Tag library compounds, compositions, kits and methods of use
Families of compositions are provided as labels, referred to as eTag reporters for attaching to polymeric compounds and assaying based on release of the eTag reporters from the polymeric compound and separation and detection. For oligonucleotides, the eTag reporters are synthesized at the end of the oligonucleotide by using phosphite or phosphate chemistry, whereby mass-modifying regions, charge-modifying regions and detectable regions are added sequentially to produce the eTag labeled reporters. By using small building blocks and varying their combination large numbers of different eTag reporters can be readily produced attached to a binding compound specific for the target compound of interest for identification. Protocols are used that release the eTag reporter when the target compound is present in the sample.
1. A method of performing a multiplexed assay for the determination of a plurality of target proteins in a sample, the method comprising the steps of:
providing a binding compound consisting of an antibody or antibody fragment thereof for each of the plurality of target proteins, each binding compound having one or more eTag reporters attached thereto by a cleavable linkage, the one or more eTag reporters of each different binding compound having a different electrophoretic mobility after cleavage so that eTag reporters of each different binding compound form distinct peaks after electrophoretic separation;
combining with the sample the binding compound and a second binding compound consisting of an antibody or antibody fragment thereof for each of the plurality of target proteins, wherein the second binding compound is conjugated with an active species producing moiety for generating an active species for oxidizing the cleavable linkage, such that in the presence of a target protein a complex is formed between each target protein and the binding compound(s) specific therefor;
cleaving each cleavable linkage by oxidation where said step includes providing the active species consisting of singlet oxygen for oxidizing the cleavable linkage thereby so that eTag reporters are released; and
electrophoretically separating and identifying the released eTag reporters to determine the plurality of target proteins in the sample.
2. The method of claim 1 further including a step prior to said step of cleaving, the step comprising separating said complexes from unbound binding compounds.
3. The method of claim 2 wherein each of said eTag reporters has a fluorescent label or an electrochemical label.
4. The method of claim 1 wherein said released eTag reporters have a charge opposite that of said complexes and said binding compounds, wherein said cleavable linkage is cleaved by oxidation, and wherein said step of cleaving includes providing an active species for oxidizing said cleavable linkage.
5. A method of performing a multiplexed assay for the determination of a plurality of target species in a sample, the method comprising the steps of:
providing a binding compound consisting of an antibody or antibody fragment thereof for each of a plurality of target species, the target species being a plurality of surface membrane proteins, each binding compound having one or more eTag reporters attached thereto by a cleavable linkage, the one or more eTag reporters of each different binding compound having a different mass/charge ratio or adsorption so that eTag reporters of each different binding compound form distinct peaks upon electrophoretic separation or chromatographic separation;
providing a second binding compound consisting of an antibody or antibody fragment thereof for each of the plurality of target species, each second binding compound being conjugated with an active species producing moiety for generating the active species for oxidizing the cleavable linkage;
combining with the sample, the binding compound and the second binding compound for each of the plurality of target species such that in the presence of a target species a complex is formed between target species and the binding compound and the second binding compound specific therefor, and such that the active species producing moiety of the second binding compound causes the generation of an active species consisting of singlet oxygen for oxidizing the cleavable linkage and cleaves the cleavable linkages to release one or more eTag reporters; and
electrophoretically or chromotographically separating and identifying the released eTag reporters to determine the plurality of target species in the sample.
6. The method of claim 5 , wherein each of said one or more eTag reporters of each said different binding compound has a different mass/charge ratio so that said eTag reporters of each of said different binding compound forms a distinct peak upon electrophoretic separation.