Elimination of shrimp endogenous peroxidase background in immunodot blot assays to detect white spot syndrome virus (WSSV)

Diseases of Aquatic Organisms

Jing Chen¹  Li Zhou¹  Zhi-Dong Zhang¹  Hideo Fukuda¹  Wen-Bin Zhan¹ 

关键词: Shrimp;    Peroxidase;    WSSV;    Immunodot blot;   

DOI  :  10.3354/dao053263

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: False positive results were obtained in immunodot blot assays to detect white spot syndrome virus when horseradish peroxidase-conjugated sheep anti-mouse immunoglobin (Ig) serum was used as a secondary antibody with 3-3¹-diaminobenzinetetrahydrochloride dihydrate as the detection substrate. The cause was considered to be a reaction of shrimp endogenous peroxidase (POD) with the substrate. In experiments designed to inhibit POD activity, 9 different reagents were used at differentconcentrations and for different treatment times. EDTA, sodium azide, HEPES-Na, NaHSO₃, H₂O₂ and phenylthiourea (PTU) were able to inhibit POD activity by 44, 60, 64, 67, 79, and 90%, respectively. Phenylmethylsulfonylfluoride did not inhibit POD, and neither periodic acid nor H₂O₂ in methanol were appropriate due to the formation of flocculant precipitates when added to shrimp extracts. It was concluded that of the treatments tested, 10 mM PTUfor 2 h yielded optimal inhibition and that such pretreatment of samples eliminates false positive results in immunodot blot assays.

【 授权许可】

Unknown