Fluorescent energy transfer ligand interaction assay on a lipid film

• US 6,235,535 B1
• Filed: 12/24/1998
• Issued: 05/22/2001
• Est. Priority Date: 06/28/1996
• Status: Expired due to Fees

• Current Assignee: Valtion Teknillinen Tutkimuskeskus
• Original Assignee: Valtion Teknillinen Tutkimuskeskus
• Inventors: Söderlund, Hans, Laukkanen, Marja-Leena, Keinänen, Kari
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): Padmanabhan, Kartic
• Application Number:
  US09/202,976
• Time in Patent Office: 880 Days
• Field of Search: 250/486.1, 250/483.1, 250/458.1, 250/459.1, 250/461.1, 422/60, 422/82.11, 422/82.07, 422/82.08, 422/82.05, 422/57, 422/58, 435/174, 435/968, 435/808, 435/291, 435/7.3, 435/7.33, 435/7.34, 435/7.35, 435/7.37, 435/7.36, 435/7.32, 435/7.2, 435/7.9, 435/7.92, 435/4, 435/7, 435/7.1, 435/5, 435/6, 435/320.1, 435/175, 435/7.94, 436/525, 436/537, 436/531, 436/805, 436/807, 436/20, 436/544, 436/530, 436/829, 436/526, 436/528, 436/534, 436/543, 436/501, 436/164, 436/172, 436/518, 436/800, 424/450, 548/159, 548/201, 549/220, 549/223, 549/288, 544/151, 544/326
• US Class Current: 436/172
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/819 Multifunctional antigen or antibody
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation

Abstract

This invention relates to a fluorescence-based immunoassay method for the detection of an analyte, or for the measurement of its concentration in a biological sample. The method is based on the ability of a multivalent analyte to induce aggregation of receptor molecules labeled with a fluorophore, which molecules are anchored to and are freely mobile on a lipid membrane, and thereby cause changes in the fluorescence.

Claims

1. A fluorescence-based immunoassay method for the detection of an analyte or for the determination of its concentration in a sample, comprising:

attaching receptor molecules specific for said analyte, one part of the molecules being labeled with a first fluorophore acting as a donor and another part with a second fluorophore acting as an acceptor, to a lipid membrane on a plane of which they are able to move freely;

bringing the sample possibly containing said analyte into contact with said receptor molecules; and

measuring the fluorescence change caused by a change of an aggregation level of the receptor molecules on the membrane as a consequence of the attachment of the analyte.

2. The method according to claim 1, wherein the analyte is an antigen and the receptor is an antibody.

3. The method according to claim 2, further comprising adding to the antigen-containing sample a known amount of molecules having two or more separate antibody-binding antigen structures when the analyte is a monovalent antigen.

4. The method according to claim 1, wherein the analyte is an antibody and the receptor is an antigen.

5. The method according to claim 1, wherein the lipid membrane is in the form of a liposome.

6. The method according to claim 1, wherein the lipid membrane is in the form of a planar membrane.

7. The method according to claim 1, wherein the receptor molecules are attached to the lipid membrane via lipid molecules, the receptor molecules are attached to the lipid molecules chemically or by genetic engineering.

8. The method according to claim 1, wherein fluorescein or its derivative is used as a fluorescence donor and rhodamine or its derivative as an acceptor.

9. The method according to claim 2, wherein the antigen to be assayed contains at least two epitopes.

10. The method according to claim 9, wherein said at least two epitopes are different.

11. The method according to claim 9, wherein said at least two epitopes are identical.