Elimination of shrimp endogenous peroxidase background in immunodot blot assays to detect white spot syndrome virus (WSSV).
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 3-3'-diaminobenzine tetrahydrochloride 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 different concentrations 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. Phenylmethylsulfonyl fluoride 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 PTU for 2 h yielded optimal inhibition and that such pretreatment of samples eliminates false positive results in immunodot blot assays.
Zhan
Wen-Bin
WB
Laboratory of Pathology and Immunology of Aquatic Animals, LMMEC, Ocean University of China, Qingdao 266003, China. wbzhan@mail.ouc.edu.cn
Chen
Jing
J
Zhang
Zhi-Dong
ZD
Zhou
Li
L
Fukuda
Hideo
H
D000818:Animals; D017756:Aquaculture; D004267:DNA Viruses / Q000276:immunology / Q000302:isolation & purification*; D005189:False Positive Reactions; D015151:Immunoblotting / Q000662:veterinary; D033561:Penaeidae / Q000201:enzymology / Q000821:virology*; D009195:Peroxidase / Q000037:antagonists & inhibitors*; D010670:Phenylthiourea / Q000494:pharmacology
D010670:Phenylthiourea; D009195:Peroxidase