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Application of soluble CD146 as blood-brain barrier injury marker in central nervous system diseases

A central nervous system, soluble technology, applied in biological testing, chemiluminescence/bioluminescence, analysis by making materials react chemically, etc., can solve the problems of elevated, insufficient specificity, low sensitivity, etc.

Active Publication Date: 2020-04-07
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the molecular weight of albumin is relatively large (67KD), and the penetration of albumin does not reflect the leakage of large-scale small molecules in the BBB 4 , so Q Alb As an indirect indicator of BBB damage, there are limitations such as low sensitivity for BBB damage detection. In addition, its value increases with age, and the specificity is insufficient; and there may be short-term BBB damage that is quickly repaired, but the central Nervous system damage has developed, in which case Q Alb Can not accurately indicate the occurrence of disease 5
Therefore, there is a general lack of sensitive and specific biomarkers for most neurological diseases

Method used

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  • Application of soluble CD146 as blood-brain barrier injury marker in central nervous system diseases
  • Application of soluble CD146 as blood-brain barrier injury marker in central nervous system diseases
  • Application of soluble CD146 as blood-brain barrier injury marker in central nervous system diseases

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Preparation of paired monoclonal antibodies

[0031] Referring to Chinese patent application numbers 200810057260.7, 201210394856.2 and 99107586, monoclonal antibodies AA1, AA4, and AA98 or their derivatives or antigen-binding fragments were prepared using hybridoma technology. The antigenic epitope it binds to, the CDR sequence of the heavy chain variable region and the CDR sequence of the light chain variable region contained in each of them are as described above, and the strains that secrete them are the strains with the preservation number of CGMCC NO.2310 and the preservation number of It is the strain of CGMCC NO.2311 and the strain of preservation number CGMCC NO.0491.

Embodiment 2

[0032] Example 2: Expression and purification of standard sCD146 protein

[0033] sCD146 is a recombinantly expressed extracellular region protein of human CD146 molecule (amino acids at positions 22-559 in SEQ ID NO: 1, refer to Chinese Patent Application No. 200810057260.7, 201210394856.2). The CD146 cDNA (SEQ ID NO: 2) sequence with signal peptide, His-tag, strep-tag, and HRV3C recognition sequence at the N-terminal was cloned into the pcDNA3.1 vector (Invitrogen), and the plasmid was transfected into CHO Lec 3.2.8.2 Cells (ATCC) were cultured in DMEM medium, cells with higher protein expression levels were screened, and the culture was expanded and the culture supernatant was collected. The cell culture supernatant was further concentrated and affinity-purified with Ni-NTA (Qiagen) and strep-tactin (IBA) to obtain a fusion protein containing His-tag and strep-tag with higher purity. After digestion with HRV3C (Sino Biological) protease and Ni-NTA affinity purification to ...

Embodiment 3

[0034] Embodiment 3: clinical sample collection and classification

[0035]The inventor collected 861 pairs of cerebrospinal fluid and serum samples from patients with central nervous system diseases who were treated at the First Affiliated Hospital of Peking University and Beijing Anzhen Hospital from 2011 to 2018. The cerebrospinal fluid sample was obtained by lumbar puncture, centrifuged at 1000rpm at 4°C for 5 minutes, and the supernatant was aliquoted and stored at -80°C. . All patients had the right to know that their own serum and cerebrospinal fluid samples were used in this retrospective study, and were approved by the ethics committees of the First Affiliated Hospital of Peking University and Beijing Anzhen Hospital. According to the clinicopathological information of the patients, the inventors divided the patients included in the analysis into 6 groups, namely the control group (control), remitting multiple sclerosis (Remitting MS), nervous system non-inflammatory...

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Abstract

The invention relates to application of soluble CD146 protein in cerebrospinal fluid to preparation of a kit or a diagnostic agent for differential diagnosis, course judgment and / or medication guidance of individual inflammatory central nervous system diseases and the prepared kit or diagnostic agent. The application and the kit or the diagnostic agent have good sensitivity, accuracy, stability and operability.

Description

technical field [0001] The present invention relates to the field of disease diagnosis and treatment, in particular to the in vitro diagnosis and treatment of human inflammatory central nervous system diseases, more specifically to the preparation of soluble CD146 (sCD146) protein in cerebrospinal fluid and / or reagents for determining the amount of soluble CD146 in cerebrospinal fluid for use in individuals Use in kits or diagnostic agents for differential diagnosis, course judgment and / or medication guidance of inflammatory central nervous system diseases. Background technique [0002] The blood-brain barrier (BBB) ​​is a natural barrier between the blood and the brain that selectively prevents macromolecules from the peripheral circulation from entering the central nervous system. It is mainly composed of tight junctions of brain microvascular endothelial cells, outer basement membrane of endothelial cells, surrounding perivascular cells and glial cells. Under normal phys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N21/76
CPCG01N21/76G01N33/577G01N33/6893
Inventor 阎锡蕴刘丹王大吉郝洪军段红霞刘铮陈雪晖冯静
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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