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Biomedical subjects

J L Guesdon

Publications and source records attributed to J L Guesdon.

At least 19 recordsLinked to original sources
PubMed1992

Immunoenzymatic techniques applied to the specific detection of nucleic acids. A review.

Numerous enzymatic and chemical methods are now available for the preparation of non-radioactive nucleic acid probes. Labels, such as enzymes, fluorophores, lumiphores can be attached to the nucleic acid probe either by covalent bonds (direct labelling) or by biospecific recognition after hybridization (indirect labelling). The principle of the latter method is based on the use of a hapten-labelled nucleic acid probe which is generally detected by an immunoenzymatic assay. Indirect labelling has several advantages: this procedure uses multienzyme complexes to increase the number of enzyme molecules associated with hybridization and hence provides an increase in detectability; moreover, haptens (biotin, dinitrophenol, acetylaminofluorene analogues, digoxigenin, brominated or sulphonylated pyrimidines) used to label nucleic acid probes are not sensitive to elevated temperatures (42-80 degrees C), extended incubation times (several hours), detergents and organic solvents currently required in hybridization techniques. The application of the immunoenzymatic and related techniques to nucleic acid probing is reviewed, focussing on the strategies of non-radioactive hybridization, hapten-labelling of nucleic acids and methods for the immunodetection of the hybrids.

Affinity Labels↗
PubMed1992

Escherichia coli heat-stable enterotoxin (STa)-biotin conjugates for the titration of STa antisera by an enzyme-linked immunosorbent assay.

The development of a new approach to the diagnosis of infectious diarrhoea, caused by Escherichia coli heat-stable enterotoxin (ST), was preceded by a preliminary study. The purpose of the latter was to establish whether three preparations of ST produced by a human isolate of enterotoxigenic E. coli (STa), obtained at different steps of the purification procedure (involving Amberlite XAD2 resin chromatography (P3), a gel filtration chromatography on a Biogel P4 (P2) or a disc-gel electrophoresis (P1)), could be employed to titrate antisera to STa using an ST-biotin enzyme-linked immunosorbent assay (ELISA). The solid-phase STa was obtained by first coupling the toxin to biotinyl-N-hydroxysuccinimide and then binding this conjugate to avidin adsorbed to flat-bottomed polystyrene microtitre plates. Using these reagents, the assay conditions were examined. Checkerboard tests determined optimal biotin-P3, P2 or P1 toxin conjugate concentrations to be used as the immunosorbent for P3, P2 and P1 antiserum titration. The immunosorbent prepared with STa purified only on Amberlite XAD2 resin was unable to differentiate significantly between P3, P2 or P1 antisera. Immunosorbent prepared with P2 or P1 detected widely differing titres between the three antisera and gave more sensitive results. Only small but questionable differences were observed between P2 and P1 toxin preparations.

Animals↗
PubMed1992

The detection of Mycobacterium tuberculosis in uncultured clinical specimens using the polymerase chain reaction and a non-radioactive DNA probe.

A Sal I-Hin dIII restriction fragment from Mycobacterium tuberculosis was found to hybridize specifically with genomic DNA from M. tuberculosis. Primers were designed from the sequence of this fragment and used to amplify uniquely M. tuberculosis-group DNA in a polymerase chain reaction. It is suggested that a combination of these primers and an acetylaminofluorene-labelled probe will prove to be a useful tool for the early diagnosis of tuberculous infections.

Base Sequence↗
PubMed1992

Detection of point mutation in bla T genes of Enterobacteriaceae by biotinylated oligonucleotide probes using microwell hybridization and enzymofluorometric method.

Point mutation in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaden their substrate spectrum towards all beta-lactams except cephamicins and imipenem. We describe here hybridization techniques for the detection of point mutations by non-radioactive oligonucleotide probes with plasmid DNA carrying bla T genes immobilized in polystyrene microwells. After hybridization in discriminating conditions with corresponding biotinylated oligonucleotide probes, the hybrids were detected by using a streptavidin-alkaline phosphatase conjugate and a fluorogenic substrate, 4-methylumbelliferyl-phosphate. The adsorption of DNA to microwells used in the present work was found to be independent of Mg2+ and Na+ concentrations. By this method, less than 3 fmols of target DNA were sufficient for the detection of point mutation.

Adsorption↗
PubMed1992

Usefulness of the ID32 staph system and a method based on rRNA gene restriction site polymorphism analysis for species and subspecies identification of staphylococcal clinical isolates.

