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Naoya Ohmura

Publications and source records attributed to Naoya Ohmura.

17 recordsLinked to original sources
PubMed2006

Simple and sensitive bacterial quantification by a flow-based kinetic exclusion fluorescence immunoassay.

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 microm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 x 10(6) and 5.20 x l0(4)cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 x l0(6) to 1.64 x l0(10)cells/mL for P. aeruginosa, and 5.20 x l0(4) to 1.04 x l0(9)cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6-2.5) x l0(5) antibodies for one P. aeruginosa cell and (2.2-2.7) x l0(8) antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.

Biosensing Techniques↗
PubMed2006

Acidianus manzaensis sp. nov., a novel thermoacidophilic archaeon growing autotrophically by the oxidation of H2 with the reduction of Fe3+.

A novel thermoacidophilic iron-reducing Archaeon, strain NA-1, was isolated from a hot fumarole in Manza, Japan. Strain NA-1 could grow autotrophically using H2 or S0 as an electron donor and Fe3+ as an electron acceptor, and also could grow heterotrophically using some organic compounds. Fe3+ and O2 served as electron acceptors for growth. However, S0, NO3-, NO2-, SO4(2-), Mn4+, fumarate, and Fe2O3 did not serve as electron acceptors. The ranges of growth temperature and pH were 60-90 degrees C (optimum: 80 degrees C) and pH 1.0-5.0 (optimum: pH 1.2-1.5), respectively. Cells were nearly regular cocci with an envelope comprised of the cytoplasmic membrane and a single outer S-layer. The crenarchaeal-specific quinone (cardariellaquinone) was detected, and the genomic DNA G + C content was 29.9 mol%. From 16S rDNA analysis, it was determined that strain NA-1 is closely related to Acidianus ambivalens (93.1%) and Acidianus infernus (93.0%). However, differences revealed by phylogenetic and phenotypic analyses clearly show that strain NA-1 represents a new species, Acidianus manzaensis, sp. nov., making it the first identified thermoacidophilic iron-reducing microorganism (strain NA-1T = NBRC 100595 = ATCC BAA 1057).

Acidianus↗
PubMed2006

Improving an immunoassay response to related polychlorinated biphenyl analytes by mixing antibodies.

Immunoassays for detection of a class of closely related antigens, e.g., PCBs, have often been too specific (responding strongly to some members of the class and missing others) and no general method for adjusting the response has been described. In this paper, the difference in the response of a model immunoassay to different Kanechlors (Japanese commercial mixtures of PCBs, analogous to Aroclors in the United States) is reduced from 20- or 50-fold (depending on which antibody is used) to 3-fold when the antibodies are mixed at the proper ratio. A mathematical model based on competitive binding of two antibodies for up to four antigens has been developed and used to describe the assay performance and to predict optimum mix ratios for the antibodies used. The model (based on separate measurement of each antibody's effective Kd for each Kanechlor) provides an excellent fit to the measured mixed antibody assay response. The model is also successful in identifying cases where mixing monoclonal antibodies will not improve the response. It is thought the method described will have applicability in a variety of cases where the analytical goal is semiquantitative screening based on the total quantity of an unknown mixture of related compounds.

Antibodies↗
PubMed2005

Simple immunoassay for detection of PCBs in transformer oil.

A rapid and inexpensive procedure to detect polychlorinated biphenyls (PCBs) in transformer oil is needed to facilitate identification and removal of PCB contaminated transformers. Here we describe a simple two-step liquid-liquid extraction using acidic dimethyl sulfoxide in conjunction with an immunoassay for detecting PCBs in transformer oil. The process described is faster and simpler than any previous immunoassay while maintaining comparable detection limit and false negative rate. Cross reactivity data, characterizing the immunoassay response to the four Kanechlor technical mixtures of PCBs in oil, are presented. Forty-five used transformer oil samples were analyzed by gas chromatography-high-resolution mass spectrometry and were also evaluated using the immunoassay protocol developed. Results presented show zero false negatives at a 1.4 ppm nominal cutoff for the transformer oils analyzed.

