WO1994017409B1 - Quantifying trace analytes by affinity capillary electrophoresis - Google Patents

Quantifying trace analytes by affinity capillary electrophoresis

Info

Publication number
WO1994017409B1
WO1994017409B1 PCT/US1994/000426 US9400426W WO9417409B1 WO 1994017409 B1 WO1994017409 B1 WO 1994017409B1 US 9400426 W US9400426 W US 9400426W WO 9417409 B1 WO9417409 B1 WO 9417409B1
Authority
WO
WIPO (PCT)
Prior art keywords
analyte
labelled
fragment
fab
agent
Prior art date
Application number
PCT/US1994/000426
Other languages
French (fr)
Other versions
WO1994017409A1 (en
Filing date
Publication date
Priority claimed from US08/006,922 external-priority patent/US5348633A/en
Application filed filed Critical
Priority to EP94908588A priority Critical patent/EP0680608A1/en
Priority to AU61625/94A priority patent/AU6162594A/en
Priority to JP51708594A priority patent/JP3530914B2/en
Publication of WO1994017409A1 publication Critical patent/WO1994017409A1/en
Publication of WO1994017409B1 publication Critical patent/WO1994017409B1/en

Links

Abstract

A method for quantitative detection of trace amounts of an analyte in a sample is disclosed. The method in the preferred embodiment includes providing an Fab' fragment of an immunoglobulin labelled at a reactive sulfhydryl group with a fluorescent dye; combining the labelled Fab' fragment with a sample that may contain the analyte; concentrating the elements of the resulting mixture in an electric field; separating the labelled analyte/agent complex formed from any unreacted labelled agent using capillary electrophoretic methods; and detecting the fluorescent signal of the labelled analyte/agent complex. The invention also is directed to a method of producing a labelled Fab' fragment that includes cleaving an immunoglobulin molecule to obtain one F(ab')2 fragment; reducing the disulfide-bonds of the F(ab')2 fragment to obtain two Fab' fragments each having at least one free, reactive sulfhydryl group; and mixing an Fab' fragment having at least one free sulfhydryl group with a fluorescent dye reactive with the free sulfhydryl to form a labelled Fab' fragment. Preferably, prior to the final mixing step, intrastrand disulfide bonds are formed by oxidation within each Fab' fragment, thereby producing individual Fab' fragments each having a single reactive sulfhydryl group. The method of quantitative detection also more broadly includes using any biospecific agent to form a complex with the target analyte.

Claims

/17409 P
AMENDED CLAIMS
I received by the International Bureau on 5 August 1994 (05.08.94); original claim 21 amended; other claims unchanged (1 page)]
20. The method of claim 18, wherein said detectable reporter group is not sensitive to pH change in the pH range of operation.
21. A method for quantitatively detecting trace amounts of an analyte in a sample comprising the steps of: mixing together a sample which may contain said analyte and a biospecific agent that can bind to said analyte to form an analyte-agent complex, said analyte-agent complex containing a detectable reporter group; concentrating elements of said mixture in an electric field by a capillary electrophoretic concentration technique; separating elements of said mixture by a capillary electrophoretic separation technique; and quantitatively detecting said reporter group on said analyte-agent complex as an indication of the amount of said analyte in said sample.
22. The method of claim 21, wherein said biospecific agent is labelled with said detectable reporter group.
23. The method of claim 21 further comprising in said mixing step, mixing a known amount of labelled said analyte, said labelled analyte containing said detectable reporter group.
24. The method of claim 21, wherein said biospecific agent is selected from the group consisting of antibody fragment, binding protein, and ligand.
25. The method of claim 21, wherein said reporter group is selected from the group consisting of fluorophore, UV or visible chromophore, radioisotope, spin label, electrochemical reporter, chemiluminescent reporter, and enzyme.
PCT/US1994/000426 1993-01-22 1994-01-12 Quantifying trace analytes by affinity capillary electrophoresis WO1994017409A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP94908588A EP0680608A1 (en) 1993-01-22 1994-01-12 Quantifying trace analytes by affinity capillary electrophoresis
AU61625/94A AU6162594A (en) 1993-01-22 1994-01-12 Quantifying trace analytes by affinity capillary electrophoresis
JP51708594A JP3530914B2 (en) 1993-01-22 1994-01-12 Determination of trace analytes by affinity capillary electrophoresis.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US08/006,922 US5348633A (en) 1993-01-22 1993-01-22 Method for quantitating trace amounts of an analyte in a sample by affinity capillary electrophoresis
US006,922 1993-01-22

Publications (2)

Publication Number Publication Date
WO1994017409A1 WO1994017409A1 (en) 1994-08-04
WO1994017409B1 true WO1994017409B1 (en) 1994-09-15

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/000426 WO1994017409A1 (en) 1993-01-22 1994-01-12 Quantifying trace analytes by affinity capillary electrophoresis

Country Status (5)

Country Link
US (1) US5348633A (en)
EP (1) EP0680608A1 (en)
JP (1) JP3530914B2 (en)
AU (1) AU6162594A (en)
WO (1) WO1994017409A1 (en)

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