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Kit for detecting anti-moesin antibody

A detection kit, membrane protein technology, applied in the biological field to achieve the effect of high sensitivity, specificity, and high-throughput detection

Active Publication Date: 2010-12-29
SHANGHAI KEXIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no international research on the involvement of moesin and its antibody in the pathogenesis of CTD-associated lung involvement

Method used

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  • Kit for detecting anti-moesin antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Determination of the AECA main target antigen moesin of CTD-related PAH

[0069] Step 1: AECA detection in patients with CTD-associated PAH

[0070] The positive rate of anti-endothelial cell antibody in patients with CTD-associated PAH was detected by cell-ELISA and Western blotting, and the relationship between AECA and CTD-associated PAH specificity and disease clinical activity indicators was analyzed.

[0071] 1. Research object and method

[0072] 1.1 Research object

[0073] The research subjects were patients with connective tissue disease-associated pulmonary arterial hypertension who were diagnosed and treated in the Department of Rheumatology and Immunology of Peking Union Medical College Hospital from January 2006 to May 2007. The experimental group selected 68 patients with CTD-associated PAH (CTD-PAH) (only with pulmonary arterial 12 patients with idiopathic pulmonary arterial hypertension (IPAH) and 61 patients with CTD without PAH in the cont...

Embodiment 2

[0127] Embodiment 2 moesin protein gene cloning, prokaryotic expression and purification

[0128] Step 1: Gene Recombination of Moesin

[0129] 1.1 Acquisition of recombinant protein moesin cloning template: RNA was extracted from gastric cancer tissue and reverse transcribed into cDNA.

[0130] 1.2 The full-length cDNA sequence of moesin protein provided by genebank is as follows,

[0131] 1.2.1 Nucleotide sequence: (for the sequence see >gi|53729335:199-1932 Homo sapiens moesin (MSN), mRNA 1734bp).

[0132] 1.2.2 Protein sequence: see SEQ ID NO: 1 and image 3 . image 3 In , the amino acid composition of Moesin and the matching amino acid sequence by mass spectrometry (the matching amino acid sequence is marked in bold and underlined).

[0133] 1.2.3 Moesin protein amplification primer sequences were designed as follows:

[0134] The sequence corresponding to Moesin-top (EcoR I) is SEQ ID NO: 2

[0135] 5'-CCGGAATTCATGCCCAAAACGATCAGT-3'

[0136] The corresponding seq...

Embodiment 3

[0152] The preparation of embodiment 3 anti-moesin antibody detection kit

[0153] The first step: exploration experiment of optimal reaction conditions

[0154] 1.1 Antigenicity evaluation and selection basis of two recombinant proteins

[0155] The antigenicity of the two recombinant protein antigens was evaluated by the indirect enzyme-linked immunosorbent method. The specific method was: the antigen was diluted to 1 μg / ml, 2 μg / ml, 4 μg / ml, 5 μg / ml, 10 μg / ml and then coated with enzyme labeling Plate, clinical collection of anti-moesin body test results are strong positive, weakly positive and negative serum samples 1 each. Within the concentration range of the above-mentioned antigen coating, the selection of antigen 1# can clearly distinguish three different serum samples, and the difference is significant. Therefore, antigen 1# is used as the antigen protein for detection, which is considered to have strong antigenicity (sensitivity) and lower background values ​​(spe...

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Abstract

The invention discloses a kit for detecting an anti-moesin antibody. The kit comprises a solid-phase carrier and a moesin antigenic protein, wherein the moesin antigenic protein is a full-length human moesin. The kit can be applied to a method for early prediction and severity evaluation of connective tissue disease (CTD)-related lung involvement and evaluation is performed by mainly sampling a biological sample of a subject and detecting the quantity of the anti-moesin antibodies in the biological sample. The moesin is prepared by proteomics technology and gene recombination technology and an enzyme-linked immunosorbent assay (ELISA) detection kit for detecting the anti-moesin antibody is provided. The detectable rate of the kit for the CTD-related lung involvement is up to 51.7 percent.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting anti-moesin antibody. Background technique [0002] Autoimmune diseases (autoimmune diseases, AID) is a kind of disease with autoimmune response leading to tissue and organ damage and corresponding dysfunction as the main pathogenesis. There are two types of autoimmune diseases: organ-specific and non-organ-specific. Diffuse connective tissue disease, referred to as connective tissue disease (CTD), is a non-organ-specific autoimmune disease. Such diseases often have the following common characteristics: high titer autoantibodies and (or) sensitized lymphocytes reacting with their own tissue components often appear in the patient's blood; The combination of corresponding self-tissue antigens causes immune damage and dysfunction of tissues and organs in different ways to cause disease. At the same time, the intensity of the autoimmune response is closely related ...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/531G01N21/31
CPCG01N2800/10G01N33/6887
Inventor 曾小峰李梦涛尹雷艾军赵久良王迁张玥黄岚杨超文李春梅孙宏彬钱杰
Owner SHANGHAI KEXIN BIOTECH
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