Metal labelling-based in-situ detection method for Abeta protein in brain tissue with Alzheimer disease

A brain tissue and methyl group technology, applied in the field of analytical chemistry, can solve the problems of complex operation, many influencing factors, inability to quantitatively analyze Aβ in situ, etc., and achieve the effects of high specificity and sensitivity, strong repeatability, and simple operation.

Active Publication Date: 2020-07-10
BEIJING UNIV OF CHEM TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional immunohistochemical staining and immunofluorescence detection can also be used to image Aβ in the brain, but there are defects such as complicated operation and many influencing factors
In particular, none of the above imaging methods can perform in situ quantitative analysis of Aβ in the brain
[0005] In short, the existing technology still lacks a fast, accurate, and intuitive method for determining the deposition of Aβ protein in the brain, thereby lacking an effective method for screening Alzheimer's disease therapeutic drugs

Method used

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  • Metal labelling-based in-situ detection method for Abeta protein in brain tissue with Alzheimer disease
  • Metal labelling-based in-situ detection method for Abeta protein in brain tissue with Alzheimer disease
  • Metal labelling-based in-situ detection method for Abeta protein in brain tissue with Alzheimer disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Ru-NHS ester and Aβ antibody-coupled chelate and its preparation

[0053] (1) Weigh the Ru-NHS ester powder with a precision balance, dissolve and mix with DMSO to make the concentration 100 μg / ml.

[0054] (2) Anti-β-Amyloid (BioLegend) was taken and dissolved in 10 mM PBS, pH 8.0, to a concentration of 2 μg / ml.

[0055] (3) Add 5 μl of the solution formed in step (1) to 100 μl of the solution formed in step (2), vortex and mix well, and shake at room temperature for 1 h in the dark.

[0056] (4) Add 2 μl of 2M glycine aqueous solution to the solution formed in step (3) to terminate the reaction, and shake at room temperature for 15 minutes in the dark.

[0057] (5) The solution formed in step (4) was dialyzed twice in 500 ml, 10 mM, pH 8.0 PBS using a 10KD dialysis tube, 30 min each time.

[0058] (6) The dialyzed solution can be stored in a centrifuge tube at 4°C. After testing, the molar ratio of Ru-NHS ester to anti-β-Amyloid in the obtained chelate w...

Embodiment 2

[0059] Example 2. Detection of Aβ protein in isolated brain tissue

[0060] (1) 6-month-old normal mice and transgenic Alzheimer's model mice were taken respectively, anesthetized, perfused with normal saline, and decapitated to obtain the brain.

[0061] (2) The brain tissue obtained in step (1) was fixed in 4% paraformaldehyde, taken out every other day, and dehydrated in gradients in 20% and 30% sucrose solutions respectively, and the brain tissue sank to the bottom of the solution before being taken out.

[0062] (3) The brain tissue obtained in step (2) was embedded with an OCT frozen section embedding agent, and then placed in a constant temperature cryostat and cut into thin slices of 10 μm.

[0063] (4) The brain tissue slices obtained in step (3) were left at room temperature for 30 min. Wash away the OCT embedding agent on the slices with ultrapure water.

[0064] (5) The slices obtained in step (4) were fixed in pre-cooled acetone at 4°C for 10 min, and then washe...

Embodiment 3

[0073] Example 3. Quantitative detection of Aβ protein in isolated brain tissue

[0074] (1) 6-month-old normal mice were taken, anesthetized, perfused with normal saline, decapitated and taken out the brain, homogenized, embedded with OCT frozen section embedding medium, placed in a constant temperature cryostat, and cut into 10 μm thin slices;

[0075] (2) Prepare methanol standard solutions of Ru-NHS esters with Ru concentrations of 0, 1, 2, 3, and 4 μg / ml, respectively.

[0076] (3) After placing the slices obtained in step (1) at room temperature for 20 minutes, soak them in the methanol standard solutions of different concentrations of Ru obtained in step (2).

[0077] (4) LA denudates the slices obtained in step (3), and ICP-MS selects 102 Ru, 13 C for signal monitoring, which uses 13 C Signal homogenization eliminates the influence of uneven thickness of brain tissue slices.

[0078] (5) Take another slice obtained in step (3), scrape off the brain tissue, weigh it...

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Abstract

The invention discloses a metal labelling technology-based imaging technology for an Abeta protein on a brain tissue section. Bis(2,2'-bipyridyl)-4'-methyl-4-carboxyl bipyridine-ruthenium N-succinimidyl ester-bis-(hexafluorophosphate) (hereinafter referred to as Ru-NHS ester) is coupled to an Abeta antibody to form a metal chelate complex; and through immunoreaction, metal is indirectly labelled on Abeta in brain tissue. The signal intensity of Ru on a brain is detected through laser ablation-inductively coupled plasma-mass spectrometry, and a distribution diagram of the Abeta on the brain tissue section is finally drawn. The method is mild in condition; the original appearance of the brain tissue section is reserved to the maximum extent; the distribution position and aggregation extent of the Abeta protein are completely and accurately represented; and the method has accuracy and traceability.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, in particular to the development and application of protein in situ analysis methods. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease, its main symptoms are memory loss and cognitive behavioral difficulties, causing great suffering to patients. Abnormalities in brain proteins generally precede the onset of symptoms. Therefore, pathological research on Alzheimer's disease is helpful for the discovery and treatment of the disease. The β-amyloid protein (amyloidβ-protein, Aβ protein) cascade hypothesis is recognized as the pathogenesis of Alzheimer's disease, that is, excessive Aβ protein deposits in the brain to form amyloid plaques and triggers degenerative changes in the nervous system. [0003] At present, the research samples for Aβ protein mainly come from cerebrospinal fluid and plasma. The change of Aβ level in cerebrospinal fluid is not specific; ...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/532G01N33/68
CPCC07K16/18G01N33/6896G01N33/532G01N33/6851G01N2800/2821
Inventor高雪罗施中冯流星
OwnerBEIJING UNIV OF CHEM TECH