Reduction of migration shift assay interference

Patent metadata

Field	Value
Publication number	US-9766216-B2
Application number	US-82165704-A
Country	US
Kind code	B2
Filing date	Apr 8, 2004
Priority date	Apr 14, 2003
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

Abstract

This invention provides methods and compositions, e.g., to reduce interference from non-specific binding sample constituents in a migration shift assay. Interference due to non-specific binding of sample constituents to an affinity substance (e.g., an affinity molecule or a conjugate of an affinity molecule and a charged carrier molecule) is prevented by, e.g., binding the constituents to charged polymers such as heparin sulfate.
The present invention also provides methods to concentrate an analyte of interest with high concentration and to detect the analyte with high sensitivity, and further to optimize the reaction conditions for easily concentrating the analyte. Such objects of the present invention are attained, for example, by concentrating a complex of the analyte and a conjugate which is formed by contacting the analyte in a sample with an affinity molecule bound to a charged carrier molecule such as DNA.

First claim

What is claimed is:

1. A method of detecting an analyte of interest in a sample, comprising:
(i) contacting
(a) a first polyanion;
(b) the sample containing the analyte, wherein the sample is a serum, a plasma, a whole blood, a sputum specimen, a stool specimen, a cerebral spinal fluid, a urine sample, a uro-genital swab, a synovial fluid, or a lymph fluid sample; and
(c) one or more affinity molecule/charged carrier molecule conjugates to form a complex of the analyte and the one or more conjugates,

wherein each affinity molecule has an affinity against the analyte, each charged carrier molecule has a net negative charge, and the charged carrier molecule causes a change in a separation property of the analyte by binding to the analyte through the affinity molecule to form a complex of the analyte and the affinity molecule/charged carrier molecule conjugate;
(ii) using a concentration medium containing a second polyanion, concentrating the complex in a concentration channel in a microfluidic device, the concentration channel having at least one microscale dimension of between about 0.1 and about 500 microns;
(iii) using a separation medium containing a third polyanion, electrophoretically separating the complex from any unbound conjugate in a separation channel in a microfluidic device, the separation channel having at least one microscale dimension of between about 0.1 and about 500 microns; and
(iv) detecting the complex to identify the presence of the analyte or to determine an amount of the analyte in the sample,

wherein the first, second, and third polyanions reduce binding of the sample's constituents to the affinity molecule, the charged carrier molecule, or the complex and reduce interference with separating the complex.

2. The method of claim 1 , wherein the first, second, and third polyanions are independently selected from one or more of polysaccharides, polynucleotides, polypeptides, synthetic macromolecular compounds, or ceramics; or a mixture thereof.

3. The method of claim 1 , wherein the first, second, and third polyanions are independently selected from one or more of poly-dIdC, heparin sulfate, dextran sulfate, polytungstic acid, polyanethole sulfonic acid, polyvinyl sulfate, polyacrylate, chondroitin sulfate, plasmid DNA, calf thymus DNA, salmon sperm DNA, DNA coupled to cellulose, glass particles, colloidal glass, or glass milk, or a mixture thereof.

4. The method of claim 1 , wherein the first, second and/or third polyanion comprises heparin sulfate.

5. The method of claim 1 , wherein at least one of the one or more affinity molecules is labeled with a detectable marker.

6. The method of claim 1 , wherein the contacting step further comprises contacting the sample with one or more non-conjugated affinity molecules, wherein each non-conjugated affinity molecule has an affinity against the analyte, to form a complex of the analyte, the at least one conjugate, and the at least one non-conjugated affinity molecule.

7. The method of claim 1 , wherein the conjugated affinity molecules bind to the analyte by an interaction selected from a protein-protein interaction, a protein-chemical interaction, or a chemical-chemical interaction.

8. The method of claim 6 , wherein the conjugated and the non-conjugated affinity molecules bind to the analyte by an interaction selected from an antigen-antibody interaction, a sugar chain-lectin interaction, an enzyme-inhibitor interaction, a protein-peptide chain interaction, a chromosome or nucleotide chain-nucleotide chain interaction, a nucleotide-ligand interaction, or a receptor-ligand interaction.

