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PubMed1986

Post-embedding direct immunogold detection of a protein antigen in insect tissue.

An immunocytochemical study was performed to localize the site of hemoglobin synthesis in larvae and embryos of the insect Chironomus thummi. Heterologous antisera specific for C. thummi hemoglobins were prepared using a highly purified hemoglobin extract. Tissue samples were prepared by glutaraldehyde fixation of whole dissected larvae or whole embryos without osmium tetroxide postfixation. Epoxy resin-embedded thin sections were labeled with a direct immunogold conjugate. Immune label was localized in rough endoplasmic reticulum of fat body cells of larvae. Immune label was also present in embryos. The technique, which did not require chemical etching of the sections, proved very useful for demonstration of this intracellular protein antigen.

Adipose Tissue↗

PubMed2006

Investigation of MR signal modulation due to magnetic fields from neuronal currents in the adult human optic nerve and visual cortex.

Neuronal currents produce weak transient magnetic fields, and the hypothesis being investigated here is that the components of these parallel to the B0 field can potentially modulate the MR signal, thus providing a means of direct detection of nerve impulses. A theory for the phase and amplitude changes of the MR signal over time due to an external magnetic field has been developed to predict this modulation. Experimentally, a fast gradient-echo EPI sequence (TR = 158 ms, TE = 32.4 ms) was employed in an attempt to directly detect these neuronal currents in the adult human optic nerve and visual cortex using a 280-mm quadrature head coil at 1.5 T. A symmetrical intravoxel field distribution, which can be plausibly hypothesized for the axonal fields in the optic nerve and visual cortex, would result in phase cancellation within a voxel, and hence, only amplitude changes would be expected. On the other hand, an asymmetrical intravoxel field distribution would produce both phase and amplitude changes. The in vivo magnitude image data sets show a significant nerve firing detection rate of 56%, with zero detection using the phase image data sets. The percentage magnitude signal changes relative to the fully relaxed equilibrium signal fall within a predicted RMS field range of 1.2-2.1 nT in the optic nerve and 0.4-0.6 nT in the visual cortex, according to the hypothesis that the axonal fields create a symmetrical Lorentzian field distribution within the voxel.

Action Potentials↗

PubMed1995

The chemistry of single-stranded 4'-DNA radicals: influence of the radical precursor on anaerobic and aerobic strand cleavage.

BACKGROUND: Deoxyribosylnucleotide radicals with a radical center at the 4'-position are important intermediates in radical-induced DNA strand cleavage. In the presence of O2, these DNA radicals yield cleavage products that are partly oxidized. In the past, the postulated peroxide intermediates could not be detected directly because they were unstable under the conditions of either radical generation, the work-up procedure, or the analytical techniques used. We set out to generate and analyze these crucial intermediates in radical-induced DNA strand cleavage under mild conditions. RESULTS: Photolysis experiments with modified single-stranded oligonucleotides generated 4'-DNA radicals that were trapped by O2. Using MALDI-MS, DNA peroxides could be detected directly. Depending upon the precursor, these peroxides are formed either before or after the cleavage of the single-stranded DNA radical. Reactions in the presence of 18O2 and/or H218O as well as subsequent transformations to the oxidized cleavage products confirmed the structure of the DNA peroxides. CONCLUSIONS: Our technique of selective DNA radical generation under mild conditions makes it possible to detect labile reaction products of single-stranded DNA radicals and to gain further insight into their cleavage reactions. In cases where a radical pair is formed, the shielding effect protects the DNA radical from external attack so that cleavage of the single strand competes successfully with trapping by O2. This shielding effect might be of general importance if the DNA radicals are generated by reagents that bind to the DNA.

Aerobiosis↗

PubMed2002

Detection of Staphylococcus aureus enterotoxin B at femtomolar levels with a miniature integrated two-channel surface plasmon resonance (SPR) sensor.

