Recombinant reduced valency carbohydrate binding ligands

• US 6,844,166 B1
• Filed: 09/10/1999
• Issued: 01/18/2005
• Est. Priority Date: 09/11/1998
• Status: Expired due to Fees

• Current Assignee: Sensory Technologies LLC (DSI Video Systems LLC)
• Original Assignee: Sensory Technologies LLC (DSI Video Systems LLC)
• Inventors: Wolf, David E.
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): GABEL, GAILENE
• Application Number:
  US09/394,265
• Time in Patent Office: 1,957 Days
• Field of Search: 436/14, 436/164, 436/147, 436/172, 436/95, 436/501, 436/531, 436/545, 436/546, 436/805, 436/807, 436/815, 436/827, 435/7.2, 435/7.92, 435/7.93, 435/14, 435/7, 435/7.1, 424/9.6, 424/9.8
• US Class Current: 435/7.93
• CPC Class Codes: A61K 49/0041 Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal in vivo G01N
• CPC Class Codes: A61K 49/0043 Fluorescein, used in vivo
• CPC Class Codes: G01N 2333/42 Lectins, e.g. concanavalin, phytohaemagglutinin
• CPC Class Codes: G01N 2333/4724 Lectins
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/827 Lectins
• CPC Class Codes: Y10T 436/104998 Glucose, ketone, nitrate standard or control
• CPC Class Codes: Y10T 436/144444 Glucose

Abstract

The invention is based on the discovery of reduced valency carbohydrate binding ligands (CBLs) that can be used to to detect or quantitate (i.e., evaluate) carbohydrates in a sample. CBLs can be used with fluorescence resonance energy transfer (FRET) to evaluate free carbohydrates or those within a carbohydrate containing compound.

Claims

1. A method of evaluating a carbohydrate in a sample comprising:

(a) contacting the sample with a specific binding pair that comprises;

(i) a reduced valency carbohydrate binding ligand (CBL) and

(ii) a glycoconjugate comprising a carbohydrate moiety, wherein the reduced valency CBL and glycoconjugate reversibly bind to each other; and

(b) determining the extent to which the carbohydrate present in the sample displaces glycoconjugate bound to the reduced valency CBL and reversibly binds to the reduced valency CBL, the extent to which the carbohydrate binds to the reduced valency CBL being correlated with the amount of carbohydrate in the sample.

2. The method of claim 1, wherein the reduced valency CBL is a monomeric form of a multimeric protein.

3. The method of claim 1, wherein the reduced valency CBL is a lectin.

4. The method of claim 3, wherein the lectin is Concanavalin A.

5. The method of claim 4, wherein the Concanavalin A is mutagenized at residues that participate in dimer-dimer interactions to produce dimers which do not assemble into tetramers.

6. The method of claim 1, wherein at least one of the reduced valency CBL and the glycoconjugate include a detectable label.

7. The method of claim 6, wherein the label is a radioactive label, a fluorescent label, an enzyme, a proximity-based signal generating label moiety, a homogeneous time resolved fluorescence (HTRF) component, or a luminescent oxygen channeling assay (LOCI) component.

8. The method of claim 1, wherein the sample is a sample of urine, blood, plasma, saliva, intracellular fluid, interstitial fluid, homogenized cells, or a cell extract.

9. The method of claim 1, wherein the carbohydrate is a monosaccharide, a disaccharide, or a polysaccharide.

10. The method of claim 1, wherein the carbohydrate is glucose.

11. The method of claim 1, wherein the carbohydrate is a component of a glycoprotein.

12. The method of claim 1, wherein the glycoconjugate comprises serum albumin.