Enzyme-immunoassay method for the determination of an analyte

• US 5,296,356 A
• Filed: 12/12/1990
• Issued: 03/22/1994
• Est. Priority Date: 12/13/1989
• Status: Expired due to Fees

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Traeger, Ulrich, Mangold, Dieter, Lange, Hans
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Spiegel, Carol A.
• Application Number:
  US07/626,257
• Time in Patent Office: 1,196 Days
• Field of Search: 422/56-60, 422/102, 435/7.9-7.95, 435/805, 435/810, 435/969, 435/970, 436/518, 436/523, 436/527-531, 436/538, 436/541, 436/810
• US Class Current: 435/7.92
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

The invention concerns a method for the detection of an analyte in a sample liquid by an enzyme-immunoassay in which an enzyme-labelled compound is partitioned between a solid and a liquid phase and the amount of enzyme label in the liquid phase outside the solid phase is determined as a measure of the concentration of the analyte. The measurement is carried out in a non-porous molding having the liquid phase contained therein in contact with the solid phase. The method is particularly suitable for carrying out in a cuvette which can be filled via the porous matrix or on a test strip having a space in contact with the solid phase.

Claims

1. Method for determining an analyte in a liquid sample by an enzyme immunoassay comprising:

(a) providing a transparent nonporous vessel comprising therein a porous solid phase having immobilized thereon a specific binding partner selected from the group consisting of a first specific binding partner which specifically binds to the analyte, the analyte, and an immunologically reactive analogue thereof;

(b) providing an enzyme-labelled immunologically active compound comprising an enzyme label conjugated to a compound selected from the group consisting of the analyte, the immunologically reactive analogue thereof and a second specific binding partner which specifically binds to the analyte or the immunologically reactive analogue thereof as either

(1) a separate liquid reagent or

(2) as part of the porous solid phase, wherein the enzyme-labelled immunologically active compound is specifically bound to the specific binding partner on the porous solid phase;

(c) contacting the liquid sample with both the enzyme-labelled immunologically active compound and the immobilized specific binding partner such that the liquid sample and the enzyme-labelled immunologically active compound form a first liquid phase wherein no more liquid volume is contacted than can be maximally absorbed by the porous solid phase and

for a time sufficient for the enzyme-labelled immunologically active compound to partition between the porous solid phase and the first liquid phase;

(d) contacting the porous solid phase of step (c) with a liquid chromogenic substrate specific for the enzyme label in a volume sufficient to displace the first liquid phase and to form a second liquid phase above the porous solid phase in the transparent nonporous vessel; and

(e) measuring the amount of chromogen produced in the second liquid phase in order to determine the amount of the analyte in the liquid sample, wherein the measurement is carried out in the transparent nonporous vessel of step (d) without prior removal of the porous solid phase.

2. The method of claim 1 wherein the contacting step (c) comprises directly contacting the liquid sample with the enzyme-labelled immunologically active compound specifically bound to the specific binding partner on the porous solid phase to form the first liquid phase thereon.

3. The method of claim 1 wherein the porous solid phase is a fleece or a fabric matrix.

4. The method of claims 1 or 2 wherein the porous solid phase is a particulate matrix.

5. The method of claims 1 or 2 wherein the transparent nonporous vessel is a cuvette and the porous solid phase is attached to the bottom of the cuvette.

6. The method of claims 1 or 4 wherein the measuring step is performed using transmission photometry.

7. The method of claim 1 wherein the enzyme-labelled immunologically active compound of step (b) is provided as a separate liquid reagent.

8. The method of claim 7 wherein the transparent nonporous vessel is a cuvette.

9. The method of claim 1 wherein the porous solid phase is a fleece matrix.

10. The method of claim 1 wherein the analyte is albumin, T₄ or TSH.

11. The method of claim 1 wherein the contacting step (c) comprises first contacting the liquid sample with the enzyme-labelled immunologically active compound provided as a separate liquid reagent to form a first liquid phase, and contacting the first liquid phase with the porous solid phase.

12. Method for determining an analyte in a liquid sample by an enzyme immunoassay comprising:

(a) providing a test strip comprising

1) a bottom foil,

2) a porous solid phase carrier having immobilized thereon the analyte or an immunologically reactive analogue thereof or a specific binding partner which specifically binds to the analyte or the immunologically reactive analogue thereof, said carrier being attached to a portion of the bottom foil,

3) an upper foil covering said bottom foil and said porous solid phase carrier, wherein a portion of said bottom foil and of said upper covering foil which is adjacent to the porous solid phase carrier form a non-porous space and wherein said upper covering foil has at least one opening for applying fluid to said porous solid phase carrier and at least one opening over the non-porous space;

(b) providing an enzyme-labelled immunologically active compound comprising an enzyme label conjugated to a compound selected from the group consisting of the analyte, the immunologically reactive analogue thereof, and a second specific binding partner which specifically binds to the analyte and the immunologically reactive analogue thereof as either

(1) a separate liquid reagent or

(2) as part of the porous solid phase carrier, wherein the enzyme-labelled immunologically active compound is specifically bound to the specific binding partner on the carrier;

c) contacting the liquid sample with both the enzyme-labelled immunologically active compound and the immobilized specific binding partner on the carrier such that the liquid sample and the enzyme-labelled immunologically active compound form a first liquid phase wherein no more liquid volume is contacted with the carrier than can be maximally absorbed by the carrier;

d) contacting a liquid chromogenic substrate for the enzyme label to the porous solid phase carrier in sufficient volume to displace unbound enzyme-labelled immunologically active compound from the carrier into the non-porous space of the test strip; and

e) measuring the amount of chromogen produced in the non-porous space in order to determine the amount of the analyte in the liquid sample.

13. The method of claim 12 wherein the measuring step is performed by transmission photometry and wherein at least one of the bottom base foil and the upper covering foil are transparent.

14. The method of claim 12 wherein the measuring step is performed by transmission photometry and both the bottom base foil and the upper covering foil are transparent.