Surface plasmon resonance (SPR) biosensors offer the capability for continuous real-time monitoring. The commercial instruments available have been large in size, expensive, and not amenable to field applications. We report here an SPR sensor system based on a prototype two-channel system similar to the single channel Spreeta devices. This system is an ideal candidate for field use. The two-channel design provides a reference channel to compensate for bulk refractive index (RI), non-specific binding and temperature variations. The SPR software includes a calibration function that normalizes the response from both channels, thus enabling accurate referencing. In addition, a temperature-controlled enclosure utilizing a thermo-electric module based on the Peltier effect provides the temperature stability necessary for accurate measurements of RI. The complete SPR sensor system can be powered by a 12V battery. Pre-functionalized, disposable, gold-coated thin glass slides provide easily renewable sensor elements for the system. Staphylococcus aureus enterotoxin B (SEB), a small protein toxin was directly detectable at sub-nanomolar levels and with amplification at femtomolar levels. A regeneration procedure for the sensor surface allowed for over 60 direct detection cycles in a 1-month period.

Animals↗

PubMed1988

Solubilisation effect of Nonidet P-40, triton X-100 and CHAPS in the detection of MHC-like glycoproteins.

We have analysed the differential solubilisation effect of three detergents on cell-membrane histocompatibility glycoproteins. Two nonionic detergents (Nonidet P-40 and Triton X-100) which are extensively used in the extraction of MHC proteins and a zwitterionic detergent (CHAPS) which is sulphobetaine derivative of cholic acid were used. An AKR (H-2k) derived spontaneous leukaemic cell line--424--was used as the experimental model. In this tumour cell line a class I-like antigen is expressed but not directly detected by cell-binding radioimmunoassay or immunoprecipitation from NP-40 or Triton X-100 solubilised glycoproteins. However, 46 kDa and 12 kDa bands consistent with the classical H-2 class I pattern were seen by SDS-PAGE after immunoprecipitation with the 34.5.8 anti-H-2Dd MoAb using CHAPS solubilised 424 glycoproteins. The H-2Dd-reactive molecule appears to be associated with at least one of the syngeneic class I specificities (H-2Kk, H-2Dk) and not accessible to react with the specific anti H-2Dd MoAb. The detergents NP-40 and Triton X-100 appear to be less efficient than CHAPS in breaking protein-protein interactions. This property of CHAPS permitted the adequate solubilisation of the novel antigen and its direct detection. The results of this study suggest that the alternative use of a non-denaturing zwitterionic detergent may contribute to the detection and characterisation of MHC-related, membrane-bound proteins of tumours and normal cells.

Animals↗

PubMed1992

Detection of respiratory syncytial virus in acute bronchiolitis in infants.

Direct detection of respiratory syncytial (RS) virus antigen in nasopharyngeal secretions (NPS) provides the most rapid diagnostic test for RS infection, but more sensitive methods might be more beneficial in the study of virus shedding. RS virus RNA was extracted from cells stored at -70 degrees C either in suspension with added RNAse inhibitor or as a pellet without inhibitor. The RNA was reverse transcribed, the resultant cDNA amplified by the polymerase chain reaction and detected by ethidium bromide staining after electrophoresis through agarose gel (RT-PCR). Of 217 specimens tested, 106 were positive by antigen detection, 99 by RT-PCR, and 92 by virus isolation. In a series of 97 sequential NPS specimens from 15 infants in whom RS virus induced bronchiolitis was confirmed, antigen detection proved most sensitive in the first week after onset and RT-PCR detected most positive specimens in the second week. Storage of the cells as a pellet proved more satisfactory than storage as a suspension. A further round of amplification using nested primers increased the number of positive results obtained by RT-PCR. The sensitivity of antigen detection using directly labelled monoclonal antibody to RS virus was slightly higher than that of RT-PCR, but the specificity was slightly lower.

Antigens, Viral↗

PubMed2005

Detection and differentiation of Vibrio vulnificus in seawater and plankton of a coastal zone of the Mediterranean Sea.