9. The method of claim 6 , wherein the conjugated and the non-conjugated affinity molecule are selected from one or more of an antibody, an Fab, F(ab′) 2 or Fab′ fragment of an antibody, an antibody variable region, a lectin, avidin, a receptor, an affinity peptide, an aptamer, or a DNA binding protein.

10. The method of claim 1 , wherein the charged carrier molecule is an anionic molecule.

11. The method of claim 10 , wherein the charged carrier molecule is an anionic molecule selected from a nucleotide chain or a sulfonated polypeptide.

12. The method of claim 1 , wherein the charged carrier molecule comprises DNA, RNA, an anionic polymer, or a sulfonated polypeptide.

13. The method of claim 12 , wherein the charged carrier molecule comprises DNA comprising one or more synthetic sequences.

14. The method of claim 13 , wherein the one or more synthetic sequences comprise one or more nucleotide analogs comprising a linker group or a linker reactive group.

15. The method of claim 14 , wherein the linker group or linker reactive group is selected from an amino group, a thiol, a carboxyl group, an imidazol group, or a succinimide group.

16. The method of claim 15 , further comprising covalently bonding a detectable marker to the linker group or linker reactive group.

17. The method of claim 1 , wherein at least one of the one or more charged carrier molecules is labeled with a detectable marker.

18. The method of claim 6 , wherein at least one conjugate or at least one non-conjugated affinity molecule is labeled with a detectable marker.

19. The method of claim 6 , wherein at least one conjugate is labeled by a detectable marker.

20. The method of claim 6 , wherein the charged carrier molecule in at least one conjugate is labeled by a detectable marker.

21. The method of claim 6 , wherein the affinity molecule in at least one conjugate is labeled by a detectable marker.

22. The method of claim 5 , 16 , 17 , 18 , 19 , 20 or 21 , wherein the detectable marker is selected from one or more of a fluorescent dye, a luminescent dye, a phosphorescent dye, a fluorescent protein, a luminescent protein or particle, a radioactive tracer, a chemiluminescent compound, a redox mediator, an electrogenic compound, an enzyme, a colloidal gold particle, or a silver particle.

23. The method of claim 1 , wherein the separation media comprises one or more of a size exclusion resin, a polyacrylamide gel, polyethylene glycol (PEG), polyethyleneoxide (PEO), a co-polymer of sucrose and epichlorohydrin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), poly-N,N-dimethylacrylamide (PDMA), or an agarose gel.

24. The method of claim 1 , wherein the third polyanion is present in the separation media at a concentration of between about 0.01 to 5% (w/v).

25. The method of claim 1 , wherein the third polyanion is present in the separation media at a concentration of between about 0.05 to 2% (w/v).

26. The method of claim 1 , wherein the separation channel has at least one cross-sectional microscale dimension of between about 0.1 and 200 microns.

27. The method of claim 1 , wherein:
at least one of the one or more conjugates is labeled by a detectable marker;
step (iii) comprises electrophoretically separating the complex from the at least one conjugate labeled by the detectable marker that is not involved in forming the complex; and
step (iv) comprises:
(a) measuring an amount of the separated complex or detecting a presence of the separated complex; and
(b) determining an amount of the analyte in the sample on the basis of the measured amount or identifying a presence of the analyte in the sample on the basis of the detected presence.

28. The method of claim 6 , wherein:
either at least one of the non-conjugated af

Assignees

• Wada H Garrett
• Kazakova Irina G
• Miki Yutaka
• Ohashi Toshinari
• Kanke Futoshi
• Wako Pure Chem Ind Ltd

Inventors

• Wada H Garrett
• Kazakova Irina G
• Miki Yutaka
• Ohashi Toshinari
• Kanke Futoshi

Classifications

• G01N30/8644 Primary

  Data segmentation, e.g. time windows · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title

• G01N33/538

  by sorbent column, particles or resin strip {, i.e. sorbent materials} · CPC title

• G01N30/34

  of fluid composition, e.g. gradient (G01N30/36 takes precedence) · CPC title

• B01L2400/0487

  fluid pressure, pneumatics · CPC title