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Antibody for detecting blood brain barrier early injury of cerebral arterial thrombosis and application of antibody

A technology for ischemic stroke and blood-brain barrier, applied in the field of disease detection, can solve problems such as the inability to rule out the influence of occludin full-length protein, misjudgment, and affect the accuracy of test results, so as to improve specificity and accuracy, eliminate effect of influence

Active Publication Date: 2018-06-22
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no method to specifically identify the degradation fragment of occludin, and the influence of the full-length protein of occludin in serum on the detection results cannot be ruled out
The recognition of the full-length protein of occludin in serum will affect the accuracy of the test results and cause misjudgment

Method used

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  • Antibody for detecting blood brain barrier early injury of cerebral arterial thrombosis and application of antibody
  • Antibody for detecting blood brain barrier early injury of cerebral arterial thrombosis and application of antibody
  • Antibody for detecting blood brain barrier early injury of cerebral arterial thrombosis and application of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The development of embodiment 1 specific antibody

[0055] This example is used to illustrate the development process of the specific antibody of the present invention, and the specific steps are as follows:

[0056] 1. According to the human occludin full-length protein sequence (AAB00195.1, as shown in SEQ ID NO.1), after hydrophobicity analysis ( figure 1 ), judge the protein as multiple transmembrane protein, and predict the suitable polypeptide sequence for antigen recognition and antibody preparation. From the N-terminal to the C-terminal, select 4 sequences for peptide synthesis.

[0057] Sequence 1#YRPDEFKPNHYAPSNC;

[0058] Sequence 2#KTRRKMDRYDKSNIL;

[0059] Sequence 3#DHYETDYTTGGESC;

[0060] Sequence 4#SKLSHIKKMVGDYDR.

[0061] 2. Synthetic antigen:

[0062] Coupling the above-mentioned 1#~4# polypeptides with KLH respectively (the coupling agent is SuLfo-SMCC) to obtain KLH-polypeptides as 1#~4# immune antigens;

[0063] The above-mentioned 1#-4# po...

Embodiment 2

[0076] Example 2 Kit for specific detection of early damage to the blood-brain barrier in ischemic stroke

[0077] This example takes the specific antibody XW-OCLN-2 obtained in step 4 of Example 1 as an example to illustrate a kit for specifically detecting early damage to the blood-brain barrier in ischemic stroke. The kit is an ELISA kit, including : Solid phase carrier coated with specific antibody XW-OCLN-2, detection antibody, enzyme-labeled antibody, standard antigen.

[0078] in:

[0079] The preparation method of the solid phase carrier is specifically:

[0080] (1) Coating: with 200mM NaHCO 3 Dilute the specific antibody XW-OCLN-2 in the buffer solution to a final concentration of 1 μg / mL, add 100 μL occludin specific antibody diluent to each well of the microtiter plate, and refrigerate overnight at 4 °C;

[0081] (2) Washing: wash each well of the microplate with PBST 4 times, 5 min each time;

[0082] (3) Blocking: add blocking solution 10% BSA, incubate at 37...

Embodiment 3

[0088] The application of embodiment 3 detection kits

[0089] This example is used to illustrate how to use the ELISA kit prepared in Example 2 to detect the level of occludin-specific degradation fragments in the serum of patients with acute ischemic stroke, so as to measure the early damage of the blood-brain barrier in patients with stroke.

[0090] In this example, the ELISA kit prepared in Example 2 was used to detect the sera of 7 acute ischemic stroke patients and 6 healthy people.

[0091] (1) Collect serum from patients with acute ischemic stroke and healthy patients; add 100 μL of human serum, incubate at 37°C for 2 hours, and wash;

[0092] (2) Add 100 μL detection antibody (Thermo Fisher Cat. No. 33-1500), incubate for 2 hours, and wash 4 times;

[0093] (3) Add 100 μL of secondary antibody (Zhongshan Jinqiao ZB-2305), incubate at 37°C for 30 minutes, and rinse;

[0094] (4) Add 90 μL of TMB substrate chromogenic solution to each well, and develop color at 37°C ...

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Abstract

The invention relates to the field of disease detection, and particularly discloses a specific antibody for detecting the blood brain barrier early injury of cerebral arterial thrombosis and application of the antibody. The antibody is characterized in that the antibody only specifically recognizes KTRRKMDRYDKSNIL in a degradation fragment of occludin protein, and does not recognize occludin total-length protein. Therefore, the antibody can be used for specifically detecting the blood brain barrier early injury of the cerebral arterial thrombosis, and can eliminate the influence of the occludin total-length protein in serum on a detection result, so that the specificity and the accuracy of detecting the blood brain barrier early injury of the cerebral arterial thrombosis are obviously improved.

Description

technical field [0001] The invention relates to the field of disease detection, in particular to a specific antibody for detecting early damage of the blood-brain barrier in ischemic stroke and its application. Background technique [0002] Stroke has become an important disease that seriously threatens human health due to its high morbidity and disability, among which 80% are ischemic strokes. According to the latest "Global Burden of Disease Report" published in "The Lancet", stroke has become the number one cause of death in China. Due to the accelerated aging of the population and the poor control of risk factors, the incidence of stroke in my country is rising rapidly at a rate of 8.7% per year; the annual cost of treating stroke patients in Beijing alone exceeds 10 billion, which is a heavy social and economic burden. [0003] Intravenous thrombolysis is currently recognized as one of the most effective methods for the treatment of acute ischemic stroke. Intravenous ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/06G01N33/68
CPCG01N33/6893C07K16/06C07K16/28C07K2317/34G01N2333/705G01N2800/2871G01N2333/90209C07K16/065G01N33/6896
Inventor 吉训明戚智锋刘克建
Owner XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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