Quantitative ELISA-polymerase chain reaction at saturation using homologous internal DNA standards and chemiluminescence revelation.
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.
Laboratoire Virus, Neurones et Immunité, Université Paris-Sud, Le Kremlin-Bicêtre, France. yassine.taoufik@pbr.ap-hop-paris.fr
Y
Y
Taoufik
D
D
Froger
S
S
Benoliel
C
C
Wallon
E
E
Dussaix
J F
JF
Delfraissy
O
O
Lantz
D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D004797:Enzyme-Linked Immunosorbent Assay; D015658:HIV Infections / Q000097:blood; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D016014:Linear Models; D008163:Luminescent Measurements; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid
D015342:DNA Probes; D004247:DNA