Immuno-amplification
A high-sensitivity, low-background immuno-amplification assay is provided, which offers a streamlined workflow suitable for high-throughput assays of clinically relevant samples, such as blood and other bodily fluids. The assay comprises the use of two proximity members that each comprise an analyte-specific binding component conjugated to an oligonucleotide. Binding an analyte brings the oligonucleotide moieties of the proximity members in sufficiently close contact that the oligonucleotides form an amplicon. The presence of the analyte then is detected through amplification of the amplicon and detection of the amplified nucleic acids. The sensitivity of the assay of the present invention is improved by preventing spurious or non-specific amplicon formation by proximity members that are not complexed with an analyte.
1. A kit for detecting an analyte in a sample, comprising:
(a) a first proximity member, comprising a first antigen that is conjugated to a single stranded first oligonucleotide comprising, from 3′ to 5′, a first portion and a second portion, wherein the first antigen is capable of forming a complex with an antigen-binding site of an analyte comprising at least two antigen-specific binding sites; and
(b) a second proximity member, comprising a second antigen, different from the first antigen, that is conjugated to a single stranded second oligonucleotide comprising, from 5′ to 3′, a first portion and a second portion, where the second antigen is capable of forming a complex with another antigen-binding site of the analyte, and wherein upon complexation of the first antigen and second antigen with the analyte, both the first oligonucleotide and the second oligonucleotide do not directly bind to the analyte and the first portion of the first oligonucleotide hybridizes to the second portion of the second oligonucleotide, leaving the first portion of the second oligonucleotide unbound to either the another antigen-binding site of the analyte or the first oligonucleotide and forming an oligonucleotide bridge from an analyte binding site of the second antigen to the second portion of the second oligonucleotide such that only the 3′ terminus of the second portion of the second oligonucleotide is capable of being extended via a polymerase to form a complement of the second portion of the first oligonucleotide.
2. A kit for detecting an analyte in a sample, comprising:
(a) a first proximity member, comprising a first peptide that is conjugated to a single stranded first oligonucleotide comprising, from 3′ to 5′, a first portion and a second portion, wherein the first peptide is capable of forming a complex with a peptide binding site of an analyte comprising at least two peptide specific binding sites; and
(b) a second proximity member, comprising a second peptide, different from the first peptide, that is conjugated to a single stranded second oligonucleotide comprising, from 5′ to 3′, a first portion and a second portion, where the second peptide is capable of forming a complex with another-peptide binding site of the analyte, and wherein upon complexation of the first peptide and second peptide with the analyte, the first portion of the first oligonucleotide hybridizes to the second portion of the second oligonucleotide, such that only the 3′ terminus of the second portion of the second oligonucleotide is capable of being extended via a polymerase to form a complement of the second portion of the first oligonucleotide.