Nucleic acid hybridization assay employing antibodies to intercalation complexes

A solid-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:

• (a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, one of the probe and the single stranded nucleic acids from the test medium being immobilized on a solid support, such contact being performed under conditions favorable to hybridization between the sequence to be detected and the complementary probe sequence,
• (b) contacting the solid support carrying resulting immobilized duplexes with a nucleic acid intercalator and an antibody, or fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator,
• (c) separating the solid support carrying resulting immobilized antibody or fragment thereof from the remainder of the reaction mixture, and
• (d) determining the separated antibody or fragment thereof on the solid support as an indication of the presence of the sequence to be detected in the liquid test medium.

A solid-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:

• (a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe, the probe comprising at least one single stranded base sequence substantially complementary to the sequence to be detected and the probe being chemically linked to a nucleic acid intercalator in the single stranded complementary region of the probe such that duplex formation in such region bearing the linked intercalator results in the formation of intercalation complexes, one of the probe and the single stranded nucleic acids from the test medium being immobilized on a solid support, such contact being performed under conditions flavorable to hybridization between the sequence to be detected and the complementary probe sequence,
• (b) adding to the reaction mixture an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator,
• (c) separating from the remainder of the reaction mixture, the solid support carrying resulting immobilized antibody or fragment thereof bound to intercalation complexes formed between the intercalator-linked probe and the sequence to be detected, and
• (d) determining the separated antibody or fragment thereof on the solid support as an indication of the presence of the sequence to be detected in the liquid test medium.

A solution-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:

• (a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, the probe comprising a binding site for a binding substance, such contact being performed under conditions favorable to hybridization between the sequence to be detected and the complementary probe sequence,
• (b) adding to the reaction mixture simultaneously or in separate steps (i) a nucleic acid intercalator, (ii) an antibody, or fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, and (iii) an immobilized form of a binding substance for the probe,
• (c) separating the resulting immobilized phase comprising antibody, or fragment thereof, bound to immobilized intercalation complexes from the remainder of the reaction mixture, and
• (d) determining the separated immobilized antibody, or fragment thereof, as an indication of the presence of the sequence to be detected in the liquid test medium.