Kits employing generalized target-binding e-tag probes

• US 20010051340A1
• Filed: 04/02/2001
• Published: 12/13/2001
• Est. Priority Date: 04/30/1999
• Status: Active Grant

• Current Assignee: Virologic, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Virologic, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Chenna, Ahmed, Matray, Tracy
• Granted Patent: US 7,001,725 B2
• Time in Patent Office: Days
• US Class Current: 435/6.16
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus

Abstract

Kits for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. Detection involves the release of identifying tags as a consequence of target recognition. The kits include sets of electrophoretic tag probes or e-tag probes, a capture agent and optionally a cleaving agent. The e-tag probes comprise a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. In using the kits, target antiligands are contacted with a set of e-tag probes and the contacted antiligands are treated with a selected cleaving agent resulting in a mixture of e-tag reporters and uncleaved and/or partially cleaved e-tag probes. The mixture is exposed to a capture agent effective to bind to uncleaved or partially cleaved e-tag probes, followed by electrophoretic separation. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification.

Claims

1. A kit for detecting for detecting the binding of or interaction between each or any of a plurality of known, selected ligands and one or more target antiligands, said kit comprising:

(a) a set of electrophoretic tag (e-tag) probes, the set comprising j members, and each of said e-tag probes having the form;

(D, M_j)—

L—

T_j,

where

(i) D is a detection group comprising a detectable label;

(ii) T_j is a ligand capable of binding to or interacting with a target antiligand,

(iii) L is a linking group connected to T_j by a bond that is cleavable by a selected cleaving agent when the probe is bound to or interacting with the target antiligand, wherein cleavage by said agent produces an e-tag reporter of the form (D, M_j)—

L′

, where L′

is the residue of L attached to (D, M_j) after such cleavage,

(iv) M_j is a mobility modifier having a charge/mass ratio that imparts a unique and known electrophoretic mobility to a corresponding e-tag reporter of the form (D, M_j)—

L′

, within a selected range of electrophoretic mobilities with respect to other e-tag reporters of the same form in the probe set; and

(v) (D, M_j)—

includes both D—

M_j— and

M_j—

D—

;

 

wherein the uncleaved or partially cleaved e-tag probes, but not the corresponding e-tag reporter, is reactive with capture agent effective to impart a mobility to the probes bound to the capture agent that prevents the probes from electrophoretically migrating within said range of electrophoretic mobilities; and

(b) a capture agent effective to bind to uncleaved or partially-cleaved probes, said uncleaved and/or partially cleaved probes produced by;

(i) reacting the antiligand(s) with the probe set under conditions that allow the probes to bind to or interact with the target antiligand(s), and

(ii) treating the reacted target sequences with the cleaving agent under conditions effective to cleave target-bound probes at the L—

T_j linkage, thereby producing a mixture of one or more corresponding e-tag reporters of the form (D, M_j)—

L′

, and uncleaved and/or partially cleaved e-tag probes, said capture agent being effective to

(i) impart a mobility to the probes bound to the capture agent that prevents the probes from electrophoretically migrating within said range of electrophoretic mobilities or

(ii) immobilize the probes on a solid support.

2. The kit of

claim 1

, wherein T_j is biotinylated and the capture agent is avidin or streptavidin.

3. The kit of

claim 1

, wherein T_j contains an antigen and the capture agent is an antibody or antibody fragment that binds specifically to the antigen.

4. The kit of

claim 1

, wherein T_j contains a particle or mass group that effectively prevents its migration under electrophoretic conditions within the range of electrophoretic mobilities of the e-tag reporters.