells resulted in further increase of the sensitivity of NC-37 cells to HSV-1. After optimization, the method proved to be as sensitive as plaque reduction. The system simplifies significantly the assay procedures and thus permits the evaluation of larger numbers of compounds for anti-HSV-1 activity.

Immunogenic properties of structurally modified human tissue plasminogen activators in chimpanzees and mice

Immunogenic properties of second generation human tissue plasminogen activator (tPA) derivatives were examined in chimpanzee and mouse systems. Five species of modified tPAs (mtPAs) (designated 2660, 2663, 2810, 8000, and 9200), recombinant native tPA or bovine serum albumin (BSA) as a positive control were subcutaneously injected nine times at suitable intervals into chimpanzees, genetically the closest species to man. These animals were tested for antigen(Ag)-specific antibodies to the corresponding proteins by means of enzyme-linked immunosorbent assay and Western blot analysis. Neither 9200, one of the five mtPAs tested, nor tPA was immunogenic, although BSA and the other four mtPAs were immunogenic under these conditions. Thus, an antigenic determinant was not exposed by the modification on 9200 and this modified tPA is expected not to be immunogenic in humans. In the mouse studies, mice were immunized with mtPAs. Serum samples from these animals were tested for antibodies to the mtPAs which did not concomitantly recognize native tPA by immune adsorption of the antibodies to tPA. The amount of such antibodies after the elimination of native tPA-reactive antibodies was little or none when the serum samples from 9200 and from the other mtPAs, except 8000, were tested. Taking into consideration the results of the chimpanzee studies, it can be concluded that Ag-specific antibodies are dominantly produced to unchanged epitopes present in modified proteins in the mouse system, in which the native protein is immunogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and characterization of monoclonal antibodies against particulate guanylate cyclase in porcine kidney

Three monoclonal antibodies (Ig G1 type) to particulate guanylate cyclase from porcine kidney cortex have been produced by fusing spleen cells from immunized BALB/c mouse with P3X63 myeloma cells. The antibodies were detected by their ability to bind immobilized antigen and by immunoprecipitation of enzyme activity. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid. The antibodies recognized a 180,000 dalton protein in Lubrol-PX extract of porcine kidney cortex membrane, and when immobilized on Sepharose 4B, they co-precipitated both [125I]human atrial natriuretic peptide (ANP)-receptor complex and guanylate cyclase activity. The antibodies caused a greater increase in generation of cGMP than that of ANP.

Establishment of the haemadsorption-based colorimetric assay for the screening of anti-influenza compounds

We established a haemadsorption-based colorimetric assay system, which may be used to screen a large number of anti-influenza compounds. Madin Darby canine kidney (MDCK) cells infected with influenza virus were cultured in the presence of test compounds and after 3 days of infection guinea pig erythrocytes (GPE) were added to infected MDCK cells. After 4 washings of the cells the adsorbed GPE were lysed with distilled water, and peroxidase activities contained in GPE were measured using o-phenylendia-mine and H2O2 as substrates. The peroxidase activity that was read as optical density of oxidized o-phenylendiamine (quinoid form) correlated well with the dose of virus and day of infection in MDCK cells. Fifty per cent inhibitory concentration (IC50) of 1-(β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin) and adamantanamine hydrochloride (amantadine) against influenza virus A/H3N2/lshikawa and influenza B/Singapore, the IC50s of ribavirin to influenza A and B were 0.5 μg ml−1 to 1.0 μg ml−1 respectively. On the other hand, the IC50 of amantadine for influenza A was 1 μg ml−1 but was more than 100 μg ml−1 for influenza B virus. By this method, ribavirin, 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide (EICAR), 3-(β-D-ribofuranosyl)-4-hydroxypyrazole-5-carboxamide (pyrazofurin) have shown inhibitory effects for virus replication at lower concentrations than their cytotoxic doses. On the other hand carbocyclic citidine (carbodine) was cytotoxic at the concentration of virus inhibition. Ribavirin and pyrazofurin showed one tenth or less EC50 for influenza A and B viruses by the haemadsorption-based colorimetric assay compared with the ECso by TCID50 method. It is possible to estimate the inhibitory effect of antiviral compounds against influenza viruses rapidly and quantitatively by this colorimetric assay system.