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Protein chip for lyme disease flagellin antigen immunoserology diagnosis and preparation method and application of protein chip

A protein chip and flagellar antigen technology, applied in the field of protein chips, can solve the problems of easy misdiagnosis and long detection time, and achieve the effects of increased accuracy, high specificity, stability and repeatability

Inactive Publication Date: 2015-02-25
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to make up for the long detection time limit and easy misdiagnosis and missed diagnosis in the clinical diagnosis of Lyme disease caused by Borrelia burgdorferi infection, a new protein chip was developed to detect the Borrelia burgdorferi flagella that causes Lyme disease. The detection of serum IgG antibodies and IgM antibodies produced by antigens will greatly improve the clinical diagnosis rate and effectively screen people in high-incidence areas of the disease, which undoubtedly has potential clinical value and social benefits

Method used

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  • Protein chip for lyme disease flagellin antigen immunoserology diagnosis and preparation method and application of protein chip
  • Protein chip for lyme disease flagellin antigen immunoserology diagnosis and preparation method and application of protein chip
  • Protein chip for lyme disease flagellin antigen immunoserology diagnosis and preparation method and application of protein chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, gold foil chip surface chemical modification

[0051] Step 1. Cleaning of gold foil chips: by NH 3 :H 2 o 2 :H 2 O=1:1:5 volume ratio mixed to obtain TL1 cleaning solution. Put the gold foil chip in a stainless steel cleaning box filled with TL1 cleaning solution, bathe in 82°C water for 6 minutes, wash twice, take it out, rinse it with ultrapure water for 2 minutes, twice, and then wash it with absolute ethanol for 2 minutes, twice , and then blow dry with nitrogen, and place it in a clean and airtight chip box for use.

[0052] Step 2. Chemically modify the surface of the cleaned gold foil chip to obtain a solid phase carrier

[0053] Immerse the cleaned gold foil chip in modification solution 1, and incubate with shaking at room temperature for 12 hours in the dark. After taking it out, wash it with absolute ethanol solution for 3 times, each time for 2 minutes, and then dry it with nitrogen gas; In the modification solution 2, shake and incubate ...

Embodiment 2

[0055] Embodiment 2 quality control experiment

[0056] Preparation of incubation solution 1: Dissolve Borrelia burgdorferi recombinant flagellar antigen in PBST-BSA solution, and configure the protein concentration gradient to be 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL mL, 3.12μg / mL, 1.65μg / mL, 0.78μg / mL, 0.39μg / mL, 0.19μg / mL flagellar antigen solution.

[0057] Prepare incubation solution 2: Dissolve rabbit anti-Borrelia burgdorferi flagellar antigen IgG antibody in PBST-BSA solution, and configure the antibody concentration gradient to be 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25μg / mL, 3.12μg / mL, 1.65μg / mL, 0.78μg / mL, 0.39μg / mL, 0.19μg / mL solution.

[0058] Prepare incubation solution 3: Dissolve Cy3-labeled goat anti-rabbit IgG antibody in PBST-BSA solution, and configure the fluorescent secondary antibody concentration gradient as 5000μg / mL, 2500μg / mL, 1250μg / mL, 625μg / mL, 312.5μg / mL, 156.2 Solutions of μg / mL, 78.1μg / mL, 39.1μg / ...

Embodiment 3

[0077] Example 3 Detection of Serum Samples from Lyme Disease Patients

[0078] The sera of 88 patients with Lyme disease who were clinically diagnosed with Borrelia burgdorferi infection were spotted on 88 wells of the chip containing the probe of recombinant flagellar antigen of Borrelia burgdorferi at an incubation concentration of 50 μg / mL, and the other 4 wells The sample solution of the wells was the serum of healthy people who were not clinically confirmed to be infected with Borrelia burgdorferi, and the sample solution of the remaining 4 wells was PBST-BSA solution, incubated at room temperature for 1 hour, and washed 3 times with PBST after taking it out, 2 minutes each time , blow dry with nitrogen. Do the same on another identical chip. Then dissolve the Cy3-labeled donkey anti-human IgG antibody diluted 1:2500 and the Cy3-labeled goat anti-human IgM antibody diluted 1:2500 in PBST-BSA solution, and apply the samples on two chips incubated with antibodies, and tak...

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Abstract

The invention discloses a protein chip for lyme disease flagellin antigen immunoserology diagnosis and a preparation method and application of the protein chip. The protein chip is characterized in that borrelia burgdorferi recombination flagellin antigen probes are fixed on the surface of a solid phase carrier in a dot matrix mode; the solid phase carrier is a 16-amino-1-hexadecyl mercaptan modified gold foil chip which is combined with 4-(N-maleinimide methyl) cyclohexane-1-carboxylic acid succinimide ester and 4-(dimethylamino) pyridine. The protein chip disclosed by the invention can accurately detect an anti-flagellin antigen IgG antibody and an anti-flagellin antigen IgM antibody in serums of lyme disease patients, the operation is simple, and the detection result is stable.

Description

1. Technical field [0001] The invention relates to a protein chip, in particular to a protein chip for detecting the relevant anti-flagellar antigen antibodies in the serum of Lyme disease patients infected with Borrelia burgdorferi and its preparation and use. 2. Technical background [0002] Lyme disease (LD) is a tick-borne infection of the human body by Borrelia sp. and is characterized by multiple organ damage. Worldwide, Lyme disease is a high incidence area in the United States and Europe, especially in Scandinavia and Central Europe. In China, Lyme disease is mainly distributed in the Greater Khingan Mountains and Dabie Mountains. The main causative agent of Lyme disease is Borrelia, which is divided into three subtypes: Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii. The incidence is higher in children than in adults. Erythema migrans (EM) is a characteristic clinical manifestation of Borrelia burgdorferi infection. Untreated patients with Lyme disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/54393G01N33/553G01N33/6893Y02A50/30
Inventor 杜卫东叶雷
Owner ANHUI MEDICAL UNIV
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