A new method for measuring reverse transcriptase activity by ELISA.
A new and sensitive assay of reverse transcriptase (RT) activity of retroviruses measures the incorporation of digoxigenin-labelled dUTP in newly synthesized DNA instead of radioactively labelled (3H- or 32P-)dTTP. To avoid difficulties associated with separation of non-incorporated nucleotides from the newly synthesized DNA, biotin-labelled dUTP is added to the reaction mixture in very low concentrations. After reverse transcription, the newly synthesized, doubly labelled DNA is immobilized on streptavidin-coated ELISA wells and evaluated photometrically by binding of peroxidase-conjugated anti-digoxigenin-antibodies (sheep) and subsequent colour development with 2,2'-azino-di[3-ethylbenzthiazolin-sulfonate(6)] (ABTSR) as substrate. For better standardization, it is suggested that RT activity is given in units (one unit of RT is the amount of enzyme incorporating one nanomole of labelled dNTP in 10 min at 37 degrees C into an acid precipitable DNA) rather than in cpm (counts per minute). The method is specific and easy to perform.
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D004279:DNA, Viral / Q000096:biosynthesis; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D016655:HIV Core Protein p24 / Q000032:analysis; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology; D015498:HIV-2 / Q000201:enzymology; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; D016655:HIV Core Protein p24; C027078:deoxyuridine triphosphate; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase