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Method for the detection of apolipoprotein e4

An apolipoprotein and binding agent technology, which can be used in measurement devices, instruments, disease diagnosis, etc., and can solve problems such as inability to measure signals

Active Publication Date: 2019-06-14
SPHINGOTEC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the estimated amount of ApoE4 in plasma / serum is only 0.02-0.5% of total plasma / serum proteins, and one would not expect to measure a signal specific for ApoE4

Method used

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  • Method for the detection of apolipoprotein e4
  • Method for the detection of apolipoprotein e4
  • Method for the detection of apolipoprotein e4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0251] The production of antibodies and the determination of their affinity constants

[0252] -Peptides / conjugates used for immunization:

[0253] A specific ApoE4 peptide and ApoE2 / 3 specific control peptide for immunization were synthesized, see Table 1 (JPT Technologies, Berlin, Germany). The ApoE4 peptide was synthesized with an additional N-terminal cysteine ​​residue for coupling the peptide to bovine serum albumin (BSA). The peptide was covalently linked to BSA using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to Perbio's manual.

[0254] Table 1:

[0255] Peptides

ApoE area

description

Affinity constant [M -1 ]

Cys-EDVRGRLVQYR

109-119 ApoE4

Immunogen

2.1x 109

EDVCGRLVQYR

109-119 ApoE2 / 3

Control peptide

-

[0256] -Immunization, immune cell fusion and screening of mice:

[0257] Balb / c mice were treated with 100 μg peptide-BSA conjugate (emulsified in 100 μl Freund's complete adjuvant) on days 0 and 14 a...

Embodiment 2

[0267] Measurement of plasma samples with known genotypes. Using plasma and whole blood matching, one can genetically analyze the ApoE status through genotyping, and use the ApoE4 assay of the present invention to measure the ApoE4 level in the matched plasma.

[0268] 2.1 Method

[0269] -Genotyping

[0270] The genetic analysis from 180 whole blood samples was performed by Medigenomix (Ebersberg, Deutschland). Isolate DNA from the sample and use Sanger sequencing technology ( Sanger&Coulson 1975 ) Sequencing the ApoE gene.

[0271] -ApoE4 assay procedure

[0272] The ApoE4 single antibody assay is performed in a one-step procedure. The plasma samples were pipetted directly into the wells of a 96-well microtiter plate (Greiner, Lumitrac 600 high binding polystyrene; 20 μl sample per well). Add ApoE4 cut-off lower limit control (recombinant ApoE4 [4μg / ml] in ApoE4-negative human plasma, 20μl per well, in duplicate) to each microplate. Add 200μl of reaction buffer (PBS, containing ...

Embodiment 3

[0276] Analysis of the binding of ApoE4 to different polymers and glass surfaces with and without detergents.

[0277] 3.1 Method

[0278] -Pre-warming bath of plasma sample on polymer or glass surface:

[0279] Divide each ApoE4 positive plasma sample into 120μl aliquots and place them in tubes of different materials (polystyrene (PS), polypropylene (PP), polyethylene terephthalate (PET) and glass) Medium temperature bath, two tubes of each material. Add 0.1% Triton X-100 to half of the aliquot before the warm bath, and treat the other half with an equal volume of PBS. Warm all tubes overnight at room temperature.

[0280] -ApoE4 single antibody assay

[0281] Pipette 20 μl of each preparation and the unpreheated control into the wells of Greiner high binding microtiter plate. The ApoE4 assay was then performed as described in Example 2. The measured ApoE4 concentration is calculated relative to the concentration of the corresponding control sample (defined as 100%).

[0282] 3.2 R...

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Abstract

The present invention provides a method for the detection of Apolipoprotein E isotype 4 (ApoE4) or fragments thereof in a blood sample of a subject, whereby said method comprises the steps of contacting said sample with a solid phase, contacting said sample with at least one binder binding specifically to ApoE4 or a fragment thereof, thereby forming an ApoE4-binder-complex, and detecting the ApoE4-binder-complex.

Description

Technical field [0001] The present invention provides a simple and cost-effective method for determining the apolipoprotein E (ApoE) status of an individual. These individuals include but are not limited to patients suffering from cognitive impairment, dementia, and / or Alzheimer's disease (AD). Background technique [0002] Among the different susceptibility genes associated with the risk of late-onset Alzheimer's disease (LOAD) and other neurological disorders, ApoE has been identified as a strong genetic determinant ( Leduc Etc., 2011 ). The human APOE gene exists as three polymorphic alleles ε2, ε3, and ε4, and their worldwide frequencies are 8.4%, 77.9%, and 13.7%. Apolipoprotein E (ApoE) is composed of 299 amino acids and binds to lipoproteins in plasma and cerebrospinal fluid ( Leduc et al. 2011 ). The differences between the three ApoE isotypes are limited to amino acids 112 and 158 (SEQ ID NO. 1), where cysteine ​​or arginine is present at the positions: ApoE2 (cys112...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
CPCG01N33/54393G01N2333/775G01N2800/2821
Inventor 安德里亚斯·伯格曼
Owner SPHINGOTEC
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