Environmental Monitoring↗
PubMed2005

Validation of accuracy of enzyme-linked immunosorbent assay in hybridoma screening and proposal of an improved screening method.

The 96-well plate format of enzyme-linked immunosorbent assay (ELISA) is the de facto standard in screening hybridomas for active antibody. Despite its widespread use, there have been few or no systematic attempts to validate its accuracy and answer the fundamental question, is it finding all the positives? We report here on a comparison between ELISA and a semiautomated flow-based kinetic exclusion assay (KinExA), both used in screening the same hybridoma cell line. Our finding is that ELISA is both overreporting (false positives) and underreporting (false negatives) compared to the KinExA system. The large number of hybridoma cells (e.g., cultured in six 96-well plates) that must be checked is daunting in considering any method other than ELISA for routine screening. To overcome this, we devised a sampling strategy in which wells are combined in a specified pattern, allowing a significant reduction in the total number of measurements required.

Animals↗
PubMed2005

Symbiotic association in Chlorella culture.

Chlorella sorokiniana IAM C-212 has long been maintained in slant culture as a mixed strain, representing an associated natural microbial consortium. In this study, the consortium was separated and five nonalgal constituents, a fungal strain (CSSF-1), and four bacterial strains (CSSB-1, CSSB-2, CSSB-3, and CSSB-4) were isolated and identified. 16S rDNA sequence analysis revealed that strains CSSB-1, CSSB-2, CSSB-3, and CSSB-4 were close to Ralstonia pickettii (99.8% identity), Sphingomonas sp. DD38 (99.4% identity), Microbacterium trichotecenolyticum (98.6% identity), and Micrococcus luteus (98.6% identity) respectively. 18S rDNA sequence analysis revealed that strain CSSF-1 resembled Acremonium-like hyphomycete KR21-2 (98.8%). The fungal strain CSSF-1 and one of the bacterial strains, CSSB-3, were found to promote the growth of Chlorella while the presence of bacterial strains CSSB-1 and CSSB-2 had no effect. Strain CSSB-4 could not be subcultured so its role was not elucidated. These results show that the interaction between Chlorella and its symbionts under photoautotrophic conditions involved both mutualism and commensalisms. The chlorophyll content of mixed strain was stable in long-term cultivation (7 months) while the chlorophyll content of a pure culture showed a marked decline. Electron microscopic analysis showed the two bacterial strains CSSB-2 and CSSB-3 were harbored on the sheath excreted by Chlorella, while the fungal strain CSSF-1 and the bacterial strain CSSB-1 directly adhered to the Chlorella cell surface. This report is the first observation of a symbiotic relationship among fungus, bacteria, and Chlorella, and the first observation of direct adhesion of fungus and bacteria to Chlorella in a consortium.

Bacteria↗
PubMed2004

Evaluation of a compact bench top immunoassay analyzer for automatic and near continuous monitoring of a sample for environmental contaminants.

A compact bench top immunoassay analyzer is evaluated and shown to possess sufficient automation to allow continuous unattended sampling and measuring while still achieving the theoretical (antibody affinity based) detection limit for analyte. The system is comprised of antigen coated particles in a disposable flow cell held at the focus of a filter fluorometer. Capture of fluorescently labeled antibody from the flow stream is inhibited by analyte in the sample, allowing analyte concentrations to be determined from the fluorescent intensity. The disposable cell was designed to allow easy end user changing of test specificity, e.g. for selection of any member of a panel of environmental contaminants. Standard curves are shown for six analytes of environmental interest, dioxin F114 (2,3,4,7,8-PeCDF), the pesticide Fenitrothion, three coplanar PCBs, including the most toxic, PCB 126, and estradiol. In each case the curves are constructed using antibody concentrations at or below the Kd of the antibody, assuring that the sensitivity shown is limited by the antibody itself rather than the analyzer. The dynamic range for the six analytes investigated ranged from a low of 5 to 340 pM for fenitrothion to a high of 0.8 to 59 nM for dioxin F114, and is correlated to the antibody Kd in every case. Data is also shown for 17 consecutive samples, including both high and low values, measured completely automatically over a period of hours. With further development and characterization, the bench top analyzer is expected to fill an important niche in environmental testing.

