通过酶联免疫吸附法（ELISA)检测CLEC5A乙型脑炎病毒

Szu-Ting Chen; Shie-Liang Hsieh

doi:10.21769/BioProtoc.268

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Peer-reviewed

Detection of CLEC5A-JEV Interaction by ELISA

通过酶联免疫吸附法（ELISA)检测CLEC5A乙型脑炎病毒

Szu-Ting Chen

Shie-Liang Hsieh email

发布: 2012年10月05日第2卷第19期 DOI: 10.21769/BioProtoc.268 浏览次数: 11950

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

May 2012

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Abstract

JEV (Japanese encephalitis virus) belonging to Flaiviridae interacts with CLEC5A (C-type lectin domain family 5, member A), a member of C-type lectin associated with DAP12 signaling protein and expressed on myeloid cell, as the same extent as Dengue virus. This protocol is used to perform and determine the interaction between purified JEV particles with human and murine CLEC5A.Fc fusion proteins by ELISA method. The different strain and batch of purified JEV as well as purity of CLEC5A.Fc fusion protein might affect the result of absorbance. Modifications are most likely needed if users intend to perform ELISA assay.

Keywords: CLEC5A (clec5a)

Japanese encephalitis virus (日本脑炎病毒)

Flavivirus (黄病毒)

ELISA (ELISA)

Materials and Reagents

The neurovirulent (RP-9) of Japanese encephalitis virus (JEV)
Aedes albopictus mosquito cell line C6/36
BHK21 cell line
20% sucrose
Phosphate buffered saline (PBS)
FreeStyle 293-F cells (Life Technologies, Invitrogen™, catalog number: R790-07 )
pcDNA3/CLEC5A_ECD.Fc plasmid (or plasmids carrying extracellular domain of other C-type lectin receptor)
293 fectin Transfection Reagent (Life Technologies, Invitrogen™, catalog number: 12347-019 )
Gibco FreeStyle 293 Expression Medium (Life Technologies, Invitrogen™, catalog number: 12338018 )
Protein A sepharose beads (GE Healthcare, catalog number: 17-0780-01 )
HRP-conjugated anti-human IgG (Fc) (Jackson ImmunoResearch Laboratories, 109-035-003 )
3,3,5,5-tetramethylbenzidine (TMB) (BD Pharmingen, catalog number: 555214 )
1x RPMI-1640 (Life Technologies, Gibco^®, catalog number: 31800-022 )
Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 11091-148 )
BSA (Sigma-Aldrich, catalog number: A2153 )
KH₂PO₄
Na₂HPO₄
Blocking buffer (see Recipes)
1 L PBS buffer (see Recipes)

Equipment

TECAN ELISA Reader
Beckman Ultracentrifugation
Pasteur pipette
Centrifuge tubes (Corning Incorporated)
Microtiter plate (Corning, Costar^®, catalog number: 9018 )

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Chen, S. and Hsieh, S. E. (2012). Detection of CLEC5A-JEV Interaction by ELISA. Bio-protocol 2(19): e268. DOI: 10.21769/BioProtoc.268.
Chen, S. T., Liu, R. S., Wu, M. F., Lin, Y. L., Chen, S. Y., Tan, D. T., Chou, T. Y., Tsai, I. S., Li, L. and Hsieh, S. L. (2012). CLEC5A regulates Japanese encephalitis virus-induced neuroinflammation and lethality. PLoS Pathog 8(4): e1002655.