A nonradioactive micro-assay for released reverse transcriptase activity of a lentivirus.
A nonradioactive micro-assay procedure for detection of released reverse transcriptase activity from cells infected with equine infectious anemia virus is described. This procedure utilizes biotinylated-dUTP in conjunction with a streptavidin-alkaline phosphatase conjugate. Detection of alkaline phosphatase is by autoradiography of the chemiluminescence produced during enzymatic dephosphorylation of Lumi Phos 530. This method, as with reverse transcriptase micro-assays employing 32P-labeled nucleotides, is suited to the processing of numerous samples, while having the advantages of safety and stability normally associated with nonradioactive methods of detection. Sensitivity is comparable to a reverse transcriptase micro-assay using 32P-dTTP.
Cook
R F
RF
Gluck Equine Research Center, Dept. of Veterinary Science, University of Kentucky, Lexington 40546-0099.
Cook
S J
SJ
Issel
C J
CJ
D000469:Alkaline Phosphatase; D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin; D002478:Cells, Cultured; D006736:Horses; D004860:Infectious Anemia Virus, Equine / Q000201:enzymology*; D008163:Luminescent Measurements; D008832:Microchemistry; D010527:Perissodactyla; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D019809:Streptavidin
D001426:Bacterial Proteins; D001710:Biotin; D019809:Streptavidin; D012194:RNA-Directed DNA Polymerase; D000469:Alkaline Phosphatase