An external ion microbeam for studies of biological samples

A new external ion-beam system was developed and combined with a light ion-microbeam system in JAERI Takasaki. The system is designed for micro-particle-induced X-ray emission analysis of biological samples in air environment with 1 Μm spatial resolution. One of the most serious problems in keeping such a high spatial resolution is multiple scattering in a beam exit window. Thin Kapton film (7.5 Μm thick) was adopted as the exit window as well as a sample backing foil to minimize the distance between the film and samples. The lifetime of the foil under ion irradiation and spatial resolution of the external microbeam were investigated. The results shows that the film can endure sufficient long-time irradiation to take elemental maps and the resolution can be kept nearly 1 Μm.     

Development of magnetic separation and quantum dots labeled immunoassay for the detection of mercury in biological samples

A rapid and sensitive immunoassays of mercury (Hg) in biological samples was developed using quantum dots (QDs) and magnetic beads (MBs) as fluorescent and separated probes, respectively. A monoclonal antibody (mAb) that recognizes an Hg detection antigen (BSA-DTPA-Hg) complex was produced by the injection of BALB/c mice with an Hg immunizing antigen (KLH-DTPA-Hg). Then the ascites monoclonal antibodies were purified. The Hg monoclonal antibody (Hg-mAb) is conjugated with MBs to separate Hg from biological samples, and the other antibody, which is associated with QDs, is used to detect the fluorescence. The Hg in biological samples can be quantified using the relationship between the QDs fluorescence intensity and the concentration of Hg in biological samples following magnetic separation. In this method, the detection linear range is 1–1000 ng/mL, and the minimum detection limit is 1 ng/mL. The standard addition recovery rate was 94.70–101.18%. The relative standard deviation values were 2.76–7.56%. Furthermore, the Hg concentration can be detected in less than 30 min, the significant interference of other heavy metals can be avoided, and the simultaneous testing of 96 samples can be performed. These results indicate that the method could be used for rapid monitoring Hg in the body.     

Development of enzyme immunoassay of amphetamines in human biological fluids

Enzyme-linked immunosorbent assay (ELISA) for detection of amphetamines in urine was developed. The conditions for ELISA using an antigen obtained from the derivative of amphetamine and ovalbumin were optimized. It was shown that the specificity of the proposed method when used in clinical conditions corresponds to that of gas chromatography with mass spectrometric detection.     

Development of an offline noncompetitive flow immunoassay for the determination of interleukin-8 in cell samples

A noncompetitive flow immunoassay system (FIA) for the analysis of interleukin-8 (IL-8) in cell samples was developed. Affinity interaction assays based on offline incubation of excess labeled antibodies and antigen (IL-8) were carried out. The residual unbound labeled antibody was trapped in an immunoaffinity column with immobilized IL-8 while the immunocomplex, labeled antibody/IL-8, was detected by a fluorescence detector. Two fluorophores, FLUOS and Cy5.5, were conjugated with IL-8 antibody. Optimization and comparison between the two fluorescent labeled antibodies were performed with regard to pH, antibody concentration, flow rate, injection volume, and association time. Additionally, a horseradish peroxidase enzyme label was used for the conjugation to the anti-IL-8. The enzyme substrate reaction was optimized with respect to temperature and length of the substrate reaction coil. The detection limits were found to be 200 amol using the FLUOS-labeled anti-IL-8 and 1 fmol using the Cy5.5 fluorescence label. The developed FIA technique was applied for the analysis of IL-8 in cell samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify IL-8 in the cell samples.     

A sensitive radioimmunoassay for adenosine in biological samples

A simple, sensitive, specific, and reproducible radioimmunoassay for the measurement of adenosine in biological materials has been developed. Adenosine antibody was obtained by immunizing rabbits with an immunogen prepared by conjugating 2′,3′-disuccinyladenosine to human serum albumin. By succinylating adenosine in samples at the 2′- and 3′-O positions with a premixed reagent consisting of succinic anhydride, triethylamine, and dioxane, the assay became sensitive enough to detect less than picomole amounts of adenosine in minute quantities of tissues. The corss-reactivity of structurally related compounds with the antibody was mostly negligible except for 2′-deoxyadenosine, whose usual concentration was very low. The use of this method made it possible to measure adenosine without any prior purification procedure. The immunoreactive materials in various biological samples disappeared during incubation of the samples with adenosine deaminase.     

