Galectin-3 Immunoassay

Inactive Publication Date: 2010-06-10
BG MEDICINE
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Benefits of technology

[0009]Methods for the detection of galectin-3 in clinical samples now have been identified and developed. It has now been discovered that the use of two binding moieties that bind specifically to at least two separate, non-overlapping epitopes on the N-terminus of galectin-3 can be used to produce various specific sandwich assay formats that can provide the

Problems solved by technology

Heart failure (HF) is a major public health problem in the United States.
Insufficient pumping leads to the congestion of blood and other fluid in the liver, abdomen, lower extremities, and lungs.
HF results in a gradual deterioration of the patient often leading to cardiovascular mortality.
Thus, a large number of patients die within one to five years after diagnosis.
Symptoms of HF include fatigue, weakness, rapid or irregular heartbeat, shortness of breath, persistent cough or wheezing, swelling of lower extremities or abdom

Method used

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Examples

Experimental program
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Example

Example 1

Enzyme-Linked Immunosorbent Assay for Quantitative Detection of Human Galectin-3

[0071]The human galectin-3 ELISA is an enzyme-linked immunosorbent assay for the quantitative detection of human galectin-3 in EDTA plasma. Human galectin-3 present in the sample or standard bound to antibodies adsorbed to the microwells. Following incubation unbound material was removed during a wash step. HRP conjugated to anti-human galectin-3 antibody was added and bound to the galectin-3 captured by the coating antibody. Following incubation unbound HRP-conjugate was removed during a wash step, and substrate solution reactive with HRP was added to the wells. A colored product was formed in proportion to the amount of human galectin-3 present in the sample or standard. The reaction was terminated by addition of acid and absorbance was measured at 450 nm. A standard curve was prepared from 7 human galectin-3 standard dilutions and human galectin-3 sample concentration determined. Plasma was r...

Example

Example 2A

A Kit for Detecting Galectin-3

[0102]Table 7 shows the components of an exemplary kit for the detection of galectin-3.

TABLE 7Galectin-3 assay reagentsQtyNameDescriptionAbbreviation1 platePlateReady-to-use microtiter plate coated with(P)anti-galectin-3 monoclonal antibody(M3 / 38)1 bottleAssay Diluent*Phosphate buffered saline with 1% bovine(AD)serum albumin (45 mL)1 bottleTMB substrateTetramethyl benzidine (15 mL)(TS)1 bottleStop solution0.5M sulfuric acid (10 mL)(ST)2 bottlesWash buffer0.5M Tris buffered saline (2 × 50 mL; 10×(WC)concentrate*concentrate)1 bottleDetectionHorseradish peroxidase (HRP) labeled(DC)concentrate*mouse anti-human galectin-3 antibody87B5 (0.4 mL)2 vialsStandardRecombinant human galectin-3, 12 ng per(S1)vial (lyophilized)2 vialsLow Quality ControlLow QC material, Recombinant human(C1)(QC)\galectin-3 in protein matrix (lyophilized)2 vialsHigh Quality ControlHigh QC material, Recombinant human(C2)(QC)\galectin-3 in protein matrix (lyophilized)2Plate seal...

Example

Example 2B

Detection of Recombinant Galectin-3 Controls

[0105]The kit of Example 2A was used in a microtiter plate-based ELISA assay to quantitate galectin-3 levels. Included in the kit were two monoclonal antibodies against galectin-3. In the assay, described in greater detail in the following paragraph, standards and quality control materials were introduced into the wells and incubated for 60 minutes. During this incubation, the galectin-3 present in the standards was bound to the capture antibody coated onto the well surface. A subsequent wash step removed all unbound material introduced with the sample including unbound galectin-3. The detection antibody was then introduced into the well and incubated for 60 minutes. During this time, an antibody-antigen-antibody complex was formed. After a wash step to remove any unbound detection antibody, the Tetramethyl benzidine (TMB) substrate was added, yielding a blue color in the presence of HRP. The color development was stopped after 2...

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Abstract

The present invention relates to methods and compositions for specifically and quantitatively detecting galectin-3 in a sample. Embodiments of the invention include a detection assay in which a capture binding moiety and a labeled binding moiety specifically recognize non-overlapping epitopes on the N-terminus of galectin-3. Further embodiments are directed to a method for establishing ranges of galectin-3 concentrations indicative of the presence and severity of heart failure in a subject and a method for predicting the clinical outcome of a subject based upon galectin-3 concentration.

Description

REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of and priority to U.S. Provisional Patent Application No. 61 / 109,366, filed Oct. 29, 2008, the complete disclosure of which is incorporated by reference herein.BACKGROUND OF THE INVENTION[0002]Heart failure (HF) is a major public health problem in the United States. Approximately 5 million people suffer from the disease and the number of patients is steadily increasing. HF is a common but severe and complex clinical syndrome, especially among elderly people. HF refers to a condition in which the heart fails to pump enough blood to meet the body's needs. Insufficient pumping leads to the congestion of blood and other fluid in the liver, abdomen, lower extremities, and lungs. Thus, HF has also been called congestive heart failure (CHF), although the term HF is preferred because not all patients exhibit fluid congestion. HF results in a gradual deterioration of the patient often leading to cardiovascular mortal...

Claims

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Application Information

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IPC IPC(8): G01N33/53
CPCG01N33/6893G01N2800/325G01N2333/4724G01N33/577G01N33/68C07K16/2851G01N33/53C07K16/18
Inventor MUNTENDAM, PIETER
Owner BG MEDICINE
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