Capillary electrophoresis enzyme immunoassay

• US 5,958,202 A
• Filed: 01/22/1997
• Issued: 09/28/1999
• Est. Priority Date: 09/14/1992
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perseptive Biosystems Incorporated (Ecovyst, Inc.)
• Inventors: Yao, Xian-Wei, Koutny, Lance, Schmalzing, Dieter, Nashabeh, Wassim, Regnier, Fred E., Fuchs, Martin, Taylor, Todd A.
• Primary Examiner(s): Warden, Robert
• Assistant Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US08/792,933
• Time in Patent Office: 979 Days
• Field of Search: 204/451, 204/455, 204/452, 204/461, 204/601, 204/605, 204/603, 204/612, 204/606, 435/4, 435/7.7, 435/6, 435/7.91, 435/7.92, 435/7.9, 436/515, 436/516
• US Class Current: 204/451
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: G01N 33/537 with separation of immune complex from unbound antigen or antibody
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors

Abstract

Disclosed are methods and apparatus for performing highly sensitive enzyme-amplified assays using channel electrophoresis of an enzyme having a biorecognition moiety. Combining a binding reaction of an analyte and an enzyme having a biorecognition moiety, and an electrophoretic separation which uses enzyme-amplified detection methodology, assay methods and apparatus are provided that offer simplicity, quantitative sensitivity and rapid analysis. Also disclosed are highly sensitive catalytically-amplified assays using a capillary electrophoresis format in which the binding reaction prior to electrophoresis is heterogeneous or homogeneous, and direct or competitive. The methods and apparatus for conducting sensitive automated catalytically-amplified assays using electrophoresis may be conducted in a microscale format.

Claims

1. A method for performing an assay comprising:

(a) introducing a first reactant and a sample containing an analyte into a channel containing an electrophoresis medium,

wherein the analyte comprises or is complexed to a competitor, said competitor comprising a biorecognition portion and a second reactant portion;

(b) applying an electric potential along the length of the channel to cause said first reactant and said second reactant portion of said competitor to form or deplete a detectable product;

(c) imposing along the length of said channel an electric potential sufficient to cause said detectable product to be separated from said first reactant by differential migration along said channel; and

(d) detecting said detectable product.

2. The method of claim 1 comprising the additional step of:

(e) quantitatively determining the amount of said detectable product formed or depleted in said channel.

3. The method of claim 1 wherein one of said first reactant or said second reactant portion comprises an enzyme and the other of said first reactant or said second reactant portion comprises an enzyme substrate.

4. The method of claim 1 comprising the additional steps before step (a) of:

(i) contacting an immobilized reactant and an analyte in a solution;

(ii) adding to said solution said competitor; and

(iii) using an aliquot of said solution, said aliquot defining said sample.

5. The method of claim 4 wherein said immobilized reactant is an immobilized antibody.

6. The method of claim 1 wherein said sample is the result of an immunological reaction.

7. The method of claim 6 wherein said immunological reaction is a competitive reaction.

8. The method of claim 1 wherein the analyte is charged.

9. The method of claim 1 wherein the analyte is complexed to the competitor which defines an analyte-competitor complex, and the analyte-competitor complex comprises a charge modifier.

10. The method of claim 1 wherein said biorecognition portion is selected from the group consisting of antibodies, antigens, enzymes, peptides, peptide nucleic acids, oligonucleotides, deoxyribonucleic acids, ribonucleic acids, biotins and their complementary binding complexes, lectins and their complementary carbohydrate binding complexes, and cellular receptor binding proteins and their complementary binding products.

11. The method of claim 1 wherein said channel used in step (a) contains said first reactant prior to the introduction of said sample.

12. The method of claim 1 including the additional step of:

subjecting said channel to zero potential by removing said electric potential after contact between said competitor and said first reactant for a time sufficient to allow said detectable product to form or to deplete.

13. The method of claim 12 further comprising, after said step of subjecting said channel to zero potential, and, the step of:

re-imposing along the length of said channel said electric potential for a time sufficient to allow said detectable product to migrate in said channel.

14. The method of claim 1 wherein said sample comprises one or more competitors comprising different biorecognition portions.

15. The method of claim 14 wherein one or more first reactants are introduced into said channel.

16. The method of claim 15 wherein plural detectable products are formed or depleted.

17. A method for performing an assay, said method comprising:

(a) conducting a heterogeneous competitive reaction in a vessel among an immobilized reactant, an analyte, and a competitor, wherein the competitor comprises a biorecognition portion and a second reactant portion;

(b) introducing an aliquot of the competitor which is unbound in step (a) into a channel containing an electrophoresis medium and a first reactant;

(c) imposing an electrical potential along the length of the channel to cause, the second reactant portion of the competitor and the first reactant to form or deplete a detectable product within the channel; and

(d) detecting the detectable product.

18. The method of claim 17 comprising the additional step of:

(e) quantitatively determining the amount of said detectable product formed or depleted in said channel.

19. The method of claim 17 wherein one of said first reactant or said second reactant portion comprises an enzyme and the other of said first reactant or said second reactant portion comprises an enzyme substrate.

20. The method of claim 17 wherein said biorecognition portion is selected from the group consisting of antibodies, antigens, enzymes, peptides, peptide nucleic acids, oligonucleotides, deoxyribonucleic acids, ribonucleic acids, biotins and their complementary binding complexes, lectins and their complementary carbohydrate binding complexes, and cellular receptor binding proteins and their complementary binding products.

21. The method of claim 17 wherein said immobilized reactant is an immobilized antibody.

22. The method of claim 17 wherein said channel used in step (b) contains said first reactant prior to the introduction of said aliquot.

23. The method of claim 17 including the additional step of:

subjecting said channel to zero potential by removing said electric potential after contact between said competitor and said first reactant for a time sufficient to allow said detectable product to form or to deplete.

24. The method of claim 23 further comprising, after said step of subjecting said channel to zero potential, and the step of:

re-imposing along the length of said channel said electric potential for a time sufficient to allow said detectable product to migrate in said channel.

25. An analytical device comprising a capillary zone electrophoresis channel, said channel comprising:

a free solution electrophoretic medium,

a first reactant in said electrophoretic medium that will react in said channel with a second reactant portion of a competitor to form or deplete a detectable product wherein the competitor further comprises a biorecognition portion; and

said analytical device further comprising electrical power means for applying a field across the ends of said channel to cause said first reactant and said second reactant portion of said competitor to migrate and react.

26. The device of claim 25 further comprising a detector for detecting said detectable product.

27. The device of claim 26 further comprising a quantitator for quantitatively determining the amount of said detectable product detected.

28. The device of claim 25 wherein said channel is a capillary.

29. The device of claim 25 wherein said competitor and said first reactant are spaced apart within said channel such that, upon the application of an electric field along the length of said channel by said electrophoresis apparatus, said competitor or said first reactant migrates in said channel to permit said first reactant to react with said second reactant portion of said competitor to form or deplete said detectable product.