Liposome-enhanced immunoaggregation assay and test device

• US 5,753,519 A
• Filed: 06/06/1995
• Issued: 05/19/1998
• Est. Priority Date: 10/12/1993
• Status: Expired due to Term

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Roberts, Matthew A., Durst, Richard Allen
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): NGUYEN, BAO THUY L
• Application Number:
  US08/467,004
• Time in Patent Office: 1,078 Days
• Field of Search: 422/56, 422/57, 422/70, 422/82.01, 422/82.03, 422/98, 422/99, 422/110, 427/213.3, 427/213.34, 427/213.35, 435/317.1, 435/7.93, 435/287.7, 435/970, 436/516, 436/518, 436/530, 436/541, 436/514, 204/182.7, 204/286, 204/288, 204/290 R, 204/297 R
• US Class Current: 436/518
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 33/586 Liposomes, microcapsules or cells
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds

Abstract

A test device for detecting or quantifying an analyte in a test sample includes an absorbent material having separate contact and measurement portions. The contact portion is positioned at or proximate to a first end of the absorbent material. The measurement portion has a receptor for a conjugate of an analyte analog and marker-encapsulating liposomes. In a method for using the test device, a binding material specific for the analyte is combined with the liposome-analyte analog conjugate and the test sample to form a test mixture. The mixture is incubated for a time sufficient to permit competition between any analyte present and the conjugate for the binding material. Following incubating, the mixture is allowed to traverse the absorbent material from the contact portion through the measurement portion of the absorbent material. Following traversal by the test mixture, the presence or amount of marker in the measurement portion of the absorbent material is then detected and correlated with the presence or amount, respectively, of the analyte in the sample. Also disclosed are liposomes encapsulating an electroactive marker used in conjunction with a test device as described, but which includes an electrochemical measurement portion in place of the measurement portion described above. Test devices and methods employing electrochemical detection or quantification of an electroactive marker corresponding to the amount of analyte in a sample may be either amperometric or potentiometric.

Claims

1. A method for detecting or quantifying an analyte in a test sample, comprising:

providing a test device comprising an absorbent material, which absorbent material comprises;

a contact portion at or proximate to a first end of said absorbent material; and

an electrochemical measurement portion at a location on said absorbent material which is positioned away from the first end;

said electrochemical measurement portion comprising a working electrode portion, a reference electrode portion, and a counter electrode portion, wherein each of said electrode portions is segregated from one another on said absorbent material, said working, reference, and counter electrode portions are electrically connected with one another, and wherein either said absorbent material further comprises a liposome lysing portion positioned between said contact portion and said working electrode portion, wherein said liposome lysing portion is segregated from said contact portion and has a liposome lysing agent bound thereto, or said working electrode portion has a liposome lysing agent bound thereto;

combining a binding material specific for the analyte with a conjugate of an analyte analog and liposomes and the test sample in an electrolyte mixture, wherein said liposomes comprise an electroactive marker;

incubating the mixture for a time sufficient to permit competition between any analyte present in the test sample and the conjugate for the binding material;

contacting the mixture with said contact portion of said absorbent material after said incubating;

allowing the mixture to migrate from said contact portion through said electrochemical measurement portion of said absorbent material after said incubating, wherein migration of aggregates of conjugate and binding material formed during said incubating is inhibited by said absorbent material, whereby said liposomes are lysed by said liposome lysing agent to release said marker, and an electrical connection between said working, reference and counter electrode portions is established causing current to flow between said working and said counter electrode portions;

detecting the presence or amount of said current flowing between said working and said counter electrode portions; and

correlating the presence or amount of said current flowing between said working and said counter electrode portions with the presence or amount, respectively, of the analyte in the test sample.

2. A method according to claim 1, wherein said working electrode portion has a marker accumulating agent bound thereto and said allowing the mixture to migrate comprises accumulating said electroactive marker in said working electrode portion.

3. A method according to claim 1, wherein said reference electrode portion is positioned between said working electrode portion and said counter electrode portion on said absorbent material, and wherein said working electrode portion is positioned between said contact portion and said reference electrode portion on said absorbent material.

4. A test device for detecting or quantifying an analyte in a test sample, said test device comprising:

an absorbent material, comprising;

a contact portion at or proximate to a first end of said absorbent material; and

an electrochemical measurement portion at a location on said absorbent material which is positioned away from the first end;

said electrochemical measurement portion comprising a working electrode portion, a reference electrode portion, and a counter electrode portion, wherein each of said electrode portions is segregated from one another on said absorbent material, said working, reference, and counter electrode portions are electrically connected with one another, and wherein either said absorbent material further comprises a liposome lysing portion positioned between said contact portion and said working electrode portion, wherein said liposome lysing portion is segregated from said contact portion and has a liposome lysing agent bound thereto, or said working electrode portion has a liposome lysing agent bound thereto.

5. A test device according to claim 4, said working electrode portion further having a marker accumulating agent bound thereto.

6. A test device according to claim 4, wherein said absorbent material has been treated with one or more blocking agents, surfactants, or mixtures thereof.

7. A test device according to claim 6, wherein said blocking agents block nonspecific binding sites on said absorbent material.

8. A test device according to claim 6, wherein said blocking agents are selected from the group consisting of proteinaceous blocking reagents which inhibit binding of molecules having a molecular weight of greater than about 1000 with said absorbent material and polymer blocking reagents which inhibit binding of molecules having a molecular weight of less than about 1000 with said absorbent material.

9. A test device according to claim 8, wherein said proteinaceous blocking reagent is selected from the group consisting of gelatin, non-fat dry milk, bovine serum albumin, and keyhold limpet hemocyanin, said polymer blocking reagent is selected from the group consisting of polyvinylpyrrolidone and polyvinylalcohol, and said surfactant is selected from the group consisting of polyoxyethylene ethers, polyoxyethylenesorbitan monolaurate, t-octylphenoxypolyethoxyethanol, sodium dodecylsulfate octylglucopyranoside, and sodium dioxycholate.

10. A test device according to claim 4, wherein said reference electrode portion is positioned between said working electrode portion and said counter electrode portion on said absorbent material, and said working electrode portion is positioned between said contact portion and said reference electrode portion on said absorbent material.