WO1994009185B1

WO1994009185B1 - Electrophoretic quantitation of specific binding complexes

Info

Publication number: WO1994009185B1
Application number: PCT/US1993/009701
Authority: WO
Filing date: 1993-10-07
Publication date: 1994-06-09
Anticipated expiration: 1995-04-14

Abstract

Quantification of analytes is achieved by gel electrophoresis, where a plurality of samples may be analyzed simultaneously. By combining an analyte with its labeled complementary binding member and electrophoretically separating the complex from the unbound labeled complementary binding member, one can calculate the amount of analyte in the sample. A plurality of spaced apart samples may be placed in a single lane, so as to greatly expand the utility of a single gel plate.

Claims

[received by the International Bureau on 9 May 1994 (09.05.94); original claims 1 and 8 amended; remaining claims unchanged (2 pages)]

1. A method for quantitating an analyte in a sample by gel electrophoresis without a calibration step, said method comprising: combining said sample with a predetermined amount of a labeled complementary binding member to provide soluble complexes of said analyte and said complementary binding member in a reaction medium; applying said reaction medium to a gel and electrophoresing said reaction medium to separate said complementary binding member from said soluble complex into separate bands in said gel; and determining the amount of said labeled conjugate in at least one of said bands as a measure of the amount of analyte in said sample.

2. A method according to Claim 1, wherein said complementary binding member is an antibody and said analyte is an antigen.

3. A method according to Claim 1, wherein said labeled complementary member is a fluorescent labeled complementary member.

4. A method according to Claim 3, wherein said fluorescent labeled complementary member is a fluorescein labeled complementary member.

5. A method according to Claim 1, wherein said sample is placed at a plurality of sites in a single lane, wherein said sites are at least about 25mm apart .

6. A method according to Claim 1, wherein said gel is an agarose gel.

7. A method according to Claim 1, wherein said gel has a plurality of lanes and said sample is placed at a plurality of sites in each lane, each of εaid sites being at least about 25mm apart.

8. A method for quantitating an antigenic analyte in a sample by gel electrophoresis without a calibration step, said method comprising: combining said sample with a predetermined amount of a fluorescent labeled antibody complementary to said analyte to provide soluble complexes of said analyte and said fluorescent labeled antibody in a reaction medium; applying said reaction medium to an agarose gel and electrophoresing said reaction medium to separate said antibody from said soluble complex into separate bands in said gel; and determining the amount of said labeled antibody in at least one of said bands as a measure of the amount of analyte in said sample.

9. A method according to Claim 8, wherein said sample is applied to said gel at a plurality of sites in a lane, said sites being at least about 25mm apart.

10. A method according to Claim 8, wherein said antibody is a monoclonal antibody.

11. A method according to Claim 8, including the additional step of applying a control sample at a site on said gel, where said control sample has a known amount of analyte.

STATEMENT UNDER ARTICLE 19

As now claimed, the subject method is novel and involves an inventive step over the reference Manian et al.. U.S. Pat. No. 5.137.609. which is cited in the International Search Report. The subject method is patentabie over Manian et al. because the method disclosed by Manian et al. requires a calibration step in order to detect and/or quantitate the amount of analyte in a sample.

In the method disclosed by Manian et al. , a calibration step is required because the various labeled bands in the gel are detected individually as they pass over a stationary metering bar during electrophoresis. In the calibration step, a quantity of unbound label is run on the gel and the time at which the label passes over the metering bar is recorded. Col. 9, lines 54-56. The calibration procedure is used to establish expected times at which both unbound labeled binding member and complex pass over the stationary metering position during the assay. Col. 2, lines 63-65. Only after these times are established can Manian use the disclosed method to detect/and or quantitate analyte in the sample.

In contrast to the method disclosed by Manian, a calibration step is not required to establish expected times in the claimed method. The claimed method measures the intensity of each band during electrophoresis by scanning the gel. The presence of two bands in the gel is representative of the presence of analyte in the sample. The intensity of one of the bands can then be used to calculate the quantity of analyte in the sample. Importantly, no calibration step is required to establish when the various bands will pass over a stationary metering position. Since no calibration step is required, the subject method is novel and comprises an inventive step over the method disclosed by Manian et al.