Diagnostic test for hepatitis B virus

Claim 3

The method of claim 1 wherein the labelled, cloned, hepatitis B virus-DNA is denatured to form single strands of said labelled, cloned, hepatitis B virus-DNA prior to said incubating step.

Claim 4

The method of claim 2 which comprises denaturing said test specimen on the substrate to form single DNA strands prior to said incubating step.

Claim 5

The method of claim 2 which comprises placing said test specimen directly on said substrate without prior purification of said test specimen.

Claim 6

The method of claim 1 wherein the test specimen is an aqueous solution or suspension of a biological material and/or wherein the substrate comprises a solid matrix, f.i. a nitrocellulose filter.

Claim 7

The method of claim 5 wherein said predetermined hybridization conditions comprise a predetermined time period, preferably between about 8 and 36 hours, and a predetermined temperature, preferably between about 5 and 10° C below the melting temperature of said HBV-DNA.

Claim 8

The method of claim 5 wherein said test specimen comprises a member selected from the group consisting of blood, blood products- serum and peripheral body fluids, and preferably comprises blood serum derived from a human subject.

Claim 9

The method of claim 6 wherein said label comprises a radioactive substance, which preferably comprises 32p or comprises 125 I, or alternatively comprises a fluorescent or an immunofluorescent substance.

Claim 10

The method of claim 7 wherein said substrate comprises a modified cellulosic paper comprising a sheet or web of cellulose fibers substituted with diazotized metaaminobenyloxy-methyl for covalent binding thereto of nucleic acid residues.

Claim 11

of 10. The method claim 6 which comprises performing said incubation step in the presence of a hybridization solution, preferably conducting said incubation in a sealed container.

Claim 12

The method of claim 10 wherein said hybridization solution comprises an aqueous salt solution between 3 and 6 times SSC and having a pH in the range between about 6,5 and 8,0, said solution also containing denatured carrier DNA or comprises a partially organic solution containing formamide.

Claim 15

The method of claim 12 wherein said hybridization conditions comprise conducting said incubation in the presence of a solution ha-ving a pH of about 7,0, said solution being between about 3 and about 6 SSC and containing denatured carrier DNA.

Claim 16

A diagnostic reagent comprising purified, cloned, hepatitis B virus-DNA, radioactively labelled to a high specific activity.

Claim 17

A diagnostic reagent comprising purified, cloned, hepatitis B virus-DNA, radioactively labelled to a high specific activity.

Claim 18

A diagnostic test kit for detecting the presence of hepatitis B virus DNA comprising the reagent of claim 11, a sealable container constructed of a material capable of withstanding boiling water temperatures and a matrix material capable for adhering DNA or RNA thereto, said matrix material preferably is nitrocellulose filter paper or diazobenzyloxymethyl-cellulose paper.

Claim 19

The diagnostic test kit of claim 15 further comprising a hybridization solution having a pH between pH 6,5 and pH 8,0, said solution being between 3 and 6 times SSC and containing denatured DNA salmon sperm or calf thymus.

Claim 20

The diagnostic test kit of claim 15 further comprising a pertially organic hybridization solution containing formamide.

Claim 21

A methof for identifying a human carrier of hepatitis B virus which comprises: collecting a blood specimen from a suspected carrier of said hepatitis B virus, isolating the serum from said specimen, applying between about 5 and about 200 ul of said serum absent any DNA purifiction pretreatment to a filter matrix made of a material that will bind RNA and DNA, placing said substrate in a sealable container with a predetermined quantity of an aqueous hybridization solution of between 3 and 6 times SSC and approximately neutral pH, and a probe prepared by cloning hepatitis B virus DNA, purifiying the cloned hepatitis B virus DNA from the r-ecombinant plasmid and labelling the DNA to high specific activity, sealing the container, incubating the sealed container for between about 8 and 36 hours at a temperature between 65 and 68° C to permit the labelled hepatitis B virus DNA to combine with any hepatitis B virus DNA present in said serum, removing uncombined DNA from the presence of said substrate, and analyzing the substrate for the presence of said radioactive label using liquid scintillatinn spectroscopy or autoradiography.

Claim 22

A methof for identifying a human carrier of hepatitis B virus which comprises: collecting a blood specimen from a suspected carrier of said hepatitis B virus, isolating the serum from said specimen, applying between about 5 and about 200 ul of said serum absent any DNA purifiction pretreatment to a filter matrix made of a material that will bind RNA and DNA, placing said substrate in a sealable container with a predetermined quantity of an aqueous hybridization solution of between 3 and 6 times SSC and approximately neutral pH, and a probe prepared by cloning hepatitis B virus DNA, purifiying the cloned hepatitis B virus DNA from the r-ecombinant plasmid and labelling the DNA to high specific activity, sealing the container, incubating the sealed container for between about 8 and 36 hours at a temperature between 65 and 68° C to permit the labelled hepatitis B virus DNA to combine with any hepatitis B virus DNA present in said serum, removing uncombined DNA from the presence of said substrate, and analyzing the substrate for the presence of said radioactive label using liquid scintillatinn spectroscopy or autoradiography.