The usefulness of the ID32 Staph System and a method based on rRNA gene restriction site polymorphism was evaluated by the study of 42 staphylococcal clinical isolates phenotypically difficult to identify. The ID32 Staph micromethod and the genomic method are adapted for recognition of 27 and 31 staphylococcal taxa, respectively. The genomic method is based on a Dice analysis of the hybridization patterns obtained by cutting the cellular DNA either with EcoRI or with HindIII and by probing with pBA2, containing the Bacillus subtilis gene encoding 16S rRNA, labeled either with [alpha-32P]dCTP or with acetylaminofluorene. This study showed that the nonradioactive labeling provided a better resolution of the hybridizing bands than radioactive labeling. Of the 42 isolates selected, only 22 could be assigned to a staphylococcal species by the ID32 Staph System, whereas 35 could be identified by the genomic method. This latter method also enabled the screening of three unclassified isolates having hybridization patterns more closely related to each other than to any of the 31 staphylococcal taxa investigated. These three isolates could belong to a staphylococcal taxon not yet described.

Animals↗
PubMed1992

Alternaria spore and mycelium sensitivity in allergic patients: in vivo and in vitro studies.

Extracts from Alternaria spores and mycelia were prepared to evaluate their allergenic potencies in allergic patients. Twelve Alternaria-sensitive patients, with histories of rhinitis or asthma, were submitted to skin prick tests and five of 12 patients received nasal challenges with spores and mycelia. In vitro, the allergenic activity of each extract was determined by RAST, basophil histamine-release and RAST-inhibition. All patients demonstrated skin reactivity to both extracts while skin reactivity to mycelia was higher than that induced by the spore extract (P < .005). Of the 12 patients, 11 had positive mycelia-RAST and 9/12 had positive spore-RAST. It was found that mycelium-IgE antibody levels were higher than spore-IgE antibody levels (P < .005). Nine RAST-positive patients had positive histamine release tests (> 50%) and basophils challenged with mycelia appeared 10-fold more sensitive compared to the spore challenge. In four of five patients subjected to nasal provocation tests, immediate-type rhinitis was elicited either after spore or mycelium challenge. The patients exhibited a higher nasal reactivity with the mycelium challenge than with spores. RAST-inhibition studies demonstrated that mycelial extracts shared common allergens with spore. These results indicated that Alternaria spore and mycelium were potent allergens in allergic patients and there was a variability in the pattern of in vivo and in vitro reactions between the patients for each allergen.

Adolescent↗
PubMed1991

[Nonradioactive molecular hybridization].

Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The new technology of non-radioactive probes (cold probes) allows the routine use of this method outside of the specialized molecular biology laboratory. Hybridization makes use of a nucleic acid probe to detect a complementary nucleic acid target present in biological fluid or in a biopsy tissue. Hybridization leads to the formation of a double-stranded molecule called hybrid or duplex, which can be detected with a great sensitivity by using high-energy radioisotope. However, the use of radioisotope labeled probes is limited by the short half-life of the isotope, radiolysis of the probe and the radioactive hazards. In order to overcome autoradiography delay and the drawbacks of techniques employing radioisotopes, various non-radioactive labels have been proposed. Labeling and detection systems are currently designed in two ways: The label molecule can be attached directly to the DNA probe (direct labelling) or it can be attached to a molecule which binds either to a modified probe or specifically to the duplex (indirect labeling). Various substances has been used to label directly a nucleic acid probe. The assay detection limit depends largely on the detection limit of the label, therefore, probe assay based on label which provides signal amplification (e.g., enzymes) is likely to be more sensitive than an assay using a label which provides only a single signal per molecule (e.g., fluorochromes). In the indirect labeling, the probe cannot be detected alone; rather, it requires the addition of a detection system.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1990

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood.

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.

Base Sequence↗
PubMed1990

An automatic modified polymerase chain reaction procedure for hepatitis B virus DNA detection.

In order to perform an efficient and reproducible diagnostic test for hepatitis B virus (HBV) infection using the polymerase chain reaction (PCR), sixteen primer couples specific for the HBV genome were selected. Primers 15-31 nucleotides in length containing between 31-73% GC permitted amplification of fragments corresponding to the whole HBV genome. The specificity and efficiency of PCR amplification were studied in detail using DNA extracted from either a viral particle preparation or from the liver of a patient with chronic active hepatitis. Three primer couples in the X, C and PreS regions, i.e. MD24/MD26, MD27/MD31 and MD19/MD18, respectively, gave satisfactory results and performed efficiently under highly stringent hybridization conditions. A modified PCR procedure was then developed using only two thermal steps with a temperature shift of 16 degrees C. This simple method was as efficient as conventional PCR and permitted detection of a single HBV DNA molecule with the X region specific primer couple. The automatization of this PCR-based procedure permitted 40 amplification cycles in 105 min.

Autoanalysis↗
PubMed1990

Biotinylated oligonucleotide probes for the detection and the characterization of TEM-type extended broad spectrum beta-lactamases in Enterobacteriaceae.

Point mutations in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaded their substrate spectrum towards all beta-lactams except cephamycins and imipenem. The presence of such variants on self-transferable plasmids accounts for the dissemination of this new type of resistance to numerous species of Enterobacteriaceae in various countries. We have synthetized biotinylated oligonucleotide probes for the detection and the discrimination of parental and mutated nucleotide sequences of TEM enzymes. Seven clinical isolates belonging to four species and harbouring TEM-1, TEM-3 or TEM-6 were studied. The results obtained indicate that detection of TEM-derived broad spectrum beta-lactamases in clinical isolates of Entero-bacteriaceae is possible with biotinylated oligonucleotide probes.

Base Sequence↗
PubMed1990

Competitive erythroimmunoassay for detecting Clostridium perfringens type A enterotoxin in stool specimens.

A competitive erythroimmunoassay (ERIA) is described for Clostridium perfringens enterotoxin (CPE) detection in stools. This technique uses sheep red blood cells sensitized by CPE and an anti-CPE-antibody-coated plate in which the results are read by eye. ERIA is simple, rapid, economic and more sensitive (2 ng/ml) than the enzyme-linked immunosorbent assay used for evaluation. ERIA is suitable for CPE detection in stool samples protected with phenylmethylsulphonylfluoride.

Clostridium Infections↗
PubMed1990

Gene heterogeneity for tetracycline resistance in Staphylococcus spp.

Nucleotide sequences related to four tet genes were studied by hybridization in 183 clinical Staphylococcus isolates. tet(K) predominated in strains resistant only to tetracycline, while tet(M) was responsible for combined tetracycline and minocycline resistance. In strains harboring both genes, they contributed additively. tet(L) was detected in only five strains, and no hybridization was observed with tet(O).

DNA, Bacterial↗
PubMed1990

Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis.

An insertion sequence-like element, IS6110, was isolated from a Mycobacterium tuberculosis cosmid library as a repetitive sequence. IS6110 shows similarities with elements of the IS3 family. This insertion sequence was found to be specific to mycobacteria belonging to the M. tuberculosis complex. For detection and identification of M. tuberculosis bacilli in uncultured specimens, oligonucleotides derived from the IS6110 sequence were used as primers and probes in polymerase chain reaction studies. The results obtained were consistent with results of classical identification procedures, bacteriological data, and clinical criteria.

DNA Transposable Elements↗
PubMed1990

[Solid phase hybridization].

Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The possibility of using them in clinical biology has been considered for several years. The transfer from basic to applied research can now take place due to the new technology of non-radioactive probes (cold probes). Hybridization features the use of a probe nucleic acid molecule and of a target nucleic acid molecule. It leads to the formation of a double-stranded molecule, also called duplex, which can be detected with great sensitivity. For this to be done, the probe can be tagged with a variety of labels such as a radio-isotope or a non-isotopic marker which can be detected for its fluorescence, luminescence or enzyme activity or by immuno-reaction(s). Hybridization can be performed either in a liquid solution or on a solid support. In the first case, hybridization is generally followed by a separation step which makes use of solid phase to isolate the hybrid (duplex) formed. This separation can be achieved by biochemical means (adsorption chromatography, differential precipitation, electrophoresis, etc.) or by immunological means (affinity chromatography, immuno-capture). In the second case, the support can be used in different ways according to the assay format employed to perform hybridization. The target DNA can be immobilized by contacting the sample to be tested with the support: simple specimen deposit with a pipette (for low volumes), or by aspiration under vacuum or by (capillary or electrophoretic) transfer from an agarose gel. In the sandwich assay format, probe DNA is immobilized onto the support to specifically extract the target DNA from the medium under assay. Support to be employed for hybridization purpose can be selected from the group consisting of: nylon (N), nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes; N+NC mixed membranes; cellulose and its derivatives; polyoside supports (Sephacryl, Sepharose, etc.), latex particles, polystyrene microplates. The coupling procedures and capacity vary according to the type of support used.

Chromatography, Affinity↗
PubMed1989

Monoclonal anti-nucleoside antibodies. Characterization and application in an enzyme immunoassay of single-stranded DNA.

Two mouse monoclonal antibodies were raised against adenosine and guanosine coupled to bovine serum albumin (BSA) by periodate oxidation. They were named A-16 and G-K21 respectively and selected for their ability to recognize single-stranded DNA. Their epitope specificities were assessed and their dissociation constants determined by an indirect ELISA method. The KD values for adenosine and guanosine coupled to BSA were 9.9 X 10(-7) M and 1.1 X 10(-10) M for G-K21 respectively, and 2.5 X 10(-8) M and 1.0 X 10(-6) M for A-16. These monoclonal anti-nucleoside antibodies were used to develop a sandwich enzyme immunoassay for single-stranded DNA. The purified IgG antibodies were coupled to beta-galactosidase and alkaline phosphatase by the one-step glutaraldehyde method and used in a test optimized for pH, saturating proteins, coating antibody, nature of the conjugate and protein concentrations. Less than 100 pg/well of single-stranded DNA could be detected, and the detection was linear over a DNA concentration ranging from 0.34 to 34 ng/ml. The assay could quantitate single-stranded DNAs of differing origin, but not RNAs. The test was compared to another titration method, and used to calibrate target DNA amounts in non-radioactive hybridization experiments.

Animals↗