Novel Smith Chimeric Peptide Antigen and Its Application in Laboratory Diagnosis

A chimeric peptide and antigen technology, which is applied in the fields of clinical immunity and medical laboratory diagnosis, can solve the problems of inability to expose antigenic determinants, difficult operation techniques, decreased diagnostic specificity, etc., and achieves good application value, low price, and easy operation. Effect

Active Publication Date: 2015-10-07
陈仁奋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But they have many disadvantages: 1. The protein is prone to denaturation during the crude extraction or purification process, the operation technology is difficult, the repeatability between batches is poor, and the price is expensive; 2. Due to the influence of the secondary space structure of the protein or polypeptide, its Some of the contained antigenic determinants cannot be exposed on the surface of protein molecules and are bound by antibodies; 3. Due to the high content of amino acids in non-antigenic determinants, they may combine with other non-specific antibodies, resulting in a decrease in diagnostic specificity
In this way, if only any synthetic peptide containing a single dominant epitope is used like some current commercial kits, a considerable part of the Sm antibody subtype will be missed, resulting in insufficient sensitivity
However, it is technically difficult to coat the above three synthetic peptides on the ELISA plate at the same time because the short peptides compete with each other for limited coating sites.

Method used

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  • Novel Smith Chimeric Peptide Antigen and Its Application in Laboratory Diagnosis
  • Novel Smith Chimeric Peptide Antigen and Its Application in Laboratory Diagnosis
  • Novel Smith Chimeric Peptide Antigen and Its Application in Laboratory Diagnosis

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Experimental program
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Effect test

Embodiment 1

[0040] 1. Sm synthetic peptide sequence and synthesis

[0041] Firstly, the serum of SLE patients was used to screen out three highly specific SLE-derived dominant antigenic epitopes (SmD-1, SmD-2 and SmB-1 synthetic peptides) derived from SmD and SmB proteins, and then a protein containing the above three antigens was designed. The Sm chimeric peptide of the epitope was synthesized and purified by Peptide Biochemical Company.

[0042] The following peptides are derived from Sm protein, which are automatically or artificially synthesized by FMOC solid-phase synthesis method. After HPLC analysis and purification, the purity of the antigen reaches more than 90%.

[0043] (1) Amino acid sequence of Sm synthetic peptide containing a single epitope

[0044] SmD-1 MKLVRFLMKLSHETVTIE;

[0045] SmD-2 GVDVSMNTHLKAVKMT;

[0046] SmB-1 PPMGIPPGRGTPMG.

[0047] (2) Amino acid sequence of Sm chimeric peptide containing multiple epitopes

[0048] MKLVRFLMKLSHETVTIEGVDVSMNTHLKAVKMTPPMGI...

Embodiment 2

[0059] ELISA plates were coated with chimeric Sm peptides containing multiple epitopes and short peptides SmD-1, SmD-2, and SmB-1 containing a single epitope, and sera from 50 cases of SLE were detected simultaneously. Such as figure 1 As shown, with ELISA optical density (OD) 0.2 as the positive cut-off value, the sensitivity of Sm chimeric peptide to detect anti-Sm antibody in SLE serum is more than 70%, far better than SmD-2 (19%), SmB -1 (29%) and SmD-1 (31%).

[0060] This experiment shows that the Sm chimeric peptide containing multiple antigenic epitopes has higher sensitivity for detecting Sm antibodies than the Sm short peptide containing a single antigenic epitope.

Embodiment 3

[0062] ELISA kit of the present invention is carried out clinical verification, have detected 170 routine SLE patients respectively, 153 routine disease contrasts: wherein comprise rheumatoid arthritis (RA) 21 examples, mixed connective tissue disease (MCTD) 11 examples, Sjogren's syndrome ( 10 cases of SS), 10 cases of psoriasis (Psoriasis), 13 cases of scleroderma (Scleroderma), 88 cases of other diseases, and 43 cases of healthy controls.

[0063] see results figure 2 . Taking 24AU / ml as the positive cut-off value (dotted line in the figure), the detection sensitivity of the kit of the present invention for SLE patients reaches 71%, and the specificity reaches 93%. However, the commercial kit (Quantalite anti-Sm ELISA) for simultaneous detection of the above sera has a sensitivity of only 28% and a specificity of 94% based on the positive cut-off value provided in the instruction manual. It shows that the ELISA kit of the present invention maintains high specificity, and ...

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Abstract

The invention discloses novel Smith chimeric peptide antigen and application thereof in experimental diagnosis. The antigen is a Sm chimeric peptide containing a plurality of antigen epitopes including three synthetic peptides of SmD-1, SmD-2 and SmB-1, wherein the Sm chimeric peptide antigen is coated on a solid phase carrier and can be used for detecting an anti-Sm antibody. The detecting method comprises an indirect ELISA (Enzyme Linked Immunosorbent Assay) method which coats the Sm chimeric peptide antigen on an elisa plate to prepare an indirect enzyme-linked immunodetection kit. The novel Smith chimeric peptide antigen disclosed by the invention is simple in preparation process, high in sensibility and specificity of detecting the anti-Sm antibody in systemic lupus erythematosus blood serum, good in repeatability, simple to operate, capable of being widely applied to clinical normal inspection and scientific research in a qualitative or quantitative manner, and has a good application value.

Description

technical field [0001] The invention belongs to the field of clinical immunity and medical experimental diagnosis, and in particular relates to a synthetic chimeric peptide (chimeric synthetic peptide) containing multiple dominant epitopes of Smith (Sm) protein antigens and its application in detecting anti-Sm in serum of systemic lupus erythematosus Antibody ELISA Kit. Background technique [0002] Systemic lupus erythematosus (SLE) is one of the most widespread and serious autoimmune diseases affecting the population, which can cause inflammation in multiple organs throughout the body. The disease develops latently, the course of the disease is protracted, repeated and exacerbated, and the clinical manifestations are complex and diverse, which is easily confused with other diseases, especially similar autoimmune diseases. Early detection, accurate diagnosis and timely treatment are decisive for improving the prognosis of SLE patients and reducing mortality. The current d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00G01N33/68
Inventor 陈仁奋魏秀妹
Owner 陈仁奋
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