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Polypeptide-ELISA kit for detecting specific antibody against envelope glycoprotein of severe fever with thrombocytopenia syndrome virus

An enzyme-linked immunosorbent adsorption and virus envelope technology, applied in the biological field, can solve the problems of long neutralization test cycle, poor sensitivity and specificity, and high requirements for operators, so as to improve detection accuracy, strong specificity, and reduce false positives. positive effect

Active Publication Date: 2016-05-25
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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  • Polypeptide-ELISA kit for detecting specific antibody against envelope glycoprotein of severe fever with thrombocytopenia syndrome virus
  • Polypeptide-ELISA kit for detecting specific antibody against envelope glycoprotein of severe fever with thrombocytopenia syndrome virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of predominant linear B cell antigen polypeptides of the envelope glycoproteins (Gn, Gc) of fever with thrombocytopenia syndrome virus

[0035] According to the characteristics of linear B cell epitopes of strong antigenicity, good surface and strong hydrophilicity, bioinformatics software is used to analyze the amino acid sequence of the envelope glycoprotein (Gn and Gc) of fever with thrombocytopenia syndrome virus, and predict and determine The dominant linear B cell epitope site on the viral envelope glycoprotein. Artificially synthesized 13-segment peptides containing linear B cell epitope sites of Gn and Gc proteins: RGGRSQVSYYPAENSYSR, GKSRTES, REHKTKWVQESSS, SESEEKAC, VNPPEQR, SSGKKSTEIHFH, NGEGNQDDVR, and KCKKSERSSS, EQKSKK, NSSEESSTA on Gc , QVFRSRTKLA, the purity after purification is more than 95%.

Embodiment 2

[0036] Example 2: Preparation of an ELISA plate pre-coated with polypeptide antigen

[0037] Use the synthesized peptides as antigens and dilute them to 10 μg / ml with coating buffer. Take 1 ml of the peptide solution and mix them. After mixing, coat 100 μl / well in a 96-well microtiter plate. , Wash the plate 3 times after overnight at 4°C. Add 200 microliters of phosphate buffer containing 1% bovine serum albumin to each well for sealing, incubate at 37°C for 1 hour, wash the plate 3 times, dry it, seal the ELISA plate in a sealed bag, and store it at 4°C for later use.

[0038] The 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 buffer (0.01mol / L PBS, pH7.4): add 8.0 g of sodium chloride (NaCl), 0.2 g of potassium dihydrogen phosphate (KH 2 PO 4 ), 2.9 grams of d...

Embodiment 3

[0043] Example 3: Preparation of other solutions in the kit

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

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

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

[0047] Enzyme substrate 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 water to make the volume to 1000 ml and mix well.

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

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

[0050] Stop solution: 2 mol / L sulfuric acid solution, add 108.7 ml of 98% concentrated sulfuric acid to 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 kit for detecting specific antibodies of the envelope glycoprotein of fever with thrombocytopenia syndrome virus. Background technique [0002] Fever with thrombocytopenia syndrome virus (severefeverwiththrombocytopeniasyndromevirus, SFTSV), also known as the new Bunya virus, is a new virus belonging to the Bunyavirus family Sandfly virus, transmitted by the bite of a tick. The disease is clinically manifested as gastrointestinal and digestive symptoms such as fever, fatigue, nausea, and vomiting, accompanied by thrombocytopenia, leukopenia, headache and even bleeding in some cases, and a small number of critically ill patients died due to multiple organ failure. The case fatality rate is about 10%. [0003] Fever with thrombocytopenia syndrome virus is a single-stranded negative-strand RNA virus whose genome is composed of three gene segments: lar...

Claims

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Application Information

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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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