A human exposure study to investigate biological monitoring methods for 2-butoxyethanol

The enzymatic activity of acetylcholinesterase (AChE) has been shown to be altered by environmental contaminants such as metals. However, the available literature illustrates a background of contradictory results regarding these effects. Therefore, the main purpose of this study was to investigate the potential of five metal ions (nickel, copper, zinc, cadmium and mercury) to inhibit AChE activity in vitro. First, to accomplish this objective, the possible interference of metals as test toxicants in the Ellman's assay, which is widely used to assess AChE activity, was studied. The potential influence of two different reaction buffers (phosphate and Tris) was also determined. The results suggest that the selected metals react with the products of this photometric technique. It is impossible to ascertain the artefactual contribution of the interaction of the metals with the technique when measuring AChE inhibition. This constitutes a major obstacle in obtaining accurate data. The presence of phosphate ions also makes enzymatic inhibition difficult to analyse. Attending to this evidence, an assay using the substrate o-nitrophenyl acetate and Tris buffer was used to investigate the effects of metals on AChE activity. O-nitrophenyl acetate is also a substrate for esterases other than cholinesterases. It is therefore only possible to use it for the measurement of cholinesterase activity with purified enzymes or after a previous verification of the absence of other esterases in the sample tissue. Under these conditions, the results indicate that with the exception of nickel, all tested metals significantly inhibit AChE activity.     

A human exposure study to investigate biological monitoring methods for 2-butoxyethanol

2-Butoxyethanol is a glycol ether widely used in printing inks, varnishes and cleaning fluids. As skin absorption can be significant, biological monitoring is useful in monitoring worker exposure. A number of analytes and matrices have been used previously, including 2-butoxyethanol in blood and free and total 2-butoxyacetic acid in urine. Using a combination of a volunteer study and samples from exposed workers, we compared the applicability of some of the biological monitoring markers available. We conclude that 2-butoxyethanol in blood is not a suitable marker for biological monitoring due to sampling problems. In view of the low-level exposures reported in occupational surveys, 2-butoxyethanol in breath is also unsuitable because of a lack of sensitivity. Measuring 2-butoxyacetic acid in blood is possible, although non-invasive urine samples are preferred. Free 2-butoxyacetic acid in urine has previously been widely used; however, we found that the extent of conjugation of 2-butoxyacetic acid in urine varied from 0 to 100% both within and between individuals and is not related to time, concentration or urine pH. Data from 48 exposed workers suggested that an estimated 57% (95% confidence interval 44-70%) of the total 2-butoxyacetic acid is excreted in the conjugated form, and that conjugation may be activated above a certain exposure level. Using total 2-butoxyacetic acid significantly reduced inter-individual variation. Elimination half-lives for free and total 2-butoxyacetic acid were similar (∼6 h) and there was no delay in excretion of the conjugated metabolite (peak excretion for both free and total was between 6 and 12 h after the end of exposure). In conclusion, we propose that total butoxyacetic acid (after acid hydrolysis) in urine is the biomarker of choice for monitoring exposure to 2-butoxyethanol. Urine samples should be collected post-shift towards the end of the working week.     

Standard reference materials for chemical and biological studies of complex environmental samples.

Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST) are often used in methods development and interlaboratory comparison studies since they are homogeneous and readily available to the scientific community. SRM 1649 (urban dust/organics), SRM 1650 (diesel particulate matter), and SRM 1597 (complex mixture of polycyclic aromatic hydrocarbons from coal tar) are three environmental samples which have been used by the scientific community for these purposes. These SRMs were originally developed to assist laboratories in validating analytical procedures for the determination of polycyclic organic compounds in complex mixtures. In addition, these SRMs have been valuable for the comparison of methodologies for bacterial bioassays and the development of bioassay-directed fractionation and bioassay-directed chemical analysis techniques. Most recently these SRMs were chosen for use as test samples in a collaborative study coordinated by the World Health Organization--International Programme on Chemical Safety. This paper provides a summary of much of the work to date (published and unpublished) on the chemical and biological characterization of these three SRMs. Information regarding the availability of other NIST SRMs that might be useful for these types of studies are provided also.     

Polymerase chain reaction for detection ofToxoplasma gondii in human biological samples

Using the polymerase chain reaction (PCR), Toxoplasma gondii from gene TGR1E with primers TGR1E-1, TGR1E-2 (standard PCR), and from B1 gene with primers TM1, TM2, TM3 (hemi-nested PCR) was detected in biological samples from 347 individuals (441 biological materials). Of the total of 441 biological materials, T. gondii DNA was detected in 5.2 %; it was positive in the following samples: blood (n = 6), blood from newborns (2), biopsies (2) and samples of progenitor cells (2) (from candidates for bone marrow transplantation). DNA of T. gondii was also revealed in 11 samples (8.3 %) of 120 cases of pregnant women during prenatal examinations. A positive result in the blood was also found in two cases of newborn babies from mothers who were infected in later pregnancy. The positive PCR examination was confirmed by serological methods (ELISA and complement fixation test). Agreement of PCR results and the detection of antibodies against toxoplasma was found in 83.3 %. Rapid PCR examination for the confirmation of acute parasitemia T. gondii is particularly important for the patients in whom the infection may cause serious consequences (e.g., for fetus in pregnant women or for patients suffering from imunosuppression).     