Vibrio vulnificus, a human and animal pathogen, is present in low numbers in the Mediterranean Sea. Seawater and plankton samples were collected from a marine coastal zone of the Straits of Messina in the Mediterranean Sea (Italy) in order to investigate V. vulnificus as free-living (>0.2 microm) and associated with small (>64 microm) and large plankton (>200 microm) utilizing cultural and molecular techniques. Characteristic colonies, grown on thiosulfate, citrate, bile salts and sucrose agar plates, were identified using a biochemical protocol system. A PCR assay was used to confirm isolates and to directly detect V. vulnificus in environmental concentrated samples. Specific primers were used to target the structural cytotoxin/hemolysin gene and the variable regions of 16S rRNA species-specific for V. vulnificus. In addition, a tri-primer PCR of 16S rRNA was used for the differentiation of V. vulnificus strains. Direct detection in marine samples was more frequent than isolation of culturable forms. All isolates were assigned to V. vulnificus biotype 1, 16S rRNA type B. These results confirm the low incidence of V. vulnificus in Mediterranean coastal waters. The isolation of cultivable forms is limited to the warmest months. 16S rRNA primers were the most sensitive molecular tool as they allowed detection of V. vulnificus in 79.1% of samples. Due to the low incidence of V. vulnificus in the Mediterranean coastal environment, its detection requires a molecular approach. The occurrence of V. vulnificus as plankton-associated confirms the role of plankton as a potential reservoir for this pathogen.

Bacterial Proteins↗

PubMed1983

Immunoassay diagnosis of adenovirus infections in children.

Direct detection of viral antigen in nasopharyngeal secretions and stool specimens by radioimmunoassay and the determination of serum antibody responses by complement fixation and immunoglobulin class-specific enzyme immunoassay against the hexon antigen were compared for diagnostic efficacy in 52 children with acute adenovirus infections. The highest diagnosis rate (85% of the cases) was obtained by antigen detection in nasopharyngeal secretions. Adenovirus antigen was also detected in stools of 72% of the 18 patients tested. The immunoglobulin G (IgG) antibody enzyme immunoassay detected 77% of the cases, being more sensitive than the complement fixation test with a 67% detection rate. The IgM antibody response was variable with no clear correlation with the age of the patient or severity of the clinical symptoms. IgM antibody response was detected in 48% of the patients and had the normal transient course, with a persistence of the IgM antibodies of approximately 2 months. Determination of IgA antibodies gave a diagnostic increase in titer in 37% of the cases and was found less suitable for serological diagnosis. Because of the clinical importance of rapid laboratory diagnosis, the direct detection of viral antigen in nasopharyngeal secretions or stool or both should be used as the primary diagnostic test in adenovirus infections.

Adenoviridae↗

PubMed1994

In vitro integration of retrotransposon Ty1: a direct physical assay.

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.

Base Sequence↗

PubMed1996

Hydroxylamine and phenol-induced formation of methemoglobin and free radical intermediates in erythrocytes.

