WO1994017409B1 - Quantifying trace analytes by affinity capillary electrophoresis - Google Patents
Quantifying trace analytes by affinity capillary electrophoresisInfo
- 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
Links
- 238000001386 capillary affinity electrophoresis Methods 0.000 title 1
- 239000012491 analyte Substances 0.000 claims abstract 11
- 239000003795 chemical substances by application Substances 0.000 claims abstract 10
- 210000001736 Capillaries Anatomy 0.000 claims abstract 3
- 238000001962 electrophoresis Methods 0.000 claims abstract 3
- 239000000203 mixture Substances 0.000 claims abstract 3
- 230000005684 electric field Effects 0.000 claims abstract 2
- 230000001809 detectable Effects 0.000 claims 4
- 238000000034 method Methods 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 102000004965 antibodies Human genes 0.000 claims 1
- 108090001123 antibodies Proteins 0.000 claims 1
- 230000027455 binding Effects 0.000 claims 1
- 102000024070 binding proteins Human genes 0.000 claims 1
- 108091007650 binding proteins Proteins 0.000 claims 1
- 239000003446 ligand Substances 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 230000035693 Fab Effects 0.000 abstract 8
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract 5
- 102000018358 Immunoglobulins Human genes 0.000 abstract 2
- 108060003951 Immunoglobulins Proteins 0.000 abstract 2
- 238000001514 detection method Methods 0.000 abstract 2
- 239000007850 fluorescent dye Substances 0.000 abstract 2
- 230000003647 oxidation Effects 0.000 abstract 1
- 238000007254 oxidation reaction Methods 0.000 abstract 1
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.
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 |
|---|---|---|---|
| US006,922 | 1993-01-22 | ||
| 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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1994017409A1 WO1994017409A1 (en) | 1994-08-04 |
| WO1994017409B1 true WO1994017409B1 (en) | 1994-09-15 |
Family
ID=21723287
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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-
1993
- 1993-01-22 US US08/006,922 patent/US5348633A/en not_active Expired - Lifetime
-
1994
- 1994-01-12 EP EP94908588A patent/EP0680608A1/en not_active Withdrawn
- 1994-01-12 JP JP51708594A patent/JP3530914B2/en not_active Expired - Lifetime
- 1994-01-12 WO PCT/US1994/000426 patent/WO1994017409A1/en not_active Application Discontinuation
- 1994-01-12 AU AU61625/94A patent/AU6162594A/en not_active Abandoned
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