Method for the recovery of retroviral reverse transcriptase (RT) activity from a sample containing unprotected cellular polymerases
A method of recovering an enzyme activity, such as reverse transcriptase (RT) activity, from enveloped viruses, such as HIV, in a biological sample containing a non-protected enzymes, is described. A cysteine-modifying substance is used to destroy the activity of the non-protected enzymes, followed by removal of the enveloped virus particles or inactivation of the cysteine-modifying substance with a chemical. Then the virus envelope is lysed and the released enzymes are recovered. Commercial packages containing written and/or data carrier instructions and some chemicals for performing laboratory steps for recovery of an enzyme activity from enveloped viruses, are also disclosed.
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.
2. Method according to claim 1 , wherein virus is selected from Lentiviruses, HTLV/BLV viruses, mammalian D-type retrovirus and mammalian C-type retrovirus.
3. Method according to claim 2 , wherein the Lentivirus is human immunodeficiency virus (HIV).
4. Method according to claim 1 , wherein the biological sample is a sample of body fluid.
5. Method according to claim 4 , wherein the sample of body fluid is a blood, plasma or serum sample.
6. Method according to claim 1 , wherein the inactivating substance is a mild reducing agent.
7. Method according to claim 6 , wherein the mild reducing agent is selected from the group consisting of cysteine, cysteamine, mercaptosuccinic acid, and glutathione.
8. The method of claim 1 , which comprises removing the enveloped virus particles from the incubation mixture using a virus binding matrix and a washing step, the virus is human immunodeficiency virus (HIV), the biological sample is a sample of body fluid and the virus-binding matrix is an anion exchanger matrix.
9. The method of claim 1 , which comprises inactivating the cysteine-modifying agent with an inactivating substance while preserving the virus envelope from lysis, the virus is human immunodeficiency virus (HIV), the biological sample is a sample of body fluid and the wherein the inactivating substance is a mild reducing agent selected from the group consisting of cysteine, cysteamine, mercaptosuccinic acid, and glutathione.