Highly sensitive histamine-sensitization test for residual activity of pertussis toxin in acellular pertussis vaccine

Histamine-sensitization test method based on histamine-sensitizing death is widely used for controlling residual activity of pertussis toxin in acellular pertussis vaccines. The test method evaluates the residual activity according to the death of mice injected with a test vaccine after histamine challenge and the test result, therefore, depends on the sensitivity of mice. A highly sensitive test method based on change in rectal temperature of mice has been used in Japan for many years but has limited feasibility in other countries. We examined the possibility of a test method using dermal temperature measured by infrared thermometer to reduce animal suffering instead of rectal temperature. The dermal temperature method was shown to be as sensitive as the rectal temperature method. Furthermore, the dermal as well as rectal temperature methods can evaluate the activity of a test vaccine in relative to a reference preparation so as to allow direct comparison of the test results among different laboratories. The activity by means of the dermal temperature method was also found to be well consistent with that by the rectal temperature method.     

Highly sensitive immunoradiometric assay for TSH and thyroid radioiodine uptake as predictors of thyroid suppression test

Suppression of TSH and thyroid radioiodine uptake by doses of either T4 or T3 were compared in 33 patients in whom Graves' thyrotoxicosis had been treated with thioamide drugs and the medication was discontinued for at least 4 months. Thyroidal radiodine uptake was suppressed in 19 patients and was not suppressed in the remaining 14 patients. Basal TSH levels before suppression were 2.07 microU/ml in the former, significantly exceeding those of the latter (0.91 microU/ml). A TSH level of at least 1.2 microU/ml before suppression is a good predictor of positive thyroid radioiodine suppression with a predictive value of 76%. A level lower than 0.7 microU/ml before suppression is a good predictor of negative thyroid radioiodine uptake suppression with a predictive value of 89%. The determination of TSH levels before the thyroid suppression test was helpful in predicting the result, but there were limitations. In the thyroid suppression test positive group, circulating T4 was depressed by doses of T3. In them, the magnitude of T4 depression correlated with the levels of thyroid radioiodine uptake before suppression. The levels of TSH correlated neither to changes in T4 nor to those in thyroid radioiodine uptake. This indicates that the thyroid glands which show high radioiodine uptake are sensitive to TSH and are also sensitive to suppression. The elevated sensitivity to TSH probably warrants the disappearance of abnormal thyroid stimulation more precisely.     

Development of a test strip reader for a lateral flow membrane-based immunochromatographic assay

A low-cost, simple strip reader system using a linear movement mechanism of CD-ROM deck has been developed to characterize a lateral flow membrane-based immunochromatographic assay. The test strip reader was assembled by a CD-ROM deck and home-made optical head especially designed for immunoassays. The optical head for detecting reflected light from the test strip surface consists of green light-emitting diode, large area silicon photodiode, and anodized aluminum mounting block providing a slit structure for cutting light from the LED. The stepping motor of the deck was operated in the full step mode, whose distance of each reading point is about 0.15 mm. The performance of the strip reader was tested by analysis of HBV (hepatitis B virus) antigen test kit. This strip reader can be useful for inexpensive, disposable, and membrane-based assays that provide visual evidence of the presence of an analyte in a liquid sample.     

Highly sensitive reaction of tryptophan with p-phenylenediamine

A simple and sensitive spectrophotometric method for the determination of tryptophan (TRP) is described. The method is based on the coupling reaction of tryptophan with diazotized p-phenylenediamine dihydrochloride (PPDD) in sulfuric acid medium to give the colored product having an absorption maximum at 520 nm. The coupled product was stable for 2h. Beer's law is obeyed in the tryptophan concentration range of 0.25-11 microg/ml. The method is applied for the analysis of pharmaceutical preparations of tryptophan and also in protein samples for tryptophan. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed method and the significant feature of the method is that most of the amino acids do not interfere in the determination of tryptophan.     

Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick

The dinoflagellate Karlodinium veneficum that is usually present at relatively low cell abundances is a globally-distributed harmful algal bloom-forming species, which negatively affects marine ecosystems, fisheries, and human health. Hence, an efficient detection platform for the rapid and sensitive identification of K. veneficum is highly demanded. In this study, a method referred to as recombinase polymerase amplification coupled with lateral flow dipstick (RPA-LFD) was developed for the rapid detection of K. veneficum. The primers for RPA and the detection probe for LFD were designed to specially target the internal transcribed spacer of K. veneficum by molecular cloning and multiple alignments of the related sequences. The developed RPA can gain an approximately 300 bp specific band from K. veneficum. Successful amplification for RPA could be achieved at a temperature range of 35 °C–45 °C. RPA for 30 min could produce enough products that could generate clearly visible electrophoresis bands and were adequate for subsequent LFD analysis. The RPA products can be visually detected by the naked eyes through an LFD after an automatic chromatography for 5 min at room temperature. The developed RPA-LFD was exclusively specific for K. veneficum and displayed no cross-reactivity with other algal species that are commonly distributed along the Chinese coast. In addition, the lowest detection limit of RPA-LFD was 10 ng μL⁻¹ of genomic DNA and 0.1 cell mL⁻¹, which was 100-fold sensitive than conventional PCR. In conclusion, the developed RPA-LFD assay in this study can be used as a rapid and sensitive method to monitor K. veneficum in the future.     

