Detection test paper for quickly diagnosing Lyme disease, and preparation method thereof

A technology for rapid diagnosis and detection of test strips, applied in the field of immunodiagnosis, can solve the problems of low sensitivity, long detection time, lack of specificity, etc., and achieve the effects of high detection accuracy, short detection time and strong specificity

Inactive Publication Date: 2019-04-09
HANGZHOU ALLTEST BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Indirect immunofluorescence (IFA) is to immobilize the bacteria on the carrier, add the patient's serum, and use the fluorescent secondary antibody to develop the color. Its sensitivity is low and there are many human interference factors; It is complicated, and it is difficult for general laboratories to implement and popularize; enzyme-linked immunosorbent assay (ELISA) mainly detects Lyme disease-specific antibodies. Traditional ELISA detection methods have non-specific reactions. Although they have high sensitivity, they lack specificity and are easy to detect. Misdiagnosis; these methods are suitable for laboratory research, the detection time is long, and are not suitable for grassroots detection. Therefore, it is urgent to develop a simpler and faster detection method

Method used

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  • Detection test paper for quickly diagnosing Lyme disease, and preparation method thereof
  • Detection test paper for quickly diagnosing Lyme disease, and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Such as figure 1 Shown, Example 1: Preparation of Lyme Disease Recombinant Antigen

[0025] (1) Expression of Lyme disease recombinant antigens

[0026] In NCBI, look for protein groups containing Lyme major antigen epitopes, BmpA (NCBI Reference Sequence: WP_002656850.1), OspC (NCBI Reference Sequence: WP_010890595.1) and VlsE (GenBank: ACC99642.1) to analyze the above three proteins Antigen epitope, the most dominant antigen epitope was selected for gene recombination expression, since the target recombinant protein needs to be expressed in E. The protein was successfully expressed in high yield and high solubility.

[0027] After the codon-optimized gene recombination sequence was double-digested with BamHI and SalI restriction endonucleases (purchased from NEB Company, namely NewEngland Biolabs), it was inserted into pET30a treated with the same two restriction enzymes (Novagen product, Cat. No. 69909 In -3), the Lyme disease recombinant expression vector plasmid...

Embodiment 2

[0030] Example 2: Preparation of Lyme disease recombinant antigen-colloidal gold conjugate

[0031] (1) Preparation of colloidal gold

[0032] Dilute 1% chloroauric acid to 0.01% (mass percentage content) with ultrapure water, add in a 100ml Erlenmeyer flask, and heat to boil with a heating magnetic stirrer. Then accurately absorb 2.0ml of 1% trisodium citrate and slowly add it to the conical flask, stir evenly, continue heating until the solution turns from black to gray and then red, and add an appropriate amount of 0.02% NaN 3 After stirring evenly, the colloidal gold solution was obtained and stored at 4°C. The newly prepared qualified colloidal gold solution should be a solution with a pure, stable, transparent appearance and no sediment and floating matter. After the preparation is completed, it is observed under an electron microscope, and colloidal gold particles with a suitable diameter are selected.

[0033] (2) Preparation of markers

[0034] Under the dark stir...

Embodiment 3

[0035] Example 3: Preparation of Colloidal Gold Immunochromatography Test Paper for Rapid Diagnosis of Lyme Disease

[0036] Such as figure 1 As shown, a colloidal gold immunochromatographic test paper for rapid diagnosis of Lyme disease includes a bottom plate 8 , a sample pad 1 , a gold standard pad 2 , a nitrocellulose membrane 6 and an absorbent pad 7 . The sample pad 1 , the gold standard pad 2 , the nitrocellulose membrane 6 , and the water-absorbing pad 7 are arranged in sequence and bonded together, and are all arranged on the bottom plate 8 . The nitrocellulose membrane 6 is provided with a first detection line 3 , a second detection line 4 and a quality control line 5 . Mouse anti-human IgM monoclonal antibody is set at the first detection line 3 . Mouse anti-human IgG monoclonal antibody is set at the second detection line 4 . Goat anti-mouse IgG polyclonal antibody is set at quality control line 5. The Lyme disease recombinant antigen-colloidal gold conjugate p...

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Abstract

The invention discloses detection test paper for quickly diagnosing the Lyme disease, and a preparation method thereof. A method for detecting the Lyme disease by ELISA (Enzyme Linked Immunosorbent Assay) is long in detection time and is not suitable for substrate detection. The colloidal gold immunochromatography detection test paper for quickly diagnosing the Lyme disease comprises a bottom plate, a sample cushion, a Jinbiao cushion, a nitrocellulose membrane and a water adsorption cushion, wherein the sample cushion, the Jinbiao cushion, the nitrocellulose membrane and the water adsorptioncushion are arranged and connected in sequence and are all arranged on the bottom plate; the nitrocellulose membrane is provided with a first detection line, a second detection line and a quality control line, wherein the first detection line is provided with a mouse anti-human IgM (Immunoglobulin M) monoclonal antibody, the second detection line is provided with a mouse anti-human IgG (Immunoglobulin G) monoclonal antibody, and the quality control line is provided with a goat anti-mouse IgG polyclonal antibody; and the Jinbiao cushion is provided with Lyme disease recombinant antigen-colloidal gold conjugate. The detection test paper has the advantages of short detection time, high detection accuracy, high specificity and convenience in operation, and does not need the help of other equipment instruments.

Description

technical field [0001] The invention belongs to the technical field of immunodiagnosis, and in particular relates to a colloidal gold immunochromatographic detection test paper for rapidly diagnosing Lyme disease and a preparation method thereof. Background technique [0002] Lyme disease is a spirochete infectious disease mediated by ticks, which is a natural foci disease caused by Borrelia burgdorferi. In 1985, my country first discovered the disease case in the forest area of ​​Heilongjiang Province, and the main clinical manifestation of the disease was nervous system damage. The most common nervous system damages are meningitis, encephalitis, cranial neuritis, motor and sensory neuritis. In the first stage of Lyme disease, only antibiotics can be effective, and in the second and third stages, it is useless to use antibiotics, especially for nervous system damage, and there is no specific treatment. In the early stage, it is characterized by chronic migratory erythema ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/577
CPCG01N33/558G01N33/577Y02A50/30
Inventor 陆维克梁伟伟陈金树高飞邵越水
Owner HANGZHOU ALLTEST BIOTECH
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