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Specific detection of clusterin isoforms

A clusterin-specific technology, applied in the field of specific detection of clusterin isotypes, can solve problems such as false positives

Active Publication Date: 2018-05-29
IDEXX LABORATORIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Failure to do so can lead to false positive test results in urine clusterin assays

Method used

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  • Specific detection of clusterin isoforms
  • Specific detection of clusterin isoforms
  • Specific detection of clusterin isoforms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] blood contamination

[0090] Normal canine serum was spiked into negative urine (ie, urine from healthy dogs) and the amount of clusterin was measured using a commercially available clusterin EIA (Biovendor). Such as Figure 1A As shown in -B, significant clusterin levels were measured even at 1:1000 dilution (1 μl per ml). Therefore, it is important to be able to detect kidney-specific clusterin isoforms while excluding any detection of serum or plasma clusterin isoforms.

Embodiment 2

[0091] Example 2: Materials

[0092] Isolation of clusterin molecules

[0093] The sequence of canine clusterin was used to design and synthesize a vector to express recombinant his-tagged canine clusterin molecule (Life Technologies). After expressing and purifying the protein, the sequence was confirmed by LC-MS. This molecule is referred to herein as recombinant clusterin or his-tagged recombinant clusterin.

[0094] Plasma Clusterin was purified by affinity chromatography from pooled plasma of 30 dogs. Madin-Darby canine kidney (MDCK) cell-derived clusterin (which is kidney-specific clusterin) was incubated in a 125 ml T-flask at 37°C, 7.5% CO 2 MDCK cells were grown to confluence in 1X MEM supplemented medium with antibiotics. The supernatant was collected and clusterin was affinity purified on an anti-clusterin column using an AKTA chromatography system (GE Healthcare).

[0095] Kidney-specific clusterin was purified by affinity chromatography from pooled urine of...

Embodiment 3

[0104] Example 3: General Clusterin Assay Protocol

[0105] Clusterin standard curves were prepared by serial dilution of 500 ng / mL standards in assay buffer (1x PBS containing 1% BSA and 0.5% Tween® (polysorbate) 20). Urine samples were diluted 1:100 in assay buffer and 100 μl were incubated in duplicate on the plate for 1 hour at ambient temperature. After washing 3 times with PetChek® buffer (IDEXX Laboratories), 100 μl of anti-clusterin antibody labeled with horseradish peroxidase was incubated for 30 minutes at ambient temperature. After washing 3 times as above, 50 μl of TMB substrate (IDEXX Laboratories) was added and the color was allowed to develop for 5 minutes. Color measurement reactions were terminated by adding 100 μl of acid (1N HCL). Plates were read immediately at 450 nm.

[0106] Clusterin-coated plates

[0107] Microplates were coated with 5 μg / ml plasma clusterin, MDCK-derived clusterin, recombinant His-tagged clusterin and BSA overnight at 4°C in 0.0...

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Abstract

The invention provides methods and compositions for the detection of specific isoforms of clusterin.

Description

[0001] priority [0002] This application claims the benefit of US Serial No. 62 / 155,175, filed April 30, 2015, which is hereby incorporated by reference in its entirety. Background of the invention [0003] Clusterin or apolipoprotein J is a disulfide-linked heterodimeric protein of 75-80 kDa. Clusterins are part of many physiological processes including sperm maturation, lipid transport, complement inhibition, tissue remodeling, membrane recycling, stabilization of stress proteins and promotion of apoptosis inhibition. Clusterin is overexpressed during renal proximal and distal duct damage, has been noted in various carcinomas, and is upregulated in renal injury. [0004] Several immunoassays have been developed and marketed for the measurement of clusterin in a variety of bodily fluids, including plasma, serum and urine. Kidney-specific clusterin can be used as a marker of kidney damage or disease. However, blood contamination of urine samples is a commonly observed even...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68G01N33/6893G01N2800/347G01N2333/42C07K16/18G01N33/523G01N33/533G01N33/534G01N33/535G01N33/54326G01N33/54386G01N2333/775
Inventor J.J.奎因M.V.S.N.耶拉米利
Owner IDEXX LABORATORIES
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