As previously shown with isolated oxyhemoglobin, methemoglobin formation can also be induced in intact erythrocytes by hydroxylamine compounds and substituted phenols such as butylated hydroxyanisole (BHA). Electron spin resonance investigations revealed that, accordingly, free radical intermediates were formed in erythrocytes from hydroxylamine, N,N-dimethylhydroxylamine, and N-hydroxyurea. Due to the low stability of the dihydronitroxyl radicals, their detection required the use of a continuous flow system and relatively high amounts of the reactants. As has already been demonstrated with the solubilized hemoglobin system, hemoglobin of intact erythrocytes also reacts with the more hydrophilic xenobiotics such as hydroxylamine. However, the reaction rate was slightly reduced, indicating the existence of an incomplete permeability barrier for these compounds. The limited solubility of phenolic compounds in the aqueous buffer of suspended erythrocytes (in combination with the strict requirement of osmolarity in order to prevent hemolysis) impeded the direct detection of the respective phenoxyl radicals previously reported in hemoglobin solutions. However, in accordance with earlier findings in homogeneous reaction systems, chemiluminescence was observed as well, indicating the existence of a further reaction intermediate, which was also obtained in pure hemoglobin solutions when mixed with the respective reactants. As has recently been demonstrated, this light emission is indicative of the existence of highly prooxidative compound I intermediates during methemoglobin formation. Prooxidant formation in erythrocytes is reflected by a significant decrease in thiol levels even with those compounds where free radical formation was not directly detectable by ESR spectroscopy. The use of the spin-labeling technique revealed membrane effects as a result of oxidative stress. Oxidative metabolism of hemoglobin with hydroxylamine caused a release of low molecular weight iron. The marked hemolysis observed in the presence of BHA results from a direct membrane effect of this compound rather than a consequence of free radical-induced oxidative stress. A correlation of the different results is discussed in terms of possible toxicological consequences.

Animals↗

PubMed1989

Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction.

Serum components inhibit DNA polymerase, thereby obviating direct detection of serum viral DNA sequences by the polymerase chain reaction (PCR). This has necessitated extraction of nucleic acid from sera before performing PCR and has resulted in loss of sensitivity. By adsorbing virus to a solid surface (microcentrifuge tubes or antibody coated microparticles) followed by proteinase K digestion, as little as three viruses per 200 microliters serum may be directly detected by PCR without nucleic acid extraction. The sensitivity is dependent on the surface area of the adsorptive surface and is increased by having antibodies on the adsorptive surface. The nucleic acid sequence of the amplified DNA fragments may be directly determined by the dideoxy method. Of 24 plasma samples from HBsAg+ volunteer blood donors, HBV DNA was detected in 7 by dot blot assay, 7 by liquid hybridization, and 9 by PCR. PCR detected DNA in every sample that was positive by another assay. Analysis of serial samples of two patients with acute self-limited hepatitis B found detectable HBsAg and pre-S2 antigenemia before HBV DNA by the PCR method. These results suggest that surface antigenemia may precede viremia during acute hepatitis.

Adsorption↗

PubMed1994

Non-radioactive DNA probe and polymerase chain reaction procedures for the specific detection of Acanthamoeba.

Acanthamoebae are potential pathogens which can cause serious infections of humans. A non-radioactive rDNA probe and polymerase chain reaction (PCR) amplification procedures which are specific, rapid, sensitive and safe for the detection of Acanthamoeba have been developed. A restriction fragment (126 bp; ArDNA-a) from a variable region of the cloned 26S rDNA unit of Acanthamoeba castellanii (from plasmid pAR2) was labelled by biotinylation. Cells and DNAs were incubated with the labelled rDNA probe to define conditions providing the highest hybridization specificity for Acanthamoeba by both colorimetric and chemiluminescent assays. Four recent isolates of Acanthamoeba, Acanthamoeba polyphaga, various bacteria, Herpes simplex virus, other eukaryotic amoebae and human cell lines, were sources of DNA for testing. The rDNA probe was found to be highly specific for Acanthamoeba and is capable of directly detecting about 250 cells without prior DNA purification. PCR primers for this unique ArDNA-a fragment have also been designed. Amplification of the targeted sequence by PCR using those primers yielded a single product which was specifically generated for Acanthamoeba template DNA and not DNA from the other control cells. This PCR procedure provided increased sensitivity with the direct detection of as few as 10 Acanthamoeba cells.

Acanthamoeba↗

PubMed1992

Screening for nonsense mutations in patients with severe hemophilia A can provide rapid, direct carrier detection.

