IMMUNO-AMPLIFICATION

摘要

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.

主权项

1. A method of detecting an analyte, comprising:
(i) combining:
(a) an analyte;(b) a first proximity member, comprising a first analyte-specific binding entity that is capable of forming a complex with the analyte and that is conjugated to a tether oligonucleotide moiety comprising a first portion and a second portion;(c) a second proximity member, comprising a second analyte-specific binding entity that is capable of forming a complex with the analyte and that is conjugated to an oligonucleotide moiety comprising a first portion;(d) a splint oligonucleotide, comprising (i) a first portion that is capable of hybridizing to the first portion of the oligonucleotide moiety and (ii) a second portion that is capable of hybridizing to the first portion of the tether oligonucleotide moiety; (ii) forming:
(a) a first hybrid comprising the first portion of the oligonucleotide moiety, the first portion of the splint oligonucleotide, and an extendable terminus of either the oligonucleotide moiety or the splint oligonucleotide; and(b) a second hybrid comprising the first portion of the tether oligonucleotide and the second portion of the tether oligonucleotide; (iii) extending the extendable terminus, thereby producing an amplicon; (iv) amplifying the amplicon to produce an amplification product; and (v) detecting the amplification product; wherein detection of the amplification product allows detection of the analyte.