Highly sensitive immunoadsorption procedure for detection of low-abundance proteins

A procedure that virtually eliminates nonspecific adsorption of radiolabeled proteins during immunoprecipitation was devised utilizing staphylococcal cells containing protein A (Staph A). Immunoprecipitates (antigen-antibody complexes) were solubilized from Staph A pellets into detergent micelles by incubation in a small volume of 1% sodium dodecyl sulfate (SDS) at 23 degrees C for 10 min. To allow re-formation of immunocomplexes and rebinding to new Staph A, the SDS-solubilized material was diluted 20-fold in buffer containing 1% Triton X-100 and 0.5% sodium deoxycholate. Specific conductance measurements revealed that this solubilization and subsequent reimmunoadsorption of antibody-antigen complexes occur at SDS concentrations that are first above and then below its critical micelle concentration. This procedure lowered the nonspecific background from approximately 2250 parts per million (ppm) to less than 25 ppm with a final recovery of 30-50% depending on the antigen and antibody. Chaotropic agents such as 2 M urea, 0.2 M KOH, and 3.5 M MgCl2 (as well as combinations of urea and SDS) can substitute for 1% SDS, although the final recovery is somewhat lower. Fluorography of radiolabeled proteins obtained in this manner displays virtually undetectable background even for exposures as long as 2 months. These methods allowed the unambiguous detection of low-abundance antigens at a high level of sensitivity, for example, mouse mammary tumor virus protein products and epidermal growth factor receptor.     

Highly sensitive detection of cocaine using a piezoelectric immunosensor

This paper describes the development of a highly sensitive competitive immunoassay with the piezoelectric sensor. The immobilized derivative of cocaine was benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO). For the immobilization of BZE-DADOO, the conjugate BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) was synthesized via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU), followed by the creation of the conjugate monolayer on the piezosensor electrodes. For the optimization of the competitive assay we used electrodes with rough or smooth gold areas and for the interaction with immobilized antigen different anti-cocaine sheep polyclonal (pAb, either whole IgG or Fab fragment) and mouse monoclonal (mAb, whole IgG) antibodies. The assay of cocaine developed achieved a detection limit (LOD) of 100 pmol/l (34 ng/l) using the sheep antibody (IgG) and piezoelectric sensors with a smooth gold surface. The total time of one analysis was 15 min and the measuring area of the sensor could be used more than 40 times without losing its sensitivity.     

Establishment and application of hyperbranched rolling circle amplification coupled with lateral flow dipstick for the sensitive detection of Karenia mikimotoi

The dinoflagellate Karenia mikimotoi is a noxious and harmful algal bloom (HAB)-forming microalga. Establishing a rapid, accurate, and sensitive method of detecting this harmful alga is necessary to provide warnings of imminent HABs through field monitoring. Here, an isothermal amplification technique combined with a rapid analytical method for nucleic acid-based amplified products, i.e., hyperbranched rolling circle amplification (HRCA) coupled with lateral flow dipstick (LFD), hereafter denoted as HRCA-LFD, was established to detect K. mikimotoi. The HRCA-LFD assay relied on a padlock probe (PLP) targeting DNA template and an LFD probe targeting PLP. The sequenced internal transcribed spacer of K. mikimotoi through molecular cloning was used as the target of PLP. The optimized HRCA conditions was determined to be as follows: PLP concentration, 20 pM; ligation temperature, 65 °C; ligation time, 10 min; amplification temperature, 61 °C; and amplification time, 30 min. The developed HRCA-LFD assay was specific for K. mikimotoi, displaying no cross-reactivity with other common microalgae. Sensitivity-comparison tests indicated that HRCA-LFD assay was 100-fold more sensitive than PCR, with a detection limit of 0.1 cell mL⁻¹ when used to analyze spiked field samples. The analysis with field samples also indicated that HRCA-LFD assay was suitable for samples with a target cell density range of 1–1000 cells mL⁻¹. All of these results suggested that HRCA-LFD assay is an alternative method for the sensitive and reliable detection of K. mikimotoi from marine water samples.     

Highly sensitive detection of Staphylococcus aureus directly from patient blood

BackgroundRapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing – polymerase chain reaction (PCR) platform as a model diagnostic system.ConclusionsWe have demonstrated a highly sensitive two-hour assay for detection of sepsis causing bacteria like S. aureus directly in 1 ml of whole blood, without the need for blood culture.     