Despite marked genetic heterogeneity in families with hemophilic patients, transition mutations in CG dinucleotides occur frequently. Of 71 CG dinucleotides in the factor VIII cDNA, a C-to-T transition in 12 would lead to a new Stop codon (CGA to TGA). Using restriction enzyme digestion of 11 amplified DNA fragments, seven point mutations were localized among 60 patients with severe hemophilia A. Five were detected as loss of a natural or introduced TaqI site at codons -5, 583, 1941, 2116, and 2209 and were confirmed as CGA (Arg) to TGA (Stop) nonsense mutations by DNA sequencing. A novel C-to-T nonsense mutation was detected as loss of the RsaI site at codon 1966 and confirmed by sequence in two unrelated individuals. Two partial gene deletions were detected as selective failure to amplify exon 1 and exons 15-22, respectively. In an additional (61st) patient who was subsequently found to have mild (instead of severe) hemophilia, digests suggested a mutation in codon 1696. Upon sequencing, this codon contained a novel missense mutation, a C-to-G transversion changing CGA (Arg 1696) to GGA (Gly). In four families with women available for testing, carrier status was rapidly determined by direct screening for the point mutation. In two of three with sporadic occurrences, the mother was a carrier as were two of four sisters. In the other family, the mother and a sister were homozygous for the TaqI cleavage site in their amplified exon 24 fragment, indicating a de novo C-to-T transition in codon 2209 in the patient's factor VIII gene. This final patient's sister was a noncarrier even though by linkage analysis she inherited the same factor VIII gene as her brother.

Base Sequence↗

PubMed1990

Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction.

We have developed a procedure that allows the detection of polymerase chain reaction (PCR) products derived from a single target DNA molecule in a human sperm without using radioactive probes. With this method, three genetic loci present in a single sperm can be amplified simultaneously. The amplification procedure is specific as well as efficient and permits detection of the PCR product by ethidium bromide staining after polyacrylamide gel electrophoresis. When allele-specific PCR primers that differ in length are used, the size of the PCR products of different alleles also vary in length, allowing the allelic state at each locus to be determined electrophoretically. Studies on individual sperm by using this procedure should facilitate the measurement of genetic recombination in humans over small physical distances. The ability to directly analyze the allelic state of PCR products from one cell rapidly and simply will also be useful for the prenatal diagnosis of genetic disease, especially in the analysis of single blastomeres taken from in vitro fertilized eggs prior to implantation.

Alleles↗

PubMed1995

[Detection of bacterial protein toxins by a bead-ELISA].

A highly sensitive bead-enzyme-linked immunosorbent assay to detect bacterial protein toxins was developed. Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as a substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG. The sensitivities of the bead-ELISA for various bacterial protein toxins were as follows: less than 40 pg/ml for cholera enterotoxin (CT), less than 20 pg/ml for VT1 and less than 6 pg/ml for VT2 of enterohemorrhagic Escherichia coli. The bead ELISA was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea. Of the 75 stool samples examined, 59 yielded biochemically and serologically confirmed strains of Vibrio cholerae O1. The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V. cholerae O1 was isolated. In addition, the bead ELISA was positive for three stool specimens which were negative by culture. These data indicate that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples.

Acute Disease↗

PubMed1997

Flow cytometric analyses of antibody binding to Chinese hamster ovary cells expressing human thyrotropin receptor.

