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  • 學位論文

抗口蹄疫病毒結構蛋白VP1單源抗體應用於酵素連結免疫吸附試驗

Establishment of ELISA with Monoclonal Antibody against Structural Protein VP1 of Foot-and-Mouth Disease Virus

指導教授 : 鄭益謙
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摘要


口蹄疫疫苗免疫後,主要是透過血清中和試驗 (serum neutralization test, SN test) 來了解免疫動物獲得中和抗體保護力的情形,但SN test必須使用口蹄疫的活病毒,因而衍生許多的不便及限制。本實驗室使用口蹄疫病毒O/TW/97所製備的單源抗體Q10E-3,已被證明能夠精準辨認口蹄疫病毒涵蓋RGD的中和抗原決定位Site 1序列;利用此單源抗體為tracer Ab,搭配原核系統所表現的口蹄疫重組結構蛋白VP1,建構一個重組結構蛋白VP1阻斷型酵素連結免疫吸附試驗 (recombinant VP1 blocking enzyme-linked immunosorbent assays, rVP1- bELISA),來檢測免疫動物體內的抗體,並以SN test作為黃金標準 (gold standard)。試驗樣本包含不同物種及不同SN力價的血清,分別為豬血清674個、牛血清100個及羊血清100個。結果顯示,以樣本PI值 (percentage inhibition value) 與SN力價所繪製的標準曲線圖,可看出兩個試驗在三種種別動物樣品皆有極顯著的相關性 ( p < 0.001)。而試驗血清所得之cut-off值、敏感性及特異性,在豬血清樣本為20%、58.8% (173/294)、92.9% (353/380),牛血清樣本為10%、66.7% (40/60)、67.5% (27/40),羊血清樣本則為30%、73.3% (44/60)、67.5% (27/40)。推測同一組SN力價血清樣本經過rVP1-bELISA檢測後PI值的差異,可能是因為血清樣本中含有抗不同中和抗原決定位的抗體,或是血清樣本影響了tracer Ab與rVP1的結合所致。本實驗的結果證明了rVP1-bELISA與SN test的正相關性,或許未來可以嘗試加入口蹄疫病毒的其他結構蛋白,再篩選出抗各個中和抗原決定位的單源抗體去搭配組合作用,以獲得更佳的敏感性及特異性,而更能精準地呈現出SN test的檢測結果。

並列摘要


To know the protection of foot-and-mouth disease virus (FMDV) vaccine, intensive serological surveillance has been implemented. And serum neutralization test (SN test) is a conventional serological assay of FMD. The test operates live virus desperately, so it should be handled in P3 class laboratory. To overcome the restriction, we try to establish a recombinant structural protein VP1 blocking enzyme-linked immunosorbent assays (rVP1-bELISA) to replace the SN test in the future hopefully. The rVP1 was expressed by prokaryotic system. The monoclonal antibody (MAb) Q10E-3 recognizing neutralization antigenic site 1 (RGD motif) on VP1 was raised by the immunization against FMDV O/Taiwan/1997. The rVP1-bELISA was checked with swine, cattle and goat sera samples with different SN titers with sample size 674, 100 and 100 respectively. SN test is the gold standard in this experiment. The current result shows the significant correlation between rVP1-bELISA and SN test (p < 0.001) among three species samples. The cut-off value, sensitivity and specificity of swine, cattle and goat samples is 20%, 58.8% (173/294), 92.9% (353/380); 10%, 66.7% (40/60), 67.5% (27/40) and 30%, 73.3% (44/60), 67.5% (27/40), respectively. The reason of the variance of results between same SN titer sample, might be the effect of antibody that induced against different antigenic site. In the future, we can try to add other FMDV structural proteins and MAb that against different antigenic sites in our ELISA kit. It may be able to obtain better sensitivity, specificity and consistency between test results of rVP1-bELISA and SN test.

參考文獻


Abu Elzein, E.M.E. and Crowther, J.R. 1978. Enzyme-labelled immunosorbent assay techniques in foot-and-mouth disease virus research. J Hyg Camb 80: 391-399.
Alexandersen, S., Zhang, Z. and Donaldson, A.I. 2002. Aspects of the persistence of foot-and-mouth disease virus in animals—the carrier problem. Microbes Infect 4: 1099-1110.
Bachrach, H.L. 1968. Foot-and-mouth disease. Ann Rev Microbiol 22: 201-244.
Bahnemann, H.G. 1990. Inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine. Vaccine 8: 299-303.
Barton, D.J., O'Donnell, B.J. and Flanegan, J.B. 2001. 5’ cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis. Embo J 20: 1439-1448.

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