Storage: −20°C
UNSPSC Code: 41105333
Components: HIV-1 Reverse Transcriptase; Incubation Buffer; Nucleotides; Template/Primer Hybrid; Lysis Buffer; Anti-Digoxigenin-POD antibody; Washing Buffer Concentrate 10x concentrated; Conjugate Dilution Buffer; Substrate Buffer; ABTS Substrate; Substrate Enhancer; Microplate Modules, precoated with streptavidin; One Strip Frame (for 8-well microplate modules); Cover Foils for Microplates
RIDADR: NONH for all modes of transport
Application:
The Reverse Transcriptase Assay is designed for the quantitative determination of RT activity in cell culture and other biological samples. The assay is used to determine the propagation of retroviruses in retrovirus-infected mammalian cells in culture. The assay is also used for in vitro screening for RT inhibitors.
General description:
Colorimetric enzyme immunoassay for the quantitative determination of retroviral reverse transcriptase activity by incorporation of digoxigenin- and biotin-labeled dUTP into DNA.
Other Notes:
For life science research only. Not for use in diagnostic procedures.
Specifications:
The assay detects the activity of natural or recombinant retroviral reverse transcriptase, including that of HIV-1, HIV-2, SIV-1, AMV, and M-MulV.
Principle:
The Reverse Transcriptase Assay, colorimetric, takes advantage of the ability of reverse transcriptase to synthesize DNA using the hybrid poly (A) x oligo (dT)15 as a template and primer. It avoids the use of [3H]- or [32P]-labeled nucleotides that are employed in standard RT assays. In place of radiolabeled nucleotides, digoxigenin- and biotin-labeled nucleotides in an optimized ratio are incorporated into the same DNA molecule by the RT activity. A template/primer hybrid is supplied, but the flexibility of the assay allows the use of a template of choice (e.g., a viral template). The detection and quantification of the synthesized DNA as a parameter for RT activity follows a sandwich ELISA protocol: biotin-labeled DNA binds to the surface of streptavidin-coated microplate modules. In the next step, an antibody to digoxigenin, conjugated to peroxidase (anti-DIG- POD), is added and bound to the digoxigenin-labeled nucleotides (Licensed by Institute Pasteur). In the final step, the peroxidase substrate ABTS is added. The peroxidase enzyme catalyzes the cleavage of the substrate to produce a colored reaction product. The absorbance of the samples is determined using a microplate (ELISA) reader, and is directly correlated to the level of RT activity in the sample.