| US 7,566,529 B2 | ||
| Method for the recovery of retroviral reverse transcriptase (RT) activity from a sample containing unprotected cellular polymerases | ||
| Clas Källander, Uppsala (Sweden); Simon Gronowitz, Uppsala (Sweden); and Tommy Gatu, Uppsala (Sweden) | ||
| Assigned to Cavidi Tech AB, Uppsala (Sweden) | ||
| Appl. No. 10/472,756 PCT Filed Mar. 27, 2002, PCT No. PCT/SE02/00612 § 371(c)(1), (2), (4) Date Oct. 03, 2003, PCT Pub. No. WO02/082088, PCT Pub. Date Oct. 17, 2002. |
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| Claims priority of application No. 0101219 (SE), filed on Apr. 05, 2001. | ||
| Prior Publication US 2004/0110265 A1, Jun. 10, 2004 | ||
| Int. Cl. C12Q 1/70 (2006.01) | ||
| U.S. Cl. 435—5 | 9 Claims |
| 1. Method of recovering a retroviral reverse transcriptase (RT) activity from enveloped retroviruses in a biological sample
containing non-protected cellular polymerases, comprising the steps of
a) incubating the biological sample with a cysteine-modifying agent selected from the group consisting of N-ethylmalemide,
Thimerosal, p-hydroxymercuribenzoate, 5,5′-dithiobis-(2-nitrobenzoic acid), 2-aminoethyl 2′-aminoethanethiolsulfonate, 2-nitro-5-thiocyanobenzoic
acid, methyl methane-thiosulfonate, phenylmercuric acetate and 2,2′-dithiodipyridine, to destroy the activity of contaminating
cellular polymerases while preserving retroviral RT activity within the virus envelope,
b1) removing the enveloped virus particles from the incubation mixture using a virus binding matrix and a washing step, or
b2) inactivating the cysteine-modifying agent with an inactivating substance while preserving the virus envelope from lysis,
c) lysing the virus envelope with a lysing buffer to release the virus enzymes, and
d) recovering the released retroviral RT enzyme from the lysing buffer.
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