Lorenzo G, Estepa A, Coll J M
INIA, CISA-Valdeolmos, Departamento de Sanidad Animal, Madrid, Spain.
J Virol Methods. 1996 Apr 26;58(1-2):1-6. doi: 10.1016/0166-0934(95)01972-3.
An enzyme-immunohistochemical procedure was employed to facilitate neutralization/diagnostic tests for viral haemorrhagic septicaemia virus (VHSV), a significant pathogen in trout farms throughout Europe. The method described can be used for trout or mice antibodies; increases speed (1 day), simplicity, and minimizes the use of reagents compared to other neutralization assays. Furthermore, the test requires a minimum handling of the cell cultures under sterile conditions, decreasing frequent contamination due to the non-sterile conditions of the fish pathological samples. Foci of 5-20 infected epithelioma papillosum carp (EPC) cells are detected and counted with an inverted microscope in under 16 h after infection of EPC monolayers using a high titre anti-N VHSV monoclonal antibody (MAb) 2C9. MAb 2C9 recognizes different viral haemorrhagic septicaemia virus serotypes and VHSV isolates from different host species (trout, salmon and barbel) and Spanish geographical locations. The high titre and specificity of MAb 2C9 favour its conjugation to peroxidase and also make it possible to use in direct immunoperoxidase staining of the VHSV infected EPC monolayers. This neutralization/immunoperoxidase assay should improve diagnostics that use currently agarose or methylcellulose plaque reduction neutralization assays.
采用酶免疫组织化学方法来促进对病毒性出血性败血症病毒（VHSV）的中和/诊断测试，VHSV是欧洲各地鳟鱼养殖场的一种重要病原体。所描述的方法可用于鳟鱼或小鼠抗体；与其他中和试验相比，提高了速度（1天）、简化了操作，并减少了试剂的使用。此外，该测试在无菌条件下对细胞培养物的处理要求最低，减少了因鱼类病理样本的非无菌条件导致的频繁污染。使用高滴度抗N VHSV单克隆抗体（MAb）2C9感染EPC单层细胞后，在16小时内用倒置显微镜检测并计数5 - 20个感染的鲤鱼上皮瘤乳头瘤（EPC）细胞灶。MAb 2C9可识别不同的病毒性出血性败血症病毒血清型以及来自不同宿主物种（鳟鱼、鲑鱼和鲃鱼）和西班牙不同地理位置的VHSV分离株。MAb 2C9的高滴度和特异性有利于其与过氧化物酶结合，也使得它能够用于VHSV感染的EPC单层细胞的直接免疫过氧化物酶染色。这种中和/免疫过氧化物酶测定法应能改进目前使用琼脂糖或甲基纤维素蚀斑减少中和测定法的诊断。