To develop a method that can be used to directly detect binding of antibodies to TSH receptor (TSHr), we employed Chinese hamster ovary (CHO) cells permanently transfected with a human TSHr complementary DNA (CHOR). These cells showed increased cAMP production when treated with either human TSH or thyroid-stimulating antibodies and decreased TSH-mediated cAMP production when treated with stimulation-blocking antibodies. We employed flow cytometry and rabbit antibodies against the extracellular domain of the TSHr (ETSHr) to test whether these cells can be used to directly detect and quantitate the binding of anti-TSHr antibodies. Rabbit anti-ETSHr bound specifically to CHOR cells, and the binding could be blocked with purified ETSHr. To test the feasibility of using these cells for epitope mapping, we tested the binding of rabbit antibodies raised against several synthetic TSHr peptides. Rabbit antipeptide 92 (amino acids 12-30) and 91 (amino acids 32-46) showed little or no binding to the CHOR cells. In contrast, antibodies raised against peptides 93 (amino acids 316-330), 95 (aa 325-345), 3A (aa 357-372), 367 (aa 367-386), and 1B (aa 362-376) showed significant binding to the CHOR cells. The specificity of binding of antipeptide antibodies was demonstrated by a complete inhibition of binding by corresponding peptides. When TSH-binding inhibitory Ig-positive sera from 15 patients with hyperthyroidism were tested, 8 of them showed specific binding to the CHOR cells compared to their relative binding to normal CHO cells; sera from all normal individuals tested did not exhibit specific binding to CHOR cells. These studies showed the usefulness of CHOR cells and flow cytometry in epitope mapping using sera with known specificities and the potential usefulness of the technique to detect anti-TSHr antibodies in patient sera.

Animals↗

PubMed2001

A novel system for convenient detection of low-affinity receptor-ligand interactions: chelator-lipid liposomes engrafted with recombinant CD4 bind to cells expressing MHC class II.

The interactions of cell surface receptors with their ligands, crucial for initiating many immunological responses, are often stabilized by receptor dimerization/oligomerization, and by multimeric interactions between receptors on one cell with their ligands or cognate receptors on the apposing cell. Current techniques for studying receptor-ligand interactions, however, do not always allow receptors to move laterally to enable dimerization/ oligomerization, or to interact multimerically with ligands on cell surfaces. For these reasons detection of low- affinity receptor-ligand interactions has been difficult. Utilizing a novel chelator-lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA), we have developed a convenient liposome system for directly detecting low-affinity receptor-ligand interactions. Our studies using recombinant soluble forms of murine CD40 and B7.1, and murine and human CD4, each possessing a hexhistidine tag, showed that these proteins can be anchored or 'engrafted' directly onto fluorescently labelled liposomes via a metal-chelating linkage with NTA-DTDA, permitting them to undergo dimerization/oligomerization and multimeric binding with ligands on cells. Fluorescence- activated cell sorter (FACS) analyses demonstrated that while there is little if any binding of soluble forms of murine CD40 and B7.1, and murine and human CD4 to cells, engrafted liposomes bind specifically to cells expressing the appropriate cognate receptor, often giving a fluorescence 4-6-fold above control cells. Such liposomes could detect directly the low-affinity interaction of murine CD40 and B7.1 with CD154- and CD28-expressing cells, respectively, and the interaction of CD4 with MHC Class II, which has hitherto defied direct detection except through mutational analysis and mAb blocking studies.

Amines↗

PubMed1987

Comparison of non-radioactive DNA hybridization probes to detect human immunodeficiency virus nucleic acid.

Simple and sensitive methods to directly detect the human immunodeficiency virus (HIV) are needed for routine use in the clinical laboratory. In this study, we compared DNA probes prepared by: (1) nick translation with biotinylated dATP; (2) direct covalent biotinylation with photobiotin; (3) direct covalent reaction with 2-acetylaminofluorene (AAF); and (4) a standard radioactive (32P) nick translation procedure. These four DNA probes were hybridized with dilutions of purified target HIV DNA blotted onto nitrocellulose strips. Hybridization was detected using a complex of strepavidin-alkaline phosphatase [for (1) and (2)], alkaline phosphatase-tagged antibodies [for (3)] and by autoradiography [for (4)]. Alkaline phosphatase was detected colorimetrically using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. After 1 h, AAF probes were most sensitive (amount detected less than 5 pg), followed by biotin (10 pg), photobiotinylated probes (20 pg) and the radioactive probe (10 pg). The AAF probes were then used to detect HIV DNA in infected CEM cells. We conclude that non-radioactive DNA labelling methods can be used to directly detect HIV DNA under conditions compatible with present clinical laboratory procedures.

2-Acetylaminofluorene↗