Method of measuring available free thyroxine bending sites

• US 5,244,786 A
• Filed: 08/13/1990
• Issued: 09/14/1993
• Est. Priority Date: 10/02/1987
• Status: Expired due to Fees

• Current Assignee: Roche Diagnostics Corporation (Roche Holding AG)
• Original Assignee: Microgenics Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Friedman, Stephen B., Picone, Teresa K.
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US07/568,644
• Time in Patent Office: 1,128 Days
• Field of Search: 435/18, 435/7.9, 435/7.7, 435/7.6, 435/975, 436/500, 436/501, 436/537, 436/517, 436/808
• US Class Current: 435/7.9
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors

Abstract

The present invention provides an improved assay for measuring thyroxine uptake by thyroxine binding globulin in which thyroxine binding globulin (TBG) activity is measured directly. The method comprises combining a sample in an aqueous solution with an enzyme donor (ED) conjugated to an analogue of polyiodothyronine that competes with thyroxine for thyroxine binding globulin binding sites. An enzyme acceptor (EA) characterized by providing a modulated enzyme activity in relation to the amount of TBG activity is combined with an enzyme donor and sample in an aqueous solution. The amount of enzyme activity in comparison to a control solution having a known amount of available TBG binding sites is determined.

Claims

1. A method for directly measuring available thyroxin binding sites in a sample, comprising:

(a) combining in an assay medium (1) a β

-galactosidase substrate, (2) said sample, (3) a β

-galactosidase acceptor fragment having substantially the same amino acid sequence as a C-terminus region of β

-galactosidase, and (4) a β

-galactosidase enzyme donor conjugate comprising a β

-galactosidase enzyme donor fragment, wherein said donor fragment has substantially the same amino acid sequence as an N-terminus region of β

-galactosidase complementary to said C-terminus region, linked to a moiety that competes with thyroxin for free thyroxin binding sites, with the proviso that said β

-galactosidase donor conjugate and said β

-galactosidase acceptor fragment are not combined in the absence of said sample, and wherein said donor and acceptor fragments form an active β

-galactosidase enzyme upon complexation, said complexation being modulated if said enzyme donor conjugate binds to a protein having a thyroxin binding site; and

(b) determining the amount of β

-galactosidase activity in said assay medium as compared to an assay medium containing a known amount of available thyroxin binding sites.

2. A method according to claim 1, wherein said moiety is a polyiodothyronine.

3. A method according to claim 1, wherein said sample is serum or plasma.