A micro-technique for preparing homogenates of biological samples

A method is described for preparing homogenates of insects, mites, nematodes, or fungal spores for electrophoretic analysis which overcomes the disadvantages of existing methods. It is also applicable to the homogenization of microsamples of animal tissue. The method involves grinding the samples in glass tubes, closed at one end, prepared from melting-point capillaries. A range of plungers is fabricated from the stainless-steel plungers from broken microsyringes, or from stainless-steel wire. The plunger is operated by hand to homogenize a sample in 2-20 microliter of buffer in a tube. The techniques for handling the samples before and after homogenization and for labeling and centrifuging them are described. Compared with existing methods, the procedure minimizes sample loss and risk of cross-contamination and eliminates the possibility of overheating the sample during homogenization.     

Development of a capillary isoelectric focusing immunoassay to measure DJ-1 isoforms in biological samples

We report on the development of a novel assay protocol for the separation and detection of charge isoforms of DJ-1 in biological samples by automated capillary isoelectric focusing followed by immunological detection. DJ-1 (PARK7) is considered as a biomarker candidate for Parkinson’s disease and may potentially support the differentiation of clinical subtypes of the disease. The new method allows for separation and subsequent relative quantitative comparison of different isoforms of DJ-1 in biological samples. The assay was successfully applied to the analysis of DJ-1 isoform patterns in brains from mice subjected to normal or high-fat diet and revealed statistically significant group differences. Furthermore, in a pooled and concentrated sample of human cerebrospinal fluid that was depleted of albumin and immunoglobulin G, four different charge variants of DJ-1 could be detected. Taken together, the capillary isoelectric focusing immunoassay for DJ-1 represents a promising tool that may ultimately serve in clinical biomarker studies.     

Development and evaluation of a new lateral flow immunoassay for serodiagnosis of human fasciolosis

Background

Human fasciolosis is a re-emerging disease worldwide and is caused by species of the genus Fasciola (F. hepatica and F. gigantica). Human fasciolosis can be diagnosed by classical coprological techniques, such as the Kato-Katz test, to reveal parasite eggs in faeces. However, although 100% specific, these methods are generally not adequate for detection of acute infections, ectopic infections, or infections with low number of parasites. In such cases immunological methods may be a good alternative and are recommended for use in major hospitals where trained personnel are available, although they are not usually implemented for individual testing.

Methodology/Principal Findings

We have developed a new lateral flow test (SeroFluke) for the serodiagnosis of human fasciolosis. The new test was constructed with a recombinant cathepsin L1 from F. hepatica, and uses protein A and mAb MM3 as detector reagents in the test and control lines, respectively. In comparison with an ELISA test (MM3-SERO) the SeroFluke test showed maximal specificity and sensitivity and can be used with serum or whole blood samples.

Conclusions/Significance

The new test can be used in major hospitals in hypoendemic countries as well as in endemic/hyperendemic regions where point-of-care testing is required.     

A new technique for staining catecholic residues in biological samples

This technique for localizing catecholic residues in biological samples is based on the condensation of Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with quinone residues obtained by the oxidation of catechols in the presence of ammonia. The product is a dark pink MBTH-quinone compound. This method is very sensitive and positive to catechol even at the 0.05 microgram level and the final product is chemically stable.     

Chromatographic and mass spectrometric methods for quantitative determination of 3-nitrotyrosine in biological samples and their application to human samples

The permanent modification of soluble and protein-associated tyrosine by nitration results in the formation of 3-nitrotyrosine, which can be used as a marker of "nitro-oxidative" damage to proteins. Based on the analysis of patient materials, over 40 different diseases and/or conditions have been linked to increased nitration of tyrosine. They include many cardiovascular diseases, conditions associated with immunological reactions and neurological diseases. In this article we review the existing chromatographic and mass spectrometric methods for quantitative measurements of 3-nitrotyrosine in different human biological samples including plasma, either from the free amino acid pool or from hydrolyzed proteins from different matrices.     

A simple diluter for flow-through toxicity studies

An original, simple, and inexpensive diluter for flow-through toxicity studies is described. The diluter is run only by water gravity and has no moving parts. The main units of the device are made of very cheap disposable components and may be mounted without qualified technical assistance. It has neither metal nor any other kind of chemically active parts contacting experimental media. The diluter may be easily modified to study toxicants independently or in mixtures, in fact, as many toxicants as are necessary. When properly calibrated, it does not require further adjustments even during long-term experiments. The main original feature of the diluter described here is its dosing system, which is based on a vacuum air lock. This key system is very simple, reliable, includes neither mechanical nor electrical devices, and ensures adequate accuracy in dosing.