Biosensing Techniques↗
PubMed2004

A combination of labeled and unlabeled antibody enables self-calibration and reduction of sample matrix effects in immunoassay.

Sample matrices interfering with analyte determinations, termed matrix effects, are one of the factors limiting the more widespread use of environmental immunoassays. Previous attempts to reduce matrix effects have focused on particular assays in specific matrices rather than on general methods. Here we describe a novel method to eliminate one class of matrix effects in immunoassay, independent of the particular matrix or analyte. The method is demonstrated with a model system detecting estradiol in either a 10% methanol or a 5% dimethyl sulfoxide matrix. Fluorescently labeled antiestradiol antibody is introduced as the detecting antibody and excess unlabeled antiestradiol antibody is included as a reference antibody. The binding of the excess reference antibody to the sample analyte artificially creates a sample containing no free analyte to bind to the detecting antibody. This allows estimation of the fluorescent signal for "zero" analyte in the actual sample matrix. The solvents employed as model systems reduce the affinity of the detecting antibody and cause false positive results at low estradiol concentrations and false negative results at high concentrations. The proposed reference method, including addition of the reference antibody, resulted in a self-calibrating assay in which the matrix effects, both positive and negative, were completely eliminated.

Antibodies, Monoclonal↗
PubMed2004

Use of excess solid-phase capacity in immunoassays: advantages for semicontinuous, near-real-time measurements and for analysis of matrix effects.

A flow-based immunoassay system using solid-phase particles with high binding capacity was used for semicontinuous, near-real-time, measurement of 17beta-estradiol (E2). The high binding capacity of the solid phase was exploited to enable (i) a quantitative determination of E2 concentration, based on rate of accumulation of fluorescently labeled anti-E2 antibody on the solid phase, and (ii) the use of a single solid phase for more than a dozen competitive binding measurements. The high binding capacity of the solid phase also permitted the immobilization of a second capture antigen. Biotin was immobilized as a second antigen and used to evaluate a biotin anti-biotin system as a control for matrix effects in the E2 immunoassay. In phosphate-buffered saline, E2 could be quantified (in the range of 10-1000 pM) by using either the summation or ratio of the signals from the labeled anti-E2 and anti-biotin antibody in the presence of biotin at a constant concentration. The same referencing system was applied to estimate the matrix effects in selected environmental samples. Matrix effects that inhibited the binding of the anti-E2 antibody to the solid phase led to false positive responses, but these matrix effects could be identified and partially corrected using the response from the anti-biotin antibody.

Antibodies, Monoclonal↗
PubMed2003

Combinational use of antibody affinities in an immunoassay for extension of dynamic range and detection of multiple analytes.

Here, we describe the coordinated use of two antibodies with different affinities in a single immunoassay to extend the dynamic range and to enable detection of multiple analytes. The combination of dual antibodies was permitted with a flow-based assay at the antibody concentration below the dissociation constant, enabling affinity to govern the antibody-antigen binding. Both high and low affinity antibodies to estriol were used in combination to extend the range. The binding of each antibody was mutually independent and individually occurred over concentration ranges of 10 pM(-1) nM and 100 pM(-1) microM. The wide dynamic range of 10 pM(-1) microM was thus achieved as summation of the proportional signals to the total binding. When a combination of antibodies toward different antigens was used, it effectively detected multiple analytes within a mixture. In simultaneous analysis of a mixture of estradiol and estriol, the total signal was the sum of the binding signals from anti-estradiol and anti-estriol antibodies. In a further refinement, the individual antibodies were flowed through the flow cell sequentially, allowing the quantification of each binding signal within the combination. With this sequential format, measurement of the individual hormones in the range of 1.6 pM(-1) nM was shown. Furthermore, the same flow format was successfully applied to assay estriol and estradiol hormones in mixtures of six related compounds.

Antibodies↗
PubMed2003

Respiratory isozyme, two types of rusticyanin of Acidithiobacillus ferrooxidans.

Among the members of the copper protein superfamily, the type I enzyme rusticyanin, which is found as an electron carrier in the oxidative respiratory chain of Acidithiobacillus ferrooxidans, is the only one to have both a high redox potential and acid stability. Here we report that two forms of the rusticyanin gene (rus) are present in the genomes of some strains of A. ferrooxidans. The more common form of rus (type-A) was found to be present in all six strains studied, including those harboring only a single copy of the gene. In addition a less common form (type-B) occurred in strains harboring multiple copies of the gene. The two genes were expressed as rusticyanin isozymes with differing surface charges due to differences in their amino acid composition. Still, the copper coordination sites were completely conserved, thereby maintaining the high redox potential necessary for an electron carrier.

Acidithiobacillus↗
PubMed2003

Isolation and characterization of phenol-catabolizing bacteria from a coking plant.

New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2. Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l. The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a Vmax of 0.321 and 0.253 mg/l/min/(mg protein), respectively. The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.

Aerobiosis↗
PubMed2002

Numerical simulation for electrochemical cultivation of iron oxidizing bacteria.

A numerical simulation model was constructed for electrochemical cultivation of iron oxidizing bacterium, Thiobacillus ferrooxidans, based on Monod's dual limitation equation. In this model, two limiting factors were examined, low supply of Fe(II) ion and dissolved oxygen, from empirical viewpoints. The simulation model was constructed taking into consideration the energy balance based on the amount of the electronic flow from the electrode to bacteria via an iron ion, and then to oxygen. The model consisted of a logarithmic bacterial growth phase during the first three days, followed by a plateau and growth limitation thereafter. The predicted results were in agreement with the actual growth under electrochemical cultivation. It was predicted the growth limiting factor would be changed from insufficient supply of Fe(II) ions to that of oxygen by decreasing the value of oxygen transfer constant K, which correlated with the aeration rate. The optimum aeration rate was determined for the ideal electrochemical cultivation. The algorithm described here can be used in any electrochemical cultivation by modifying the parameters for each system.

Bioreactors↗
PubMed2002

Electrochemical regeneration of Fe(III) to support growth on anaerobic iron respiration.

Here we describe artificial help for the respiratory electron flow supporting anaerobic growth of Thiobacillus ferrooxidans through exogenous electrolysis. Flux between H(2) and a anode through cells was accomplished with electrochemical regeneration of iron. The electrochemical help resulted in a 12-fold increase in yield compared with the yield observed in its absence.

Anaerobiosis↗
PubMed2002

Anaerobic respiration using Fe(3+), S(0), and H(2) in the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans.

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has been known as an aerobe that respires on iron and sulfur. Here we show that the bacterium could chemolithoautotrophically grow not only on H(2)/O(2) under aerobic conditions but also on H(2)/Fe(3+), H(2)/S(0), or S(0)/Fe(3+) under anaerobic conditions. Anaerobic respiration using Fe(3+) or S(0) as an electron acceptor and H(2) or S(0) as an electron donor serves as a primary energy source of the bacterium. Anaerobic respiration based on reduction of Fe(3+) induced the bacterium to synthesize significant amounts of a c-type cytochrome that was purified as an acid-stable and soluble 28-kDa monomer. The purified cytochrome in the oxidized form was reduced in the presence of the crude extract, and the reduced cytochrome was reoxidized by Fe(3+). Respiration based on reduction of Fe(3+) coupled to oxidation of a c-type cytochrome may be involved in the primary mechanism of energy production in the bacterium on anaerobic iron respiration.

Anaerobiosis↗