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Polypeptide-ELISA (enzyme linked immunosorbent assay) kit for detecting H7N9 subtype avian influenza virus NA (neuraminidase) specific antibody

An enzyme-linked immunosorbent, neuraminidase technology, applied in biological testing, measuring devices, material testing products, etc., can solve the problems of high operator requirements and expensive instruments, and achieve high sensitivity, easy operation, specificity and so on. strong effect

Inactive Publication Date: 2016-03-30
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of H7N9 is mostly carried out by a combination of clinical diagnosis and laboratory diagnosis. Laboratory diagnosis is mainly based on reverse transcription polymerase chain reaction (RT-PCR). more demanding

Method used

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  • Polypeptide-ELISA (enzyme linked immunosorbent assay) kit for detecting H7N9 subtype avian influenza virus NA (neuraminidase) specific antibody
  • Polypeptide-ELISA (enzyme linked immunosorbent assay) kit for detecting H7N9 subtype avian influenza virus NA (neuraminidase) specific antibody
  • Polypeptide-ELISA (enzyme linked immunosorbent assay) kit for detecting H7N9 subtype avian influenza virus NA (neuraminidase) specific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Predict and determine the linear B cell resistance of H7N9 subtype avian influenza virus neuraminidase (N9)

[0032] pro-epitope

[0033] (1) Download the neuraminidase amino acid sequence of H7N9 avian influenza virus Zhejiang strain from GenBank, and use the Protean program in the bioinformatics software DNAStar to analyze the antigenicity (AntigenicIndex), hydrophilicity (HydrophilicityPlot) and surface accessibility of the sequence Surface ProbabilityPlot, calculate the weighted average of each index, and predict the potential linear B cell epitopes 1-SQPETTNTS, 2-NYYNETN, 3-IQMEERTSRNFNNLTK, 4-IRGKHSNGTIHDRSQY, 5- See Table 1 for DNPRPNDPNIGKCNDPYPGNNNN, 6-ALTDDRSKPIQ, and 7-GRPKEDK.

[0034] Table 1 is the peptide information of the predicted H7N9 neuraminidase linear B cell epitope

[0035]

Embodiment 2

[0036] Example 2 Preparation of an ELISA microtiter plate pre-coated with H7N9 avian influenza virus neuraminidase linear B cell antigen epitope polypeptide

[0037] (1) Dilute the artificially synthesized N9 antigen polypeptide to 10-30 micrograms / ml with coating solution, add to 96-well ELISA plate, 100 microliters / well, react overnight at 4°C, wash the plate 3 times with washing solution;

[0038] (2) Add 200 microliters of phosphate buffer solution containing 1% bovine serum albumin to each well for blocking, incubate at 37°C for 2 hours, wash the plate 3 times with washing solution, and dry it in the air.

[0039] The solution formula used is as follows:

[0040] Coating buffer (0.05 mol / L carbonate buffer, pH9.6): 1.59 grams of sodium carbonate (Na 2 CO 3 ), 2.93 g of sodium bicarbonate (NaHCO 3 ), add 1000 ml of distilled water, dissolve and mix well.

[0041] Phosphate buffered saline (0.01 mol / L PBS, pH7.4): 8.0 g sodium chloride (NaCl), 0.2 g potassium dihydrogen...

Embodiment 3

[0043] Preparation of other solutions in the third kit

[0044] (1) Sample diluent: 1% bovine serum albumin and 0.1% Tween-20 were added to PBS to adjust the pH to 7.4.

[0045] (2) Enzyme-labeled antibody: commercially available HRP-labeled IgG, used after dilution of 1:5000.

[0046] (3) Concentrated washing solution: 100 mmol / L PBS containing 1% Tween-20, pH 7.4, diluted 10 times for use.

[0047] (4) Chromogenic solution: composed of enzyme substrate A solution, B solution and substrate C. A solution: 5.1 grams of citric acid (C 6 h 8 o 7 ·H 2 O), 18.4 grams of disodium hydrogen phosphate (Na 2 HPO 4 12H 2 O), add 1000 milliliters of distilled water and mix; B solution: 30% hydrogen peroxide (H 2 o 2 ) 1 ml; Substrate C: 1 g of o-phenylenediamine (OPD) powder.

[0048] (5) Stop solution (2 mol / L sulfuric acid solution): Take 108.7 ml of 98% concentrated sulfuric acid and add it to 891.3 ml of water, mix and cool.

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Abstract

The invention relates to a detection kit, in particular to a polypeptide-ELISA (enzyme linked immunosorbent assay) kit for detecting a H7N9 subtype avian influenza virus NA (neuraminidase) specific antibody. The kit comprises a 96-hole ELISA plate precoated with H7N9 NA linear B cell epitope polypeptide, positive serum, negative serum, an antibody marked with HRP (horse radish peroxidase), a sample diluent, a concentrated cleaning solution, a color development solution and a stop solution. NA linear B cell epitope of a Zhejiang strain of the H7N9 subtype avian influenza virus is predicted with bioinformatics software, corresponding polypeptides are artificially synthesized and then the ELISA plate is coated with the polypeptides; the NA specific antibody in serum is detected in a polypeptide-ELISA manner, and the polypeptide-ELISA kit is established. The polypeptide-ELISA kit for detecting the H7N9 subtype avian influenza virus NA specific antibody does not require expensive equipment such as a PCR (polymerase chain reaction) instrument and the like, has the advantages of quickness, simplicity, convenience, sensitivity, high specificity and the like and can be widely used in grassroots units.

Description

technical field [0001] The invention relates to a detection kit, in particular to a polypeptide-enzyme-linked immunosorbent (ELISA) kit for detecting the specific antibody of H7N9 subtype avian influenza virus neuraminidase (N9). Background technique [0002] Avian influenza virus is a negative-strand RNA virus of type A influenza virus. It can be divided into different subtypes according to the antigenic differences of hemagglutinin (HA) and neuraminidase (Neuraminidase, NA). HA has 16 antigenic types. , NA has 9 antigenic types, and different HA and NA form different influenza virus subtypes. NA is a glycoprotein that constitutes an important component of the viral envelope fibrils, which can hydrolyze the sialic acid residues at the sugar end to release the virus particles from the host cell receptors. The subtypes of avian influenza viruses that have been confirmed to infect humans include H5N1, H9N2, H7N7, H7N2, H7N3, H5N2, H7N9, H10N7 and H6N1, among which H5N1 is the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/68G01N33/543
CPCG01N33/56983G01N33/543G01N33/68G01N2333/11
Inventor 高孟罗永能谢珲沈立军姜立民顾春燕高丽美倪红霞毛子安
Owner ZHEJIANG PUKANG BIOTECH
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