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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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A Neutravidin-based Assay for Reverse Transcriptase Suitable for High Throughput Screening of Retroviral Activity

  • Brennan, Lyndall E. (Institute of Medical Microbiology) ;
  • Sune, Carlos (Institute of Medical Microbiology) ;
  • Klimkait, Thomas (Institute of Medical Microbiology)
  • Published : 2002.05.31
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Abstract

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-asociated HIV-p24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.

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References

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