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Protein combined chip for Lyme disease immunoserology diagnosis and preparation method and application thereof

A protein and chip technology, applied in the field of biomedical detection, to achieve the effect of reducing non-specificity, avoiding false positives, simple and reliable detection methods

Inactive Publication Date: 2016-05-25
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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  • Protein combined chip for Lyme disease immunoserology diagnosis and preparation method and application thereof
  • Protein combined chip for Lyme disease immunoserology diagnosis and preparation method and application thereof
  • Protein combined chip for Lyme disease immunoserology diagnosis and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] The selection and surface chemical treatment of embodiment 1 carrier

[0081] 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.

[0082] Step 1. Cleaning of gold foil chips:

[0083] 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 6 minutes, rinse with deionized water 4-5 times, ethanol twice, each time for 3 minutes; air-dry with nitrogen, dry and store.

[0084] Step 2. Chemically modify the surface of the cleaned gold foil chip to obtain a solid phase carrier

[0085]Spot the modification solution, that is, a solution of dithiobis(succinimidyl undecanoate) (DSU) in dimethyl sulfoxide (D...

Embodiment 2

[0087] Embodiment 2 quality control experiment

[0088] Preparation of incubation solution 1: Dissolve VlsE, Flagellin, and OspC antigens in PBST-BSA solution, respectively, and prepare concentration gradients of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL mL, 3.13μg / mL, 1.56μg / mL, 0.78μg / mL, 0.39μg / mL, 0.19μg / mL antigen solution.

[0089] Prepare incubation solution 2: Dissolve rabbit anti-VlsEIgG antibody, rabbit anti-FlagellinIgG antibody, and rabbit anti-OspCIgG antibody in PBST-BSA solution, respectively, and prepare antibody concentration gradients of 200 μg / mL, 100 μg / mL, 50 μg / mL, and 25 μg / mL , 12.5 μg / mL, 6.25 μg / mL, 3.13 μg / mL, 1.56 μg / mL, 0.78 μg / mL, 0.39 μg / mL, 0.19 μg / mL solution.

[0090] Preparation of incubation solution 3: Dissolve Cy3-labeled goat anti-rabbit IgG antibody in PBST-BSA solution to prepare a solution with a concentration of fluorescent secondary antibody of 2.5 μg / mL.

[0091] Prepare incubation solution 4: Dissolve equal...

Embodiment 3

[0103] Example 3 Detection of Serum Samples from Lyme Disease Patients

[0104] Sera from 56 clinically diagnosed patients with Lyme disease were selected and divided into two groups, with 28 serum samples from clinically diagnosed patients with Lyme disease in each group, which were detected by the combined protein chip and detection method of the present invention.

[0105]In the first group, the sera of 28 patients with clinically confirmed Lyme disease were spotted on 168 wells of the chip containing VlsE, Flagellin, OspC recombinant antigen, and three VlsEIR6 polypeptide probes at an incubation concentration of 50 μg / mL. , the other 12 wells were sampled with serum from healthy people who were clinically confirmed not infected with Borrelia burgdorferi, and the other 12 wells were sampled with PBST-BSA solution, incubated at room temperature for 1 hour, washed with PBST 3 times after removal, Blow dry with nitrogen gas for 2 minutes each time. In the second group, the se...

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Abstract

The invention belongs to the field of biomedicine detection and particularly relates to a protein combined chip for Lyme disease immunoserology diagnosis and a preparation method and application thereof. The protein combined chip comprises a solid phase carrier and capture molecules fixed to the surface of the solid phase carrier, and the capture molecules comprise Borrelia burgdorferi flagellin, Borrelia burgdorferi outer membrane protein C (OspC) and Borrelia burgdorferi variable main protein sequence expression E protein (VlsE). The protein combined chip can further comprise capture molecules VlsE IR6 polypeptide. Compared with a traditional method, the protein combined chip can conveniently distinguish infection types on the basis of greatly increasing the positive rate.

Description

technical field [0001] The invention belongs to the field of biomedical detection, and in particular relates to a protein combination chip for immunoserological diagnosis of Lyme disease 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 tissue and organ damage. The main pathogen of Lyme disease is Borrelia burgdorferi, which is mainly divided into three subtypes: Borrelia burgdorferi sensustricto, Borrelia afzelii and Borrelia garnii. Lyme disease strains in my country are also divided into three genotypes, among which the ratios of B.burgdorferisensuestricto, 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 Europe; in China, the incidence of Lyme disease is mainly distributed...

Claims

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Application Information

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Patent Type & Authority Applications(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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