錦鯉疱疹病毒抗體力價與中和效力之相關性分析

Analysis the Correlation between ELISA Titer and Neutralizing Effect of Antibody of Koi Herpesvirus

陳玫伶(Mei-Ling Chen)

指導教授 ： 陳媺玫

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10343

摘要

錦鯉疱疹病毒 (koi herpesvirus, KHV) 為一新興起的魚類病毒性疾病，會造成錦鯉 (Cyprinus carpio koi) 及鯉魚 (Cyprinus carpio carpio) 80~100% 的死亡率。此病毒性疾病具有季節性、在水溫18-28℃間最具感受性。KHV基因體為線性、具有雙股DNA且其大小約為295 kbp。經親源性分析結果，KHV目前已被歸類至Alloherpesviridae下之鯉科疱疹病毒第三型 (Cyprinid herpesvieus 3, CyHV-3)。KHV於1998年最初發生於以色列，我國則於2002年12月出現首例。許多文獻指出耐過KHV感染的錦鯉可能會成為帶原KHV並長時間表現潛伏性感染的帶原者。這些帶原者可能會在季節變化時，將體內潛在的KHV傳播給健康的錦鯉或新進的魚隻，導致高死亡率及大規模的疾病爆發。本實驗為了解潛在性的帶原者，將收集各養殖場錦鯉血清，先進行免疫點墨法的初步篩檢，再另外作KHV間接型ELISA的建立，最佳化後的陽性陰性比顯現可有效區分陽性及陰性的血清，並用以測試錦鯉體內是否有抗體的產生。同時再配合血清中和試驗，比較兩種抗體力價的結果，發現同一血清之ELISA及中和力價並非成正比的關係。我們依據各血清兩種抗體力價的表現可分成三組，分別為ELISA與中和力價皆高、ELISA低而中和力價高以及ELISA力價高而中和力價低的組別，我們從中選擇具代表性的血清來作西方墨點法的分析，可見不同組別的血清，其蛋白質表現皆有所不同。在純化病毒的抗原上，中和力價高的血清傾向於較大的蛋白質表現，而ELISA力價高的血清則傾向於有較小的蛋白質訊號。

關鍵字

抗體 ； 間接型酵素連結免疫吸附法 ； 錦鯉疱疹病毒 ； 中和力價

並列摘要

Koi herpesvirus (KHV), is a newly recognized virus associated with 80~100% mortality in common (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) carp. This disease is seasonal, afflicting fish when the water temperature is 18 to 28℃. The genome of KHV comprises a linear, double-stranded DNA sequence of 295 kbp. Phylogenetic analysis of the KHV genome sequence led to its classification in the new family Alloherpesviridae under the species name Cyprinid herpesvirus 3. KHV was found firstly in Israel in 1998, and the first case in Taiwan has been identified in December 2002. Some reports indicated that koi survived from KHV infection could be the carrier harboring KHV, presenting latent infection for a long period. These suspected carriers could spread KHV to naive koi or new fish through the seasonal change, cause high mortality and large scale of illness. To understand the titer of antibody in serum of these potential carriers, dot blot assay can be used to primarily screen the koi serum collected from fisheries, optimize the reagents, then established an indirect ELISA method for the diagnosis of KHV. Our results revealed good positive-to-negative ratio (P/N ratio) that could effectively differentiate the positive from negative serum and detect the titer of antibodies in koi. Comparison of serum neutralization (SN) test and ELISA value indicated that the titers of ELISA and serum neutralization were not positive correlation. Based on the correlation of two kind of titers, sera could be seperated to three groups as high ELISA and SN titer, low ELISA but high SN titer and high ELISA but low SN titer as well. We choose one most remarkable serum from each groups to analyze by western blot assay, and the protein patterns of each groups were different. Serum with high SN titer tended to express strong signals to larger proteins while serum with high ELISA titer express most protein signals to smaller proteins on purified KHV virion.

並列關鍵字

antibody ； ELISA ； koi herpesvirus ； neutralizing

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