Kit and method for detecting ophthalmopathy

A kit and eye disease technology, applied in biological testing, biochemical equipment and methods, disease diagnosis, etc., can solve problems such as time-consuming and complicated operation, and achieve the effects of low cost, easy preparation and easy preservation

Active Publication Date: 2016-02-17
宁波美康盛德医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional detection of the expression of ICAM-1 has the disadvantages of complex operation and time-consuming

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1, Screening and Preparation of Nucleic Aptamers

[0019] Design a random nucleic acid library comprising approximately 20 nucleotides at both ends and 39 nucleotides in the middle as follows:

[0020] 5'-ACGACTAACGTGTAACAG(N39)CAGTGCATGATGCTACGAA-3'; N39 represents 39 random nucleotides.

[0021] The single-stranded DNA library was amplified into double-stranded DNA, and the product was subjected to 2% agarose gel electrophoresis and then gel-cut to recover and purify; the recovered double-stranded DNA was used as a template to transcribe a single-stranded RNA random library in vitro, and the transcript was purified by PAGE . The 75 μg RNA library was reverse-screened with a nitrocellulose membrane to remove membrane-bound RNA molecules, and then expressed with 2ugsICAM-1 protein (through the method disclosed in Chen Zhihong et al., Eukaryotic Cell Expression and Identification of Human Intercellular Adhesion Molecule-1). Target protein), incubated at 37°C fo...

Embodiment 2s

[0037] Example 2 Performance determination of sICAM-1 protein binding to suitable gametes

[0038] 2.0 μg of the RNA aptamers were digested with calf intestinal alkaline phosphatase (CIP) at 37°C for 1 hour, and the dephosphorylated RNA was purified and recovered; [γ-32P]ATP was dephosphorylated by T4 polynucleotide kinase labeling the ends of the RNA molecules. 10nmol radioactively labeled RNA aptamers were incubated with different concentrations (1-200nM) of sICAM-137°C for 30min, each group of reaction solution was filtered through nitrocellulose membrane, the filter membrane was washed, dried, and liquid scintillation counter To measure the radioactivity remaining on the filter membrane, the same sample is measured twice in parallel. The dissociation constant of each aptamer with sICAM-1 was calculated. The result is as follows:

[0039] name Dissociation constant Kd (unit nM) sICAM-1-1 8.8 sICAM-1-2 8.9 sICAM-1-3 9.0 sICAM-1-4 7.9 ...

Embodiment 3

[0040] Adapter specificity analysis and stability analysis described in embodiment 3

[0041] Human serum albumin, immune serum globulin, Vibrio cholerae VgrG3C protein, Escherichia coli outer membrane protein A, Lp-PLA2 protein, and 15 aptamers were used for specific detection. After binding experiments, it was found that these aptamers Neither binds to these proteins, but only binds to sICAM-1 with high specificity.

[0042] 0.2ug of the aptamer was taken and placed in normal temperature serum and aqueous solution respectively for two weeks. Through RT-PCR detection, it was found that its structure was stable and not degraded after two weeks of storage.

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PUM

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Abstract

The invention discloses an aptamer specifically bound with sICAM-1 and a kit. The aptamer and the sICAM-1 have the good affinity. By means of the aptamer, the sICAM-1 in a solution can be captured to be prepared into the corresponding kit for ophthalmopathy screening. The kit has the advantages of being high in sensitivity and low in cost, and is suitable for large-scale screening.

Description

technical field [0001] The invention relates to a kit for detecting eye diseases and a detection method thereof. Background technique [0002] Thyroid associated ophthalmopathy (TAO) is one of the common diseases in endocrinology department, and it is also one of the most difficult diseases to treat in the world. TAO has a long course of disease, can occur in all age groups, and is more common in women. In mild cases, it can affect the quality of life and work, and damage appearance. In severe cases, it can lead to vision loss or even blindness, which brings heavy economic and psychological burdens to patients. In recent years, due to changes in lifestyle, increased work pressure, changes in social environment, popularization of iodized salt, and irregular drug use, the incidence of TAO has increased significantly, and the hospitalization rate has been high. At present, the cause of this disease is unknown, and the clinical treatment is mainly based on hormones, immunosupp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCC12N15/115C12N2310/16G01N33/68G01N33/6893G01N2333/47G01N2333/70525G01N2800/046G01N2800/16
Inventor 李倩
Owner 宁波美康盛德医学检验所有限公司
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