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Echinococcosis diagnostic kit

A diagnostic kit and technology for hydatid disease, applied in biological tests, antibody mimics/stents, material inspection products, etc. The detection accuracy needs to be further improved to achieve the effect of high accuracy and accurate diagnosis

Active Publication Date: 2021-10-29
WEST CHINA HOSPITAL SICHUAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection accuracy of echinococcosis diagnostic reagents prepared by recombinant antigens needs to be further improved
[0005] In addition, the existing diagnostic reagents can only roughly do qualitative detection, it is difficult to distinguish between AE and CE, and it is difficult to evaluate the severity of echinococcosis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1 recombinant antigen

[0030] The specific Eg95 (GenBank ID number: LN901900.1), AgB8 / 1 (GenBank accession number: AF143813.1) and Em18 gene sequences of Sichuan strain (GenBank accession number: AY513264.1) were obtained from the GenBank database. After the analysis of the sequence characteristics, the signal peptide and the transmembrane region were removed, and the different proteins were connected by a linker (linker) GGGGSGGGG to construct the recombinant sequence and sequence. The amino acid sequence (SEQ ID NO.1) expressed by the gene is as follows:

[0031]

[0032] Note: The sequence marked with single solid line is AgB8 / 1 protein, the sequence marked with double solid line is Em18 protein, and the sequence marked with dotted line is EG95 protein.

[0033] Using software (ExpOptimizer tool), the DNA sequence corresponding to the above amino acid sequence was codon-optimized according to the codon preference of Escherichia coli,...

Embodiment 2

[0039] Embodiment 2 A kind of echinococcosis diagnostic kit

[0040] This kit is an indirect ELISA diagnostic kit, using T3 in Example 1 as an antigen (protein concentration 1.5 mg / mL).

[0041] The kit includes the following components:

[0042] Antigen, PBST buffer, secondary antibody (goat anti-human IgG-HRP), substrate TMB, enzyme plate.

[0043] The method of using the kit is as follows:

[0044] 1) The new recombinant antigen T3 was prepared by coating the microplate with concentrations of 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL and 2 μg / mL, staying overnight at 4 degrees Celsius, washing the plate with PBST, and sealing the plate with 5% skimmed milk powder for 2 hours. 96T test board spare;

[0045] 2) Determine the optimal antigen dilution concentration and enzyme reaction working concentration by checkerboard titration (secondary antibody: conventional goat anti-human IgG-HRP diluted 1:4000);

[0046] 3) The serum to be tested is diluted with 1:10, 1:100, 1:200, 1:400, 1:...

experiment example 1

[0051] Comparison of experimental example 1 with the clinical detection effect of rEm18-Eg95 (T2) antigen and commercially available ELISA detection kit

[0052] 1. Materials and methods

[0053] 1. Materials

[0054] 1.1 rEm18-Eg95 antigen

[0055] On the basis of Example 1, the rAgB8 / 1 antigen in T3 was omitted, and only the echinococcosis rEm18 and Eg95 proteins were fused to obtain the rEm18-Eg95 antigen, referred to as T2.

[0056] 1.2 Commercially available ELISA detection kits

[0057] Purchased from Zhuhai Haitai Biological Co., Ltd., the trade name is hydatid IgG antibody detection kit (enzyme-linked immunoassay), the antigen is mainly cystic fluid crude antigen (AGB), referred to as commercial H reagent.

[0058] 1.3T3 antigen

[0059] Namely T3 of Example 1.

[0060] 2. Serum

[0061] The research protocol was approved and approved by the Ethics Committee of West China Hospital of Sichuan University and the Ethics Committee of Ganzi Prefecture People's Hospita...

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Abstract

The invention discloses an echinococcosis diagnostic kit, and belongs to the field of disease diagnostic kits. According to the kit, the fusion protein obtained by removing signal peptides from echinococcosis proteins Eg95, AgB8 / 1 and Em18 and connecting through connecting peptide is used as the antigen for capturing the anti-echinococcosis antibody of a body, so that the kit not only has good echinococcosis detection performance, but also can distinguish two types of echinococcosis, namely cystic echinococcosis (CE) and alveolar echinococcosis (AE), and can evaluate the size of a focus.

Description

technical field [0001] The invention belongs to the field of disease diagnosis kits. Background technique [0002] Echinococcosis, also known as echinococcosis, is divided into cystic echinococcosis (CE) and alveolar echinococcosis (AE). China is one of the high-incidence areas in the world, and Sichuan Province has the highest prevalence. Echinococcosis lacks sensitive and efficient means of early diagnosis. In addition, there are no obvious symptoms in the early stage of the disease. Most patients are already in the middle and late stages when they are diagnosed, making treatment extremely difficult. [0003] Early screening and early diagnosis are one of the most important means to prevent and control echinococcosis. The current screening method is only ultrasonography, which has a low early detection rate, poor objectivity, high manpower and material resources requirements, and takes a long time. The specificity and sensitivity of the existing serological detection met...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62G01N33/68G01N33/53
CPCC07K14/43554G01N33/5308G01N33/6893C07K2319/00G01N2333/43543Y02A50/30
Inventor 杨先伟王文涛沈舒陈颖
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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