Polypeptide-ELISA (enzyme-linked immunosorbent assay) kit for detecting specific antibody of N (nucleocapsid) protein of SFTSV (severe fever with thrombocytopenia syndrome virus)

An enzyme-linked immunosorbent assay and nucleocapsid protein technology, applied in the biological field, can solve the problems of long test period, poor sensitivity and specificity, and high cost of neutralization test methods, and achieve improved detection accuracy, strong specificity, and easy operation. easy effect

Active Publication Date: 2016-04-20
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods for detecting fever with thrombocytopenia syndrome virus mainly include immunological detection method, nucleic acid molecular biology detection method, etc. Among them, reverse transcription polymerase chain reaction (RT-PCR) and other methods rely on sophisticated instruments, and the experimental cost is high; And the test method has a long test cycle, poor sensitivity and specificity

Method used

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  • Polypeptide-ELISA (enzyme-linked immunosorbent assay) kit for detecting specific antibody of N (nucleocapsid) protein of SFTSV (severe fever with thrombocytopenia syndrome virus)
  • Polypeptide-ELISA (enzyme-linked immunosorbent assay) kit for detecting specific antibody of N (nucleocapsid) protein of SFTSV (severe fever with thrombocytopenia syndrome virus)
  • Polypeptide-ELISA (enzyme-linked immunosorbent assay) kit for detecting specific antibody of N (nucleocapsid) protein of SFTSV (severe fever with thrombocytopenia syndrome virus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Predominant Linear B Cell Antigen Polypeptide Preparation of Nucleocapsid Protein N of Fever with Thrombocytopenia Syndrome Virus

[0035]According to the characteristics of strong antigenicity, good surface and strong hydrophilicity of the linear B cell epitope, the amino acid sequence of nucleocapsid protein N of fever with thrombocytopenia syndrome virus was analyzed using bioinformatics software, and the core of the virus was predicted and determined. Predominant linear B-cell epitope sites on capsid proteins. The six-segment polypeptides KKLKETGGDDWVKDTK, ASGKMSNSGSKRL, ERAETRL, LKVENYPP, GVSEATT and KMRGASKTEVYNSFRDP containing the linear B cell epitope site of N protein were artificially synthesized, and the purity after purification was more than 95%.

Embodiment 2

[0036] Example 2: Preparation of ELISA plate coated with polypeptide antigen in advance

[0037] Use the synthesized peptides as antigens, dilute them to 10 μg / ml with coating buffer, take 2 ml of the peptide solutions for mixing, and coat them in 96-well microtiter plates at an amount of 100 μl / well , wash the plate 3 times after overnight at 4°C. Add 200 microliters of phosphate buffer saline containing 1% bovine serum albumin to each well to block, incubate at 37°C for 1 hour, wash the plate three times, dry it, seal the microplate in a sealed bag, and store it at 4°C for later use.

[0038] Described solution formula is as follows:

[0039] Coating buffer (0.05 mol / L carbonate buffer, pH9.6): add 1.59 g of sodium carbonate (Na 2 CO 3 ) and 2.93 grams of sodium bicarbonate (NaHCO 3 ), dissolve and mix well.

[0040] Phosphate buffered saline (0.01 mol / L PBS, pH7.4): Add 8.0 grams of sodium chloride (NaCl) to 1000 milliliters of distilled water, 0.2 grams of potassium d...

Embodiment 3

[0042] Embodiment 3: preparation of other solutions in the kit

[0043] Sample diluent: add 1% bovine serum albumin and 0.1% Tween-20 to PBS, pH7.4.

[0044] Enzyme-labeled antibody: HRP-labeled antibody, commercially available, diluted 5000 times with sample diluent.

[0045] Concentrated washing solution: 100 mmol / L PBS containing 1% Tween-20, pH 7.4, diluted 10 times for washing.

[0046] Enzyme substrate A solution: 5.1 g 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 water to the volume to 1000 milliliters and mix.

[0047] Enzyme substrate B solution: 1 ml of 30% hydrogen peroxide.

[0048] Enzyme substrate C: o-phenylenediamine (OPD) powder 1 g.

[0049] Stop solution: 2 mol / L sulfuric acid solution, take 108.7 ml of 98% concentrated sulfuric acid and add 891.3 ml of water, mix and cool.

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Abstract

The invention relates to detection kits in the technical field of biology, in particular to a polypeptide-ELISA (enzyme-linked immunosorbent assay) kit for detecting a specific antibody of an N (nucleocapsid) protein of an SFTSV (severe fever with thrombocytopenia syndrome virus). The kit comprises an ELISA plate pre-coated with synthetic SFTSV N protein linear B-cell antigenic polypeptide, a sample diluent, negative control serum, positive control serum, an HRP (horse radish peroxidase) marked antibody, a concentrated cleaning solution, an enzyme substrate solution and a stop buffer. The kit has the advantages of high specificity, good repeatability and simplicity, convenience and rapidness in operation, and can be widely used for evaluating SFTSV infection conditions.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a polypeptide-enzyme-linked immunosorbent assay kit for detecting specific antibodies to nucleocapsid protein of fever with thrombocytopenia syndrome virus. Background technique [0002] Severe fever with thrombocytopenia syndrome virus (Severefeverwiththrombocytopeniasyndromevirus, SFTSV), also known as novel bunyavirus, is a new virus of the genus Phlebovirus in the family Bunyaviridae, which is transmitted by tick bites. The virus is a single-stranded negative-strand RNA virus, and its genome consists of three gene segments: a large segment (L), a medium segment (M) and a small segment (S). The nucleocapsid protein N is encoded by the S fragment, wraps the genomic RNA, and forms a ribonucleoprotein complex, which plays an important role in gene transcription, replication, and virus assembly. N protein has good antigenicity and can induce the body to produce specific antibodies. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 罗永能高孟孙培蓓姜立民陈军顾春燕张锋倪红霞毛子安
Owner ZHEJIANG PUKANG BIOTECH
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