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A succinyl-β-cyclodextrin-modified protein chip for Lyme disease detection and its preparation and application

A protein chip, Lyme disease technology, applied in the field of biomedical testing, to avoid false positives, high specificity, improve screening efficiency, positive detection rate and accuracy

Active Publication Date: 2017-09-19
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preferred method for detection of Lyme disease recommended by the US National Centers for Disease Control and Prevention is to use ELISA or direct immunofluorescence to detect antibodies produced by Borrelia in serum, but the results still need to be confirmed by Western blot method

Method used

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  • A succinyl-β-cyclodextrin-modified protein chip for Lyme disease detection and its preparation and application
  • A succinyl-β-cyclodextrin-modified protein chip for Lyme disease detection and its preparation and application
  • A succinyl-β-cyclodextrin-modified protein chip for Lyme disease detection and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] The selection and surface chemical treatment of embodiment 1 carrier

[0082] The present invention selects a gold foil chip from Interactiva Company (Ulm, Germany). Its substrate is a glass sheet covered with a layer of pure gold (purity 99.9%) with a thickness of 10nm. On the gold foil is a regionalized 50μm TEFLON Membrane array (96 holes*2, 8 rows*12 columns), the array aperture is 1.25mm, such as figure 1 shown.

[0083] Step 1. Cleaning of gold foil chip

[0084] Prepare TL1 solution (H 2 O:H 2 o 2 :NH 3 ·H 2 O=5:1:1) into a stainless steel box, put the chip into the box, bathe in 82°C water for 5 minutes, rinse with deionized water 4 to 5 times, ethanol twice, each time for 3 minutes; air-dry with nitrogen, dry and store.

[0085] Step 2. Chemically modify the surface of the cleaned gold foil chip to obtain a solid phase carrier

[0086] After cleaning the gold foil chip, immerse it in the aforementioned modification solution 1, and incubate with shaking at...

Embodiment 2

[0089] Embodiment 2 quality control experiment

[0090] Preparation of incubation solution 1: Dissolve VlsE antigen in PBST-BSA solution, and prepare concentration gradients of 0.5μg / ml, 0.25μg / ml, 0.125μg / ml, 0.0625μg / ml, 0.0313μg / ml, 0.0156μg / ml , 0.0078μg / ml, 0.0039μg / ml, 0.0019μg / ml, 0.00095μg / ml, 0.000475μg / ml antigen solution.

[0091] Preparation of incubation solution 2: Dissolve human IgG in PBST-BSA solution, and configure the concentration gradient to be 0.5μg / ml, 0.25μg / ml, 0.125μg / ml, 0.0625μg / ml, 0.0313μg / ml, 0.0156μg / ml, 0.0078μg / ml, 0.0039μg / ml, 0.0019μg / ml, 0.00095μg / ml, 0.000475μg / ml solution;

[0092] Preparation of incubation solution 3: Dissolve Flagellin antigen in PBST-BSA solution, and prepare a concentration gradient of 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, 0.0313 μg / ml, 0.0156 μg / ml, 0.0078μg / ml, 0.0039μg / ml, 0.0019μg / ml, 0.00095μg / ml, 0.000475μg / ml solution;

[0093] Prepare incubation solution 4: dissolve the OspC antigen in PBST-BSA ...

Embodiment 3

[0118] Embodiment 3 repeatability experiment

[0119] Table 1 shows the repeatability experiment within a group. Each 16 wells is regarded as a group, and there are three groups in total. On the same day, the VlsE antigen with a concentration of 0.0156 μg / ml and the rabbit anti-VlsE antigen IgG antibody with a concentration of 50 μg / ml are incubated according to the steps and Cy3-labeled goat anti-rabbit IgG antibody at a concentration of 2.5 μg / ml.

[0120] Table 1

[0121]

[0122] Table 2 shows the repeatability experiment between groups. Each 24 wells is regarded as a group, and there are three groups in total. The VlsE antigen with a concentration of 0.0156 μg / ml and the rabbit anti-VlsE antigen IgG with a concentration of 50 μg / ml were incubated according to the steps within three days. Antibody and Cy3-labeled goat anti-rabbit IgG antibody at a concentration of 2.5 μg / ml.

[0123] Table 2

[0124]

[0125] In Tables 1 and 2, SD is the standard deviation, and CV...

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Abstract

The invention belongs to the field of biomedicine detection, and particularly relates to a protein chip modified by succinyl-beta-cyclodextrin for detecting lyme disease and preparation and application of the protein chip. On the one hand, the invention provides a protein chip for detecting the lyme disease, comprising a solid phase carrier and a capture molecule fixed on the solid phase carrier, wherein the capture molecule contains a principal protein-like sequence expression E protein (VisE) of borrelia burgdorferi variable. In detection, each chip can detect multiple serum samples simultaneously, and is relatively high in flexibility and specificity, so that the occurrence probability of false positive detection and false negative detection among groups is reduced. Compared with a traditional method, the protein chip is applied to screening population at high-risk areas on a large scale; relatively simple, convenient and reliable detection methods can be provided; the screening efficiency as well as the positive detecting rate and accuracy are enhanced.

Description

technical field [0001] The invention belongs to the field of biomedical detection, and in particular relates to a succinyl-β-cyclodextrin-modified protein chip for Lyme disease detection and its preparation and application. Background technique [0002] Lyme disease (Lyme disease, LD) is a disease caused by spirochete infection transmitted by ticks, involving skin, nerves, joints, heart and other multiple tissue and organ damage, also known as Lyme borreliosis (Lymeborreliosis). The main pathogen of Lyme disease is Borrelia burgdorferi, which is divided into three subtypes: Borrelia burgdorferi sense stricto, Borrelia afzelii and Borrelia burgdorferi garinii). Lyme disease strains in my country are also divided into three genotypes, among which the ratios of B.burgdorferisensue stricto, B.afzelii, and B.garinii genotypes are 5.81%, 23.26%, and 66.28%, respectively. The incidence of Lyme disease is high in the United States and Europe, especially in Scandinavia and Central ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/553
CPCG01N33/553G01N33/56911G01N33/68G01N2333/20Y02A50/30
Inventor 杜卫东黄娜丽叶雷
Owner ANHUI MEDICAL UNIV
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