Abstract: The present invention relates to a method for assessing susceptibility of a virus to treatment with a drug inhibiting an enzyme of the wild-type virus comprising the steps: a) extracting the viral enzyme from a sample containing the virus; b) measuring the viral enzyme activity both in the absence of the drug and in the presence of the drug at a single predetermined concentration; and c) determining the virus' susceptibility to treatment with the drug from the relation between the enzyme activities in the presence of the drug and the absence of the drug. The invention further relates to a system for performing the method.

Abstract: A method of testing phenotypic drug susceptibility in an enveloped virus-infected mammalian individual by testing on an enzyme packed into an enveloped virus, such as HIV, recovered from a biological sample, such as blood or plasma, from said individual is described. The method comprises the steps of a) adding an enzyme inactivating agent to the sample for inactivating polymerase activity other than that present in the enveloped virion, b) removing the enzyme inactivating agent, enzyme activity blocking antibodies, endogenous enzyme activity inhibitors and antiviral drugs, c) lysing the virus particle to release the enzyme, d) recovering the concentrated purified viral enzyme, such as a HIV reverse transcriptase (RT), resulting from c) and determining the drug sensitivity profile of the individual from the recovered enzyme by using sensitive enzyme assays. The drug sensitivity profile may be used for selecting drug treatment therapy. A commercial package is included.

Abstract: 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.

Abstract: 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.

Abstract: A method for measuring DNA-dependent DNA polymerisation in a biological sample, is described. The method comprises the steps of providing a primer with a single stranded short specific sequence, which is unable to base pair internally, bound to a solid phase; contacting the primer construct with a reaction mixture containing a single stranded deoxynucleotide template with a part of the sequence complementary to the primer and the four deoxynucleoside triphosphates, one of which is modified so that it is specifically recognized by a labeled antibody; adding a biological sample comprising the DNA polymerase, such as retrovirus reverse transcriptase (RT), to the mixture; allowing the polymerase reaction to proceed; incubating the immobilized reaction product with the labeled antibody; detecting the amount of bound labeled antibody; and measuring the amount of incorporated modified deoxynucleoside triphosphate, as a measure of the DNA polymerisation, which may be used drug susceptibility testing.

Abstract: A reverse transcriptase (RT) assay kit for analysis of RT activity in biological samples is described. The kit comprises solid phase bound prA and/or pdA template(s) obtainable by contacting a polystyrene-based solid phase with a 1-methylimidazole-containing coupling solution, and RT-type adapted assay components selected from a buffer, divalent metal ion, chelator, polyamine, RNase inhibitor, reducing agent, salt, stabilizing agent, and detergent, and deoxynucleotide triphosphate, primer, protective agent and concentrated washing buffer, and optionally lyophilized reference enzyme(s), and further optionally lyophilized alkaline phosphatase conjugated anti-BrdU monoclonal antibody, alkaline phosphatase substrate buffer and alkaline phosphatase substrate, and written instructions for use of the assay kit.

Abstract: A method of testing phenotypic drug susceptibility in an enveloped virus-infected mammalian individual by testing on an enzyme packed into an enveloped virus, such as HIV, recovered from a biological sample, such as blood or plasma, from said individual is described. The method comprises the steps of a) adding an enzyme inactivating agent to the sample for inactivating polymerase activity other than that present in the enveloped virion, b) removing the enzyme inactivating agent, enzyme activity blocking antibodies, endogenous enzyme activity inhibitors and antiviral drugs, c) lysing the virus particle to release the enzyme, d) recovering the concentrated purified viral enzyme, such as a HIV reverse transcriptase (RT), resulting from c) and determining the drug sensitivity profile of the individual from the recovered enzyme by using sensitive enzyme assays. The drug sensitivity profile may be used for selecting drug treatment therapy. A commercial package is included.

Abstract: A method for measuring DNA-dependent DNA polymerisation in a biological sample, is described. The method comprises the steps of providing a primer with a single stranded short specific sequence, which is unable to base pair internally, bound to a solid phase; contacting the primer construct with a reaction mixture containing a single stranded deoxynucleotide template with a part of the sequence complementary to the primer and the four deoxynucleoside triphosphates, one of which is modified so that it is specifically recognized by a labeled antibody; adding a biological sample comprising the DNA polymerase, such as retrovirus reverse transcriptase (RT), to the mixture; allowing the polymerase reaction to proceed; incubating the immobilized reaction product with the labeled antibody; detecting the amount of bound labeled antibody; and measuring the amount of incorporated modified deoxynucleoside triphosphate, as a measure of the DNA polymerisation, which may be used drug susceptibility testing.

Abstract: 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.

Abstract: 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.

Abstract: A method for determining the activity of a nucleotide polymerizing enzyme in a sample, and use of the method for determining HIV 1 RT- and Herpes Simplex DNA-polymerase activity. The enzyme is captured by means of a nonoclonal antibody which is immobilized to a solid carrier and is capable of binding the enzyme without detrimentally effecting the enzyme activity. Contaminants and disturbing factors are removed and the nucleotide polymerization starts by the addition of a reaction solution containing a primer/template construct and nucleotides substrate, the reaction conditions being chosen such that they promote permanent association between antibody enzyme- and primer/template constructs. When necessary a nucleotide substrate, primer/template and reaction solution are washed away from the newly synthesized polymer, and the amount of nucleotide which as been incorporated into the polymer is determined, and the activity of the enzyme is determined with the guidance of this determination.