A neutravidin-based assay for reverse transcriptase suitable for high throughput screening of retroviral activity.
A non-isotopic neutravidin-based reverse transcriptase (RT) assay adapted for high throughput screening of HIV activity is described. Using a 96-well microtitre plate, HIV particles are lysed and the RT enzyme released into a reaction mixture containing poly(A) RNA, biotinylated oligo d(T) and fluorescein-labelled dUTP (FI-dUTP). With poly(A) as a template and oligo d(T) as primer, the viron RT incorporates FI-dUTP into an elongating DNA strand. The resulting product is captured on a neutravidin-coated 96-well plate and the unincorporated nucleotides removed by a series of washing steps. A simple ELISA is subsequently performed using a monoclonal antifluorescein antibody conjugated to alkaline phosphatase. Quantification of RT activity is facilitated by a colorimetric readout. The assay was validated in the context of a diagnostic HIV-1 phenotyping assay. Using supernatants from HIV-1 infected lymphocyte cultures the assay was shown to be as sensitive as a radioactive assay and the RT activity correlated well with levels of cell-associated HIVp24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.
Brennan
Lyndall E
LE
Institute of Medical Microbiology, Basel, Switzerland.
Suñé
Carlos
C
Klimkait
Thomas
T
D001360:Avidin / Q000737:chemistry; D003124:Colorimetry; D024921:Drug Resistance, Multiple, Viral; D015683:Gene Products, gag / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000378:metabolism; D015658:HIV Infections / Q000175:diagnosis* / Q000188:drug therapy / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000302:isolation & purification*; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019469:Indinavir / Q000494:pharmacology; D008403:Mass Screening; D011498:Protein Precursors / Q000378:metabolism; D019438:Ritonavir / Q000494:pharmacology; D019258:Saquinavir / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000382:microbiology*
D015683:Gene Products, gag; D016655:HIV Core Protein p24; D017320:HIV Protease Inhibitors; D011498:Protein Precursors; C093483:neutravidin; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D001360:Avidin; D019469:Indinavir; D054303:HIV Reverse Transcriptase; D019258:Saquinavir; D019438:Ritonavir