Quantitative ELISA-polymerase chain reaction at saturation using homologous internal DNA standards and chemiluminescence revelation. 1998

Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
Laboratoire Virus, Neurones et ImmunitĂ©, UniversitĂ© Paris-Sud, Le Kremlin-BicĂȘtre, France. yassine.taoufik@pbr.ap-hop-paris.fr

In this report, we describe the development and validation of a convenient, versatile and high throughput quantitative polymerase chain reaction (PCR) method. This assay is based on the use of only one concentration of an internal homologous standard (IS) easily obtained by replacing an 18 nt specific sequence using recombinant PCR. Target and IS amplicons are quantitated at the PCR plateau phase using ELISA which includes a hybridization step with either target or IS specific probes and luminometric revelation. Luminometry allows measurement of amplicon levels without the need for serial dilutions. Experimental values were obtained by comparing their target/IS signal ratios to those of an external scale. A linear dynamic range over four orders of magnitude and good reproducibility were obtained. We used this assay to investigate variations of IL-13 mRNA expression in HIV-infected patients under highly active antiretroviral therapy. Furthermore, we also report a variant of this method using Taqman assay in the ABI PRISM 7,700 apparatus.

UI MeSH Term Description Entries
D007963 Leukocytes, Mononuclear Mature LYMPHOCYTES and MONOCYTES transported by the blood to the body's extravascular space. They are morphologically distinguishable from mature granulocytic leukocytes by their large, non-lobed nuclei and lack of coarse, heavily stained cytoplasmic granules. Mononuclear Leukocyte,Mononuclear Leukocytes,PBMC Peripheral Blood Mononuclear Cells,Peripheral Blood Human Mononuclear Cells,Peripheral Blood Mononuclear Cell,Peripheral Blood Mononuclear Cells,Leukocyte, Mononuclear
D008163 Luminescent Measurements Techniques used for determining the values of photometric parameters of light resulting from LUMINESCENCE. Bioluminescence Measurements,Bioluminescent Assays,Bioluminescent Measurements,Chemiluminescence Measurements,Chemiluminescent Assays,Chemiluminescent Measurements,Chemoluminescence Measurements,Luminescence Measurements,Luminescent Assays,Luminescent Techniques,Phosphorescence Measurements,Phosphorescent Assays,Phosphorescent Measurements,Assay, Bioluminescent,Assay, Chemiluminescent,Assay, Luminescent,Assay, Phosphorescent,Assays, Bioluminescent,Assays, Chemiluminescent,Assays, Luminescent,Assays, Phosphorescent,Bioluminescence Measurement,Bioluminescent Assay,Bioluminescent Measurement,Chemiluminescence Measurement,Chemiluminescent Assay,Chemiluminescent Measurement,Chemoluminescence Measurement,Luminescence Measurement,Luminescent Assay,Luminescent Measurement,Luminescent Technique,Measurement, Bioluminescence,Measurement, Bioluminescent,Measurement, Chemiluminescence,Measurement, Chemiluminescent,Measurement, Chemoluminescence,Measurement, Luminescence,Measurement, Luminescent,Measurement, Phosphorescence,Measurement, Phosphorescent,Measurements, Bioluminescence,Measurements, Bioluminescent,Measurements, Chemiluminescence,Measurements, Chemiluminescent,Measurements, Chemoluminescence,Measurements, Luminescence,Measurements, Luminescent,Measurements, Phosphorescence,Measurements, Phosphorescent,Phosphorescence Measurement,Phosphorescent Assay,Phosphorescent Measurement,Technique, Luminescent,Techniques, Luminescent
D012015 Reference Standards A basis of value established for the measure of quantity, weight, extent or quality, e.g. weight standards, standard solutions, methods, techniques, and procedures used in diagnosis and therapy. Standard Preparations,Standards, Reference,Preparations, Standard,Standardization,Standards,Preparation, Standard,Reference Standard,Standard Preparation,Standard, Reference
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004797 Enzyme-Linked Immunosorbent Assay An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. ELISA,Assay, Enzyme-Linked Immunosorbent,Assays, Enzyme-Linked Immunosorbent,Enzyme Linked Immunosorbent Assay,Enzyme-Linked Immunosorbent Assays,Immunosorbent Assay, Enzyme-Linked,Immunosorbent Assays, Enzyme-Linked
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012689 Sequence Homology, Nucleic Acid The sequential correspondence of nucleotides in one nucleic acid molecule with those of another nucleic acid molecule. Sequence homology is an indication of the genetic relatedness of different organisms and gene function. Base Sequence Homology,Homologous Sequences, Nucleic Acid,Homologs, Nucleic Acid Sequence,Homology, Base Sequence,Homology, Nucleic Acid Sequence,Nucleic Acid Sequence Homologs,Nucleic Acid Sequence Homology,Sequence Homology, Base,Base Sequence Homologies,Homologies, Base Sequence,Sequence Homologies, Base
D015203 Reproducibility of Results The statistical reproducibility of measurements (often in a clinical context), including the testing of instrumentation or techniques to obtain reproducible results. The concept includes reproducibility of physiological measurements, which may be used to develop rules to assess probability or prognosis, or response to a stimulus; reproducibility of occurrence of a condition; and reproducibility of experimental results. Reliability and Validity,Reliability of Result,Reproducibility Of Result,Reproducibility of Finding,Validity of Result,Validity of Results,Face Validity,Reliability (Epidemiology),Reliability of Results,Reproducibility of Findings,Test-Retest Reliability,Validity (Epidemiology),Finding Reproducibilities,Finding Reproducibility,Of Result, Reproducibility,Of Results, Reproducibility,Reliabilities, Test-Retest,Reliability, Test-Retest,Result Reliabilities,Result Reliability,Result Validities,Result Validity,Result, Reproducibility Of,Results, Reproducibility Of,Test Retest Reliability,Validity and Reliability,Validity, Face
D015342 DNA Probes Species- or subspecies-specific DNA (including COMPLEMENTARY DNA; conserved genes, whole chromosomes, or whole genomes) used in hybridization studies in order to identify microorganisms, to measure DNA-DNA homologies, to group subspecies, etc. The DNA probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the DNA probe include the radioisotope labels 32P and 125I and the chemical label biotin. The use of DNA probes provides a specific, sensitive, rapid, and inexpensive replacement for cell culture techniques for diagnosing infections. Chromosomal Probes,DNA Hybridization Probe,DNA Probe,Gene Probes, DNA,Conserved Gene Probes,DNA Hybridization Probes,Whole Chromosomal Probes,Whole Genomic DNA Probes,Chromosomal Probes, Whole,DNA Gene Probes,Gene Probes, Conserved,Hybridization Probe, DNA,Hybridization Probes, DNA,Probe, DNA,Probe, DNA Hybridization,Probes, Chromosomal,Probes, Conserved Gene,Probes, DNA,Probes, DNA Gene,Probes, DNA Hybridization,Probes, Whole Chromosomal

Related Publications

Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
December 1996, Analytical biochemistry,
Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
January 1998, Hepato-gastroenterology,
Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
October 1998, Analytical chemistry,
Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
January 2008, Analytical biochemistry,
Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
March 1996, Analytical chemistry,
Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
January 2001, Analytical biochemistry,
Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
January 2018, Scientific reports,
Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
March 2021, Molecular therapy. Methods & clinical development,
Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
December 2014, British journal of hospital medicine (London, England : 2005),
Y Taoufik, and D Froger, and S Benoliel, and C Wallon, and E Dussaix, and J F Delfraissy, and O Lantz
January 2007, Methods in molecular biology (Clifton, N.J.),
Copied contents to your clipboard!