Method of concentrating and recovering a viral enzyme activity from biological samples
A method of concentrating and recovering an enzyme activity from enveloped viruses present in a biological sample, is described. The method comprises contacting the biological sample in a first buffer solution with a virus-binding matrix, such as an anion exchanger matrix, to attach virus particles present in the sample to the matrix, washing the matrix carrying the virus particles with a second buffer solution to remove components interfering with viral enzyme activity, lysing the immobilized virus particles in a third buffer solution and recovering the concentrated viral enzyme activity from the third buffer solution. Additionally, a commercial package containing written and/or data carrier instructions for performing laboratory steps for concentration and recovery of an enzyme activity from enveloped viruses present in a biological sample and at least one component necessary for the assay, is disclosed.
1. Method of concentrating and recovering a reverse transcriptase (RT) enzyme activity from enveloped retroviruses present in a biological sample
comprising the steps of
contacting the biological sample in a first buffer solution at a concentration in range of 100-500 mM of buffering substance and having a pH of 4.0-8.5 and optionally containing chaothropic ions at an ionic strength of up to 2 M with a virus-binding matrix, to attach virus particles present in the sample to the matrix,
washing the matrix carrying the virus particles with a second buffer solution at a concentration of 1-100 mM of buffering substance and containing cations at a concentration of 0.1-1 M and having a pH of 4-9, to remove components interfering with viral enzyme activity,
lysing the immobilized virus particles in a third buffer solution at a concentration of 10-500 mM of buffering substance and containing a non denaturing detergent and having a pH of 4-9 which prevents the enzyme from binding to the virus-binding matrix, and
recovering the concentrated viral enzyme activity from the third buffer solution.
2. Method according to claim 1 , wherein the virus-binding matrix is an anion exchanger matrix.
3. Method according to claim 2 , wherein the anion exchanger matrix contains tertiary and/or quaternary amine groups.
4. Method according to claim 1 , wherein the first buffer is selected from the group consisting of 150 mM MES pH 6.0, 200 mM Potassium iodide (KI).
5. Method according to claim 1 , wherein the second buffer is selected from the group consisting of 10 mM MES pH 6.0, 500 mM Potassium acetate (KAc).
6. Method according to claim 1 , wherein the third buffer is selected from the group consisting of enzyme assay compatible buffers including a detergent and a buffering substance.
7. Method according to claim 1 , wherein the biological sample is selected from the group consisting of serum and plasma samples.
8. Commercial package containing written and/or data carrier instructions for performing laboratory steps for concentration and recovery of a reverse transcriptase enzyme activity from enveloped retroviruses present in a biological sample, and
the components
a virus-binding matrix,
a first buffer solution at a concentration in range of 100-500 mM of buffering substance and having a pH of 4.0-8.5 and containing chaothropic ions at an ionic strength of up to 2 M,
a second buffer solution at a concentration of 1-100 mM of buffering substance and containing cations at a concentration of 0.1-1 M and having a pH of 4-9,
a third buffer solution at a concentration of 10-500 mM of buffering substance and containing a non denaturing detergent and having a pH of 4-9, and optionally
Mini columns, and
Plastic tubes.
9. Commercial package according to claim 8 , wherein the virus-binding matrix is an anion exchanger matrix,
the first buffer is selected from the group consisting of 150 mM MES pH 6.0, 200 mM
Potassium iodide (KI),
the second buffer is selected from the group consisting of 10 mM MES pH 6.0, 500 mM
Potassium acetate (KAc),
the third buffer is selected from the group consisting of enzyme assay compatible buffers including a detergent and a buffering substance.
10. Commercial package according to claim 9 , wherein the anion exchanger matrix contains tertiary and/or quaternary amine groups.
11. Method according to claim 2 , wherein the first buffer is selected therefor the group consisting of 150 mM MES pH 6.0, 200 mM Potassium iodide (KI); the second buffer is selected from the group consisting of 10 mM MES pH 6.0, 500 mM Potassium acetate (KAc); and wherein the third buffer is selected from the group consisting of enzyme assay compatible buffers including a detergent and a buffering substance.
12. Method according to claim 11 , wherein the biological sample is selected from the group consisting of serum and plasma samples.
13. Method according to claim 12 wherein the RT activity is quantitated with a sensitive assay.