Enzyme linked immunosorbent assay kit for detecting sever fever with thrombocytopenia syndrome virus antigen

A technology of enzyme-linked immunosorbent reagents and virus antigens, which is applied in the field of disease detection to achieve the effects of improving detection efficiency, high sensitivity, and strong specificity

Pending Publication Date: 2020-04-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the enzyme-linked immunosorbent assay kit for detection of fever with thrombocytopenia syndrome virus antigen based on horseradish peroxidase and antibody double-labeled gold probe has not been reported yet for the detection of fever with thrombocytopenia syndrome virus surface antigen

Method used

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  • Enzyme linked immunosorbent assay kit for detecting sever fever with thrombocytopenia syndrome virus antigen
  • Enzyme linked immunosorbent assay kit for detecting sever fever with thrombocytopenia syndrome virus antigen
  • Enzyme linked immunosorbent assay kit for detecting sever fever with thrombocytopenia syndrome virus antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The ELISA coated plate coated with the capture antibody was made by the following method: the rabbit polyclonal antibody (Jingtiancheng Biotechnology Co., Ltd.) against the NP antigen of SFTSV was mixed with 0.05M sodium carbonate buffer solution of pH=9.6 Dilute to 40 μg / mL, add to 96-well ELISA plate, 100 μL per well, stand at 4°C for 16 hours and wash with washing buffer; add 5% skimmed milk of final mass, block at 37°C for 2 hours, wash with washing buffer, Pat dry on clean absorbent paper, put in a sealed bag, vacuumize and store at 4°C; SFTSV is the abbreviation for fever with thrombocytopenia syndrome virus, and NP is the abbreviation for nucleoprotein.

[0032] Wash buffer was 0.01M PBS buffer pH 7.4.

Embodiment 2

[0034] The antigen NP solution with a concentration of 15.6ng / mL was prepared by the following method:

[0035]The nucleotide sequence of the NP of the fever with thrombocytopenia syndrome virus of GENBANK: MF574213.1 was artificially synthesized, and cloned into the pET30a expression vector (commercial product), transferred into BL21 (DE3) competent cells (commercial product), and spread evenly Spread in solid LB medium containing 50 μg / mL kanamycin resistance, culture overnight at 37°C, pick out the correct clones and inoculate them into 2×YT medium containing 50 μg / mL kanamycin resistance, at 37°C Cultivate until the OD value reaches 0.6, then add IPTG to a final concentration of 1 mM, culture overnight at 30°C at 200 rpm; centrifuge at 4°C, 8000 rpm for 20 min, discard the culture supernatant, resuspend the bacteria in PBS, centrifuge, discard the supernatant; Suspension liquid, sonicate in ice bath, power 800W, sonicate for 5s, stop for 5s, repeat 200 times; centrifuge, 4...

Embodiment 3

[0037] AuNP-Ab at a gold nanoparticle concentration of 100 μg / mL 2 -HRP Probe Concentrate was prepared as follows:

[0038] The newly prepared 50 μg / mL aqueous solution of gold nanoparticles (Gold nanoparticles, AuNPs) was adjusted to a pH of 7.0 (or any value between 7.0 and 8.0) with 0.2 M potassium carbonate aqueous solution, and the final concentration was 15 μg / mL. mL horseradish peroxidase-labeled antibody, mixed and stirred at 4°C for 12h, added PEG20000 with a final mass concentration of 0.2%, stirred at room temperature for 30min, centrifuged at 10000rpm for 40min and discarded the supernatant to obtain AuNP-Ab 2 -HRP probe, washed twice with double distilled water with pH 7.0 (or any value between 7.0 and 8.0), redissolved in storage buffer, and prepared AuNP with a gold nanoparticle concentration of 100 μg / mL -Ab 2 -HRP probe concentrate, store at 4°C for later use;

[0039] The antibody labeled with horseradish peroxidase is a monoclonal antibody (1H10) against ...

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Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting sever fever with thrombocytopenia syndrome virus antigen. The enzyme linked immunosorbent assay kit comprises a reagent A, a reagent B, a reagent C, a reagent D, a reagent E, a reagent F, and a reagent H. The reagent A is an ELISA coated plate coated with a capture antibody; the reagent B is a PBS buffer solution; the reagent C is an antigen NP solution with the concentration of 15.6 ng/mL; the reagent D is an AuNP-Ab2-HRP probe concentrated solution with the gold nanoparticle concentration being 100 microgram/mL; for the reagent E, a 0.01 M PBS buffer solution containing 5% of BSA and having a pH value of 7.4 is used as a probe diluent; the reagent F is skim milk powder; the reagent G is 3, 3', 5, 5'-tetramethyl benzidine; and the reagent H is 0.1 M of HCL. The kit is high in sensitivity and specificity, and the aggregation concentration of enzymes on each immune sandwich structure is enhanced. The absorbance value under a specific wavelength is measured, and the sever fever with thrombocytopenia syndrome virus antigen NP can be qualitatively and quantitatively detected.

Description

technical field [0001] The invention relates to the technical field of disease detection, in particular to an ELISA kit for detecting viral antigens of fever with thrombocytopenia syndrome. Background technique [0002] Between 2007 and 2010, severe febrile illnesses associated with gastrointestinal symptoms, thrombocytopenia, and leukopenia emerged in eastern and central China, with a high mortality rate. The disease, known as febrile thrombocytopenia syndrome (SFTS), is caused by a newly identified bunyavirus (SFTSV). Subsequently, SFTS was diagnosed in South Korea and Japan one after another. It has been reported that SFTSV is a negative-strand segmented RNA virus consisting of three segments (L, M and S). The L, M and S segments encode RNA-dependent RNA polymerase, precursors of glycoproteins (Gn and Gc), nucleocapsid protein (NP) and nonstructural protein (NS), respectively. The nucleocapsid protein (NP) is closely related to viral replication and is highly immunogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/56983G01N33/54306G01N2469/10
Inventor 王志云段宇琴赵秋子王涛
Owner TIANJIN UNIV
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