A polypeptide-enzyme-linked immunosorbent assay kit for detecting antibodies specific to the envelope glycoprotein of fever with thrombocytopenia syndrome virus

A virus envelope and syndrome technology, applied in the biological field, can solve the problems of long neutralization test experiment period, poor sensitivity and specificity, and high operator requirements, so as to improve the detection accuracy, strong specificity, and reduce false positives. Effect

Active Publication Date: 2017-03-29
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RT-PCR technology relies on precision instruments, has high requirements for operators, and high experimental costs, so it is limited in the detection of a large number of samples; the neutralization test has a long experimental cycle, poor sensitivity and specificity, and has problems in practical applications. Certain limitations

Method used

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  • A polypeptide-enzyme-linked immunosorbent assay kit for detecting antibodies specific to the envelope glycoprotein of fever with thrombocytopenia syndrome virus
  • A polypeptide-enzyme-linked immunosorbent assay kit for detecting antibodies specific to the envelope glycoprotein of fever with thrombocytopenia syndrome virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of the dominant linear B cell antigen polypeptide of the envelope glycoprotein (Gn, Gc) 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 the envelope glycoprotein (Gn and Gc) of fever with thrombocytopenia syndrome virus was analyzed using bioinformatics software, and predicted and determined Predominant linear B-cell epitope site on the viral envelope glycoprotein. Artificially synthesized thirteen-segment polypeptides containing linear B cell epitope sites of Gn and Gc proteins: RGGRSQVSYYPAENSYSR, GKSRTES, REHKTKWVQESSS, SESEEKAC, VNPPEQR, SSGKKSTEIHFH, NGEGNQDDVR on Gn and KCKKSSS, EELKSKK, SSEESARTIK, RFERSHDSQ, HSSKNST on Gc , QVFRSRTKLA, the purity after purification is over 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 1 ml of the peptide solutions for mixing, and coat them in a 96-well microtiter plate at an amount of 100 μl / well , wash the plate 3 times after overnight at 4°C. Add 200 microliters of phosphate buffered 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, pH 9.6): add to 1000 ml of distilled water

[0040] 1.59 grams of sodium carbonate (Na 2 CO 3 ) and 2.93 grams of sodium bicarbonate (NaHCO 3 ), dissolve and mix well.

[0041] Phosphate buffered saline (0.01 mol / L PBS, pH 7.4): Add 8.0 g of sodium chloride (NaCl) to 1000 ml of disti...

Embodiment 3

[0043] Embodiment 3: preparation of other solutions in the kit

[0044] Sample diluent: add 1% bovine serum albumin and 0.1% Tween-20 to PBS, pH7.4.

[0045] Enzyme-labeled antibody: HRP-labeled antibody, commercially available, diluted 5000 times with sample diluent.

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

[0047] 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.

[0048] Enzyme substrate B solution: 1 ml of 30% hydrogen peroxide.

[0049] Enzyme substrate C: o-phenylenediamine (OPD) powder 1 g.

[0050] 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 the field of biotechnology, especially to a polypeptide-ELISA kit for detecting a specific antibody against the envelope glycoprotein (Gn and Gc) of the severe fever with thrombocytopenia syndrome virus (SFTSV). The kit comprises an enzyme-labeled plate coated with SFTSV envelope glycoprotein dominant linear B cell antigen polypeptide, a sample diluents, negative control serum, positive control serum, a horseradish peroxidase (HRP) labeled antibody, a concentrated washing solution, an enzyme substrate solution and a stopping solution. The kit has the advantages of high specificity and good repeatability, can simply and conveniently detect the specific antibody against SFTSV, reduces false positive results, and is applicable to large-scale serological detection and epidemiological investigation and assessment the infection condition of SFTSV.

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 the envelope glycoprotein of fever with thrombocytopenia syndrome virus. Background technique [0002] Severe fever with thrombocytopenia syndrome virus (Severe fever with thrombocytopenia syndrome virus, SFTSV), also known as novel bunyavirus, is a new virus of the genus Phlebovirus in the family Bunyaviridae, which is transmitted by tick bites. Clinically, the disease manifests as acute fever, fatigue, nausea, vomiting and other gastrointestinal symptoms, accompanied by thrombocytopenia and leukopenia. Some cases have symptoms such as headache and even bleeding. A small number of severe patients died due to multiple organ failure. The case fatality rate is about 10%. [0003] FTSV is a single-stranded negative-strand RNA virus, and its genome consists of three gene segments: large segment (L),...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 罗永能孙培蓓蒋秀梅陈卓许玲敏严峥高孟倪红霞毛子安
Owner ZHEJIANG PUKANG BIOTECH
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