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Methods and reagents for the assessment of gestational diabetes

A technology for kits and subjects, applied in diseases, metabolic diseases, blood diseases, etc., can solve problems such as increased oxygen affinity and decreased responsiveness

Active Publication Date: 2016-09-07
哈佛大学董事会
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, amino-terminal glycation of the β-chain of hemoglobin produces glycated hemoglobin (HbA1c) with reduced responsiveness to 2,3-bisphosphoglycerate and increased oxygen affinity

Method used

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  • Methods and reagents for the assessment of gestational diabetes
  • Methods and reagents for the assessment of gestational diabetes
  • Methods and reagents for the assessment of gestational diabetes

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0249] Example 1. Reductive Synthesis of Surrogate Compound Synthesis

[0250] Reduced synthetic proteins used as surrogate compounds were synthesized according to the following method. Equimolar amounts of Ac-ACNFNDVTTRLRENELTYYCAAK-NH including a disulfide bond between cysteine ​​residues 2 and 20 (corresponding to residues 39 and 63 of mature CD59) 2 (SEQ ID NO:5) and also known as 24-N-Hydroxysuccinimidyl-75-N-(3-maleimidopropionyl)-amino-4,7 of 24-NHS ester (Quanta Biodesign, Powell, Ohio) ,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73-tetraoxa Pentane heptacetyl ester, molecular weight: 1394.55, in which the single compound is discrete poly-(ethylene glycol) spacers (82 atoms and ) was dissolved in dimethyl sulfoxide (DMSO). Triethylamine was added portionwise to bring the pH to 7.0, and the reaction mixture was stirred at room temperature for 1 hour.

[0251] Analytical high performance liquid chromatography (HPLC) monitoring indicated the...

example 2

[0252] Example 2. Synthesis of Alternative Compounds Containing Amadori

[0253] Amadori-containing synthetic protein standards including surrogate compounds were synthesized according to the following method. Equimolar amounts of Ac-ACNFNDVTTRLRENELTYYCAAK-NH comprising a disulfide bond between cysteine ​​residues 2 and 20 (corresponding to residues 39 and 63 in mature CD59) 2 (SEQ ID NO:5) and 24-NHS ester (molecular weight: 1394.55; single compound The spacer is 82 atoms and is 95.2A, Quanta Biodesign) dissolved in DMSO. Triethylamine was added portionwise to bring the pH to 7.0, and the reaction mixture was stirred at room temperature for 1 hour.

[0254] Analytical HPLC monitoring indicated completion of the reaction. One equivalent of Ac-NKAWKFEHANFNDC-OH (SEQ ID NO: 11), where K5 (corresponding to K41 of mature CD59) is glycated, including the Amadori product, was added to the above reaction mixture and stirring was continued for one hour. Analytical HPLC monit...

example 3

[0255] Example 3. GCD59 sandwich ELISA with samples pretreated with reducing agents

[0256] GCD59 sandwich enzyme-linked immunosorbent assay (ELISA) measures serum or plasma GCD59. Basic elements include anti-CD59 mouse monoclonal antibody 4466 (10A7) as capture antibody, anti-glucitol lysine rabbit monoclonal antibody as detection antibody, goat anti-rabbit IgG-H&I cross-adsorbed antibody, Horseradish peroxidase (HRP)-conjugated (Bethyl Laboratories, Montgomery, TX) and synthetic glycated CD59 surrogate compound used as a protein standard comprising two CD59 constructs connected by a linker area. Plates were coated with capture antibody. For this work, capture antibodies were diluted in 1X Dulbecco's phosphate buffered saline (DPBS; Lonsar, Basel, Switzerland) to a final concentration of 3 μg / ml. Wells of Immulon 4HBX plates (Thermo Fisher, Waltham, MA) were coated with 100 μl of 3 μg / ml capture antibody solution. The plates were then incubated overnight at 4°C with sh...

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Abstract

The invention involves assays, diagnostics, kits, and assay components for determining levels of glycated CD59 in the assessment of gestational diabetes mellitus and / or related disorders and / or conditions. Some kits comprise a capture antibody capable of associating with a capture epitope on CD59, wherein said capture epitope may lack lysine residue number 41 (K41 ). Kits may also comprise a detection antibody capable of associating with a detection epitope on CD59. Such detection epitopes may comprise glycated K41.

Description

[0001] governmental support [0002] This invention was made with government support under Grant No. DK095429-01 awarded by the National Institutes of Health, entitled "Glycated CD59 as a novel biomarker for gestational diabetes." The US Government may have certain rights in this invention. Background of the invention [0003] Diabetes is characterized by elevated blood sugar levels. Sustained increases in blood sugar levels can affect protein through a process called glycation. Glycation is the non-enzymatic attachment of glucose to proteins and is believed to be the primary pathophysiological mechanism responsible for tissue damage in diabetic subjects. Glycation involves the reaction of glucose and / or other reducing sugars with amino groups in proteins, resulting in the formation of Schiff bases or aldimines. This unstable adduct can undergo tautomerization through the Amadori rearrangement to generate more stable ketoamines. [0004] Different glycated proteins includi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/566G01N2333/70596G01N2800/042G01N2400/02G01N2440/38G01N2800/368G01N33/689A61P15/00A61P15/08A61P7/00A61P3/10G01N33/577G01N33/581G01N33/6854G01N33/6893
Inventor 迈克尔·乔雷夫约瑟·阿尔伯特·哈普林
Owner 哈佛大学董事会
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