家禽流行性感冒病毒H5亞型ELISA診斷試劑之開發及垂直傳播試驗

Development of ELISA Kit for Detecting H5 Subtype Avian Influenza Viruses and Vertical Transmission Test

陳奕成(Yi-Cheng Chen)

指導教授 ： 王金和

國立臺灣大學/生物資源暨農學院/獸醫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02147

摘要

本研究目的以sandwich ELISA快速診斷H5亞型抗原，以blocking ELISA快速診斷H5亞型抗體。其方法為將3233/04之濃縮純化病毒與其HA重組蛋白免疫小鼠進行單源抗體的製備，並將製備好的單源抗體以免疫墨點法 (immunodot blot assay)、西方轉漬法 (western blot assay) 及血球凝集抑制試驗 (hemagglutination inhibition test) 分析其特性，接著進行單源抗體純化與過氧化氫酶標示後成為追蹤子 (tracer)，最後以blocking ELISA的架構進行野外H5亞型陽性血清檢測，以sandwich ELISA的架構進行H5亞型病毒檢測。在sandwich ELISA結果方面，只能偵測到3233/04 (H5N2) 病毒株，且病毒力價於106 EID50/0.1 mL以上時皆可被檢測到。在blocking ELISA結果方面，以228個H5陰性的血清樣本進行檢測，其特異性為97.36％ (222/228)；以234個H5陽性的血清樣本進行檢測，其敏感性為98.29％ (230/234)。
家禽流行性感冒病毒是否會經由母雞垂直傳染給小雞的證據目前仍缺乏，因此本實驗為將宜蘭雞場分離到的H6N1亞型病毒株2838K/00，一方面進行母雞攻毒試驗，視其病毒是否會經由母雞垂直傳染給小雞，而另一方面則進行無特定病原雞胚胎蛋攻毒試驗，視其小雞是否會將病毒直接帶毒出來。其方法為取108.65 EID50/0.1 mL的病毒量，以鼻內接種法攻毒於產蛋母雞，另將病毒液稀釋成1EID50/0.1 mL的劑量，直接將病毒液經由尿囊腔接種於無特定病原的雞胚胎蛋中，接著收取攻毒後母雞所生小雞與直接攻毒於SPF雞胚胎蛋出生小雞之氣管、腎臟與卵黃囊，以SPF雞胚胎蛋分離兩代後，抽取尿囊液以RT-PCR的方式進行AIV NP基因的檢測。其結果可從卵黃卵白中檢測到病毒核酸，但出生小雞檢體其病毒分離結果為陰性，依此結果推論家禽流行性感冒病毒不會有垂直傳播的情形發生。

關鍵字

家禽流行性感冒病毒 ； 酵素連結免疫吸附法

並列摘要

In this study, we used sandwich ELISA to detect H5 virus and blocking ELISA to detect H5 antibody. The concentrated purified virus (3233/04) and hemagglutinin (HA) recombinant proteins were used for the production of monoclonal antibodies. These monoclonal antibodies were confirmed to be specific to the H5 HA by immunodot blot assay, western blot assay and hemagglutination inhibition test. These monoclonal antibodies were purified and then labeled with horseradish peroxidase to become tracer. The tracer was used for the detection of H5 antibody in serum by blocking ELISA and for the H5 subtype virus by sandwich ELISA. The results showed that sandwich ELISA detected the H5 virus above the titer of 106 EID50/0.1 mL. The specificity and sensitivity for blocking ELISA were 97.36% (222/228) and 98.29% (230/234), respectively.
Little information about AIV vertical transmission is available. This study aimed to prove the existence of vertical transmission of AIV from hens to embryos. We used a H6N1 low pathogenetic AIV Taiwanese isolate (Yilan, Taiwan), 2838K/00, in the vertical transmission test. After challenging the hens with 108.65 EID50/0.1 mL of 2838K/00, and inoculating the specific-pathogen-free embryonated eggs with 1EID50/0.1 mL of 2838K/00, the embryos were hatched. The tracheas, kidneys, and yolk sacs were collected from the hatched chicks for the virus isolation by the allantoic sac route. After two passages, we harvested the allantoic fluid and tested for the detection of NP gene of AIV by RT-PCR. The results showed that we detected the nucleic acid of AIV in the yolk and albumen mixture from unfertilized eggs. But we didn’t detect the AIV in the hatched chicks. According to above results, AIV seems to be no vertical transmission in chickens.

並列關鍵字

Avian influenza virus ； ELISA

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