Homogeneous specific binding assay element and lyophilization production method

• US 4,461,829 A
• Filed: 09/14/1981
• Issued: 07/24/1984
• Est. Priority Date: 09/14/1981
• Status: Expired due to Fees

• Current Assignee: Miles Laboratories, Inc. (Bayer AG)
• Original Assignee: Miles Laboratories, Inc. (Bayer AG)
• Inventors: Greenquist, Alfred C.
• Primary Examiner(s): Marantz, Sidney
• Application Number:
  US06/302,218
• Time in Patent Office: 1,044 Days
• Field of Search: 435/4, 435/7, 435/177, 435/188, 435/805, 435/810, 23/230 B, 422/55-58, 424/8, 424/12, 252/408, 436/530, 436/810
• US Class Current: 435/7.72
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

A homogeneous specific binding assay element, a method for its preparation, and a method for its use in determining a ligand in or the ligand binding capacity of a liquid sample are disclosed. The test element comprises a solid carrier incorporated with reagents for a homogeneous specific binding assay system which produces a detectable response, usually an electromagnetic radiation signal, that is a function of the presence or amount of the ligand in or the ligand binding capacity of the sample. There is further disclosed a method for preparing a homogeneous specific binding assay element for determining a ligand in or the ligand binding capacity of a liquid sample by incorporating a carrier with a composition which includes a label conjugate, comprising a label component coupled to a ligand moiety or a specific binding analog thereof, and a reagent reactive with the label conjugate, which method comprises (a) incorporating the carrier with the reagent reactive with the label conjugate in a first liquid; (b) subjecting the carrier of (a) to conditions effective to reversibly suspend or reduce the activity of the reagent therein; (c) incorporating the carrier of (b) with the label conjugate; (d) subjecting the carrier of (c) to a temperature effective to freeze the reagent reactive with the label conjugate and the label conjugate; and (e) lyophilizing the reagent and label conjugate in the carrier of (d).

Claims

1. A test device for determining a ligand in, or the ligand binding capacity of, a test sample, the device comprising

a support member having a substantially planar side,

a carrier member affixed to the substantially planar side, the carrier member comprising a substantially bibulous material, and

a reagent system homogeneously incorporated with, but not bound to, the carrier member, the reagent system comprising ingredients capable of participating in a specific binding assay and including a label conjugate and a specific binding partner to the conjugate and the ligand, the conjugate comprising a label bound to the ligand or a specific binding analogue thereof, the conjugate and specific binding partner therefor being present in the carrier substantially unbound to one another.

2. The test device of claim 1 in which the label conjugate is a fluor bound to the ligand or specific binding analogue thereof.

3. The test device of claim 1 in which the label conjugate is unbelliferone-ligand.

4. The test device of claim 1 in which the reagent system additionally includes a component capable of cleaving the label from the conjugate.

5. The test device of claim 4 in which the label is an enzyme substrate and the compound is an enzyme reactive with the substrate.

6. The test device of claim 5 in which the label is a β

-galactoside moiety and the enzyme is β

-galactosidase.

7. The test device of claim 6 in which the label conjugate is β

-galactosyl-umbelliferone-ligand or specific binding analogue thereof.

8. A method for preparing a test device for determining a ligand in, or the ligand binding capacity of, a test sample, the method comprising the sequential steps of

incorporating a carrier member with a reagent system in a first solvent, the reagent system being capable of producing a detectable response to the presence of a label conjugate, and including a specific binding partner to the conjugate and a component capable of cleaving the label from the conjugate to produce a detectable response,

drying the carrier member, to remove the first solvent

cooling the dried carrier member to a temperature sufficient to substantially prevent the component from cleaving the label,

further incorporating the carrier member with the label conjugate in a second solvent, the conjugate comprising the label coupled to the ligand or specific binding analogue thereof, the label being a moiety cleavable by the component to produce a detectable response,

cooling the carrier member to a temperature sufficient to freeze the second solvent at a rate sufficient to prevent substantial cleavage of the label by the component, and lyophilizing the carrier member.

9. The method of claim 8 in which the label is a substrate for an enzyme, and the component is the enzyme for which the label is a substrate.

10. The method of claim 8 in which the ligand is theophylline, the label conjugate is theophylline-umbelliferone-galactose, the component is β

-galactosidase and the specific binding partner is antibody to theophylline.

11. The method of claim 8 in which the label is a β

-galactoside moiety and the component is β

-galactosidase.

12. The method of claim 11 in which the label conjugate is β

-galactosyl-umbelliferone-ligand or ligand binding analogue.