Highly sensitive proteome analysis of FACS-sorted adult colon stem cells

In proteomics, multidimensional liquid chromatography combined with mass spectrometry has become a standard technique to reduce sample complexity and tackle the vast dynamic range. Such fractionation is necessary to obtain a comprehensive analysis of biological samples such as tissues and cell lines. However, extensive fractionation comes at the expense of sample losses, hampering the analysis of limited material. We previously described a highly sensitive multidimensional chromatographic strategy based on a combination of hydrophilic interaction liquid chromatography and reversed phase chromatography, which allows proteomic analysis with minimal sample losses. Here we apply this strategy to the analysis of a limited number of FACS-sorted colon stem cells extracted from mouse intestine, obtaining a proteome coverage comparable to current methods that generally require 100-fold more starting material. We propose that this alternative multidimensional chromatographic technology will find ample application such as in the analysis of distinct cellular populations obtained by laser microdissection.     

Highly sensitive glycosylation analysis of membrane glycoproteins avoiding polymeric contaminants

We established a ‘seize-and-release’ purification method to eliminate polyhexose contaminants for a highly sensitive glycan profiling. Pig liver membrane lysates were pretreated with sodium dodecyl sulfate (SDS) surfactant and subsequently dialyzed to remove polyhexose contaminants. From the purified membrane glycoproteins, glycans were released and identified by mass spectrometry. As a result, we clearly obtained N- and O-glycan profiles of a pig liver, which were not achieved without any pre-treatments. This technique demonstrates a powerful approach for enhancing the sensitivity of MS-based glycan profiling.     

Highly sensitive fluorescence imaging of cancer with avidin-protease probe conjugate

It is a long-term goal of cancer diagnosis to develop tumor-imaging techniques that have sufficient specificity and sensitivity to detect small tumor nodules during surgery or endoscopic surgery. Here, we introduce an avidin-conjugated fluorescence probe, Avidin-Leu-HMRG, which consists of a cancer-targeting macromolecule (avidin) and a protease-activatable probe. The conjugate has a high affinity for lectin on cancer cells and undergoes endocytosis, followed by irreversible fluorescence activation due to cleavage by lysosomal leucine aminopeptidase. In a mouse model of peritoneal ovarian metastases, the probe could detect submillimeter-sized tumor nodules with a high S/N ratio at 1 h after intraperitoneal injection.     

Highly sensitive detection of target gene by electrochemical method.

A highly sensitive DNA sensing method was developed using electrochemically active ligand. This method is based on the detection of electric current generated by electrochemically active ligand concentrated on the electrode. Electrochemically active, intercalating ligand can bind to the double stranded DNA of target gene sequence on the electrode, where the complementary single strand is immobilized as a probe. We succeeded in the detection of 0.1 amol target gene. The technique was applicable to the detection of 0.1-10 amol gene.     

Highly sensitive amperometric detection of genomic DNA in animal tissues

A simple and highly sensitive method for the detection of genomic DNA in tissue samples is described. It is based on amperometric detection of target DNA by forming an analyte/polymeric activator bilayer on a gold electrode. The biotinylated target DNA is hybridized to oligonucleotide capture probes immobilized on the gold electrode, forming the first layer. A subsequent binding of glucose oxidase– avidin conjugate to the target DNA and the introduction of a second layer of a redox polymer to the electrode, via layer-by-layer electrostatic self-assembly, allow for electrochemical detection of the catalytic oxidation current of glucose in a PBS solution. Less than 2.0 fg of rat genomic DNA, for both regulated and house-keeping genes, can be easily detected in 2.5 µl droplets. The proposed procedure shows very high specificity for genomic DNA in a RT–PCR mixture.     

Highly sensitive chemiluminescent method for the detection of cell contamination

Minisatellite analysis is commonly used in forensic disputes but can also be applied to the investigation of cell contamination. Such a problem arises, for example, when transplantation is performed. The presence of contamination has been investigated by other authors using radioactive methods. In the present study we describe a method that allows the detection of contamination with high sensitivity without using radioactive substances. Our technique is based on the use of polymerase chain reaction (PCR) amplification of minisatellite sequences (VNTR), followed by chemiluminescent detection. In particular, biotin-labelled dCTP is included in the PCR mixture and detection of PCR products is obtained following the CSPD chemiluminescent protocol (Southern-Light Nucleic Acid Detection Systems). We applied this method to artificial mixes of DNA of two individuals with alleles of different sizes. We performed progressive dilutions of an individual DNA into the other's DNA and revealed a contamination of 1 in 2500 cells. We also tested our technique searching for maternal contamination in cord blood samples in 60 cases and revealed a 18.3% contamination. The technique that we set up proves to be a very sensitive one which could be applied not only to the detection of maternal cells in cord blood but also in studying any other kind of contamination. © 1998 John Wiley & Sons, Ltd.