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Anti-human AQPs autoantibody detection kit and preparation method thereof

An autoantibody and kit technology, which is applied in the field of anti-human AQPs autoantibody detection kits and preparation, achieves the effects of low requirements for testing personnel, simple operation, and simple result interpretation.

Pending Publication Date: 2021-02-02
BEIJING TIANTAN HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no detection method and application for other AQPs antibodies other than AQP4, including AQP1

Method used

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  • Anti-human AQPs autoantibody detection kit and preparation method thereof
  • Anti-human AQPs autoantibody detection kit and preparation method thereof
  • Anti-human AQPs autoantibody detection kit and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0034] The present embodiment provides a method for preparing a detection component of an indirect immunofluorescence kit for detecting human AQPs autoantibodies, which is rich in microvessels and vascular endothelium with high expression of AQPs brain regions, comprising the following steps:

[0035] 1) 7-week-old male C57 mice were reared in a standard environment in the SPF animal room for one week to adapt to the environment, and continued to be reared for one week for standby;

[0036] 2) Preparation of brain slices with high expression of AQPs rich in microvessels and vascular endothelium: rat brain coronal slices, and slices of brain areas with high expression of AQPs rich in microvessels and vascular endothelium ( figure 2 area indicated by the gray border); specific operations include:

[0037] 2.1) The mice were anesthetized and perfused with heart, blood was evacuated with normal saline, perfused with paraformaldehyde fixative solution, and the brain was taken afte...

Embodiment 2

[0041] This embodiment provides an indirect immunofluorescence kit for detecting human AQPs autoantibodies. The kit includes microvessels and vascular endothelium-rich AQPs high-expression brain slice detection components, blocking solution, PBST, mounting agent, negative control, Anti-human fluorescent secondary antibody.

[0042] Indirect immunofluorescence kit for detection of human AQPs autoantibodies AQPs high-expression brain slices rich in microvessels and vascular endothelium The detection component is prepared from slices of AQPs high-expression brain areas rich in microvessels and vascular endothelium and a gridded slide , wherein the slide is a detachable slide with 8 squares.

Embodiment 3

[0044] This embodiment provides an application method of a kit for detecting human AQPs autoantibodies, comprising the following steps:

[0045] 1) Place the prepared microvessel and vascular endothelial highly expressed brain region detection components at room temperature for 20 minutes, add 100 μl of PSBT blocking solution containing 5% BSA to each square and block at 37°C for 20 minutes;

[0046] 2) Discard the blocking solution. Add 100 μl of diluted serum or cerebrospinal fluid sample to be tested, and set up a grid as a negative control, add an equal amount of diluted normal human serum, and incubate at 37°C for 1H. The serum sample to be tested was diluted with PBST 1:10; the cerebrospinal fluid sample to be tested was not diluted; the negative control was diluted with PBST 1:10;

[0047] 3) Discard the liquid; wash with PBST 3 times, 5 minutes each time;

[0048] 4) Dilute the Alexa Fluor488-labeled anti-human fluorescent secondary antibody in PBST 1:500, and incuba...

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Abstract

The invention discloses preparation of an AQPs high-expression brain region slice detection assembly with abundant capillaries and vascular endothelium required by a human AQPs autoantibody detectionkit, an indirect immunofluorescence kit and application thereof. The preparation method of the microvascular and vascular endothelium-rich AQPs high-expression brain region slice detection assembly comprises the following steps: obtaining rat brain tissues; preparing a specific brain area tissue slice; and detection assembly preparation. The kit comprises the microvascular and vascular endothelium-rich AQPs high-expression brain region slice detection assembly, a confining liquid, PBST, a mounting medium and a fluorescent secondary antibody. The kit can be used for detecting anti-AQP4, AQP1 and other AQPs autoantibodies in clinical serum and cerebrospinal fluid samples. The kit is easy to operate, high in sensitivity, strong in staining form specificity, simple in result interpretation, accurate and reliable, and capable of supplementing the detection capability on AQP1 and other AQPs antibodies on the basis of ensuring that the detection on the AQP4 spatial conformational antibody isnot lower than the specificity and sensitivity of other means and meeting the requirements of auxiliary clinical diagnosis.

Description

technical field [0001] The invention relates to the field of disease molecular detection in the field of biomedicine, in particular to an indirect immunofluorescence detection technology for detecting human AQPs autoantibodies, including the preparation of detection kits and the detection of AQPs antibodies in clinical blood samples and cerebrospinal fluid samples. The method for rapid and effective detection of human AQPs antibodies can meet the needs of clinical differential diagnosis of neuromyelitis optica spectrum disease and other neurological diseases. Background technique [0002] Aquaporins (Aquaporins, AQPs) are proteins located on the cell membrane (intrinsic membrane protein), which form "pores" on the cell membrane and have the physiological function of controlling the transport of water and certain ions. AQPs have multiple subtypes such as AQP0-AQP12, among which the ones expressed in the central nervous system are: AQP1, AQP3, AQP4, AQP5, AQP6, AQP7, AQP8, AQP...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/533G01N21/64
CPCG01N33/6872G01N33/533G01N21/6408
Inventor 张星虎尹琳琳苌浩晓要雅君王化冰丛衡日魏玉帧周衡马越涛杜利
Owner BEIJING TIANTAN HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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