Solid-phase hybridization assay using anti-hybrid antibodies

Claim 1

A method for determining a particular polynucleotide sequence present in a virus or cell contained in a test sample, wherein nucleic acids from the virus or cell are contacted with a polynucleotide probe comprising at least one single stranded base sequence which is substantially complementary to the base sequence to be determined, such contact being performed under conditions favorable to hybridization between the sequence to be determined and the complementary probe sequence, and wherein resulting hybrids are detected, characterized in that the test sample is treated to release a significant detectable amount of nucleic acids from the virus or cell in single stranded form and to immobilize such sample nucleic acids on a solid support, the polynucleotide probe being (i) substantially composed of RNA when the sequence to be determined is DNA, or (ii) substantially composed of DNA or RNA when the sequence to be determined is RNA, and the hybridized probe is detected by adding an antibody reagent capable of binding to DNA'RNA or RNA'RNA duplexes formed between the sequence to be determined and the complementary probe sequence and determining the antibody reagent that becomes bound to such duplexes.

Claim 2

A method for determining a particular polynucleotide sequence present in a virus or cell contained in a test sample, wherein nucleic acids from the virus or cell are contacted with a polynucleotide probe comprising at least one single stranded base sequence which is substantially complementary to the base sequence to be determined, such contact being performed under conditions favorable to hybridization between the sequence to be determined and the complementary probe sequence, and wherein resulting hybrids are detected, characterized in that the test sample is treated to release a significant detectable amount of nucleic acids from the virus or cell in single stranded form and to immobilize such sample nucleic acids on a solid support, the polynucleotide probe being (i) substantially composed of RNA when the sequence to be determined is DNA, or (ii) substantially composed of DNA or RNA when the sequence to be determined is RNA, and the hybridized probe is detected by adding an antibody reagent capable of binding to DNA'RNA or RNA'RNA duplexes formed between the sequence to be determined and the complementary probe sequence and determining the antibody reagent that becomes bound to such duplexes.

Claim 4

The method of any of Claims 1 and 2 wherein the virus or cell is immobilized on the solid substrate and then treated to release and denature a significant detectable amount of nucleic acids therefrom, the resulting single stranded nucleic acids becoming immobilized on such substrate.

Claim 5

The method of any of Claims 1 to 3 wherein the test sample is a biological fluid and the substrate is a filter through which the biological fluid sample is passed to immobilize such virus or cell.

Claim 6

The method of any of Claims 1 to 4 wherein the single stranded sample nucleic acids are immobilized on the solid substrate by adsorption.

Claim 8

The method of any of Chains 1 to 6 wherein the antibody reagent is labeled with a detectable chemical group, preferably an enzymatically active group such as an enzyme, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.

Claim 9

The method of any of Chains 1 to 6 wherein the antibody reagent is labeled with a detectable chemical group, preferably an enzymatically active group such as an enzyme, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.

Claim 11

The method of any of Claims 1 to 8 wherein the particular polynucleotide sequence to be determined is RNA or DNA and said probe is substantially composed of RNA.