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Development of diagnostic reagents for detecting antibodies to structural and non-structural proteins of foot-and-mouth disease virus
Foot-and-mouth disease virus,Structural protein,Non-structural protein,Antibody detection,Enzyme-linked immunosorbent assay,Luminex assay,
|Publication Year :||2014|
|Abstract:||口蹄疫 (FMD)、豬水疱病 (SVD)、水疱性口炎 (VS) 及水疱疹 (VE) 皆屬高度傳染性之動物水疱性疾病，這些疾病於臨床上常被疑惑而不易區別診斷。近年來陸續研發建立能檢測口蹄疫抗體檢測方法且能有效區別其他水疱性疾病，包括sandwich ELISA、sigleplex Luminex及multiplex Luminex (xMAP) 等三種方法。使用的試驗重組蛋白產製於原核大腸桿菌 (E.coli.) 表現系統，經純化而高度保留口蹄疫病毒 (FMDV) O/TW/1997 病毒株的VP1結構蛋白及O/TW/1999病毒株的3ABC非結構蛋白等抗原決定位區域，藉此利用特異性的重組蛋白量產製作單株抗體，以間接結合方式分別建立盤式及微珠等複合型界面檢測平台，應用在血清樣品中的FMDV標的抗體如結構蛋白 (SP-VP1) 及非結構蛋白 (NSP-3ABC) 等評估與分析。在第一項研究中，sandwich ELISA經試驗檢測結果於診斷敏感性 (Dsn) 大致可達98.4%，及於健康和免疫豬隻所測試之診斷特異性 (Dsp) 可達100%，從NSP抗體檢測能力顯示相當於prioCHECK和UBI等商品化試劑，以sandwich ELISA進一步分別與prioCHECK、UBI及CHEKIT等商品化試劑比較分析後，發現一致性分別為97.5%、93.4%及66.6%，其中Kappa統計分析值各為0.95、0.87及0.37。且sandwich ELISA除了可檢測台灣O型口蹄疫病毒株之外，也可檢測其他六種血清型如A、C、Asia 1、SAT 1、SAT 2、SAT 3等牛源血清中的NSP抗體。第二項研究以sigleplex Luminex分析檢測64支感染、307支免疫及280支健康等豬隻血清中的FMDV-NSP抗體，於感染樣品之Dsn可高達100%， Dsp之分析結果顯示免疫樣品可達98.7%，及於健康樣品可達97.5-100%。比較sigleplex Luminex 及商品化3ABC polypeptide blocking ELISA二者之一致性為96.3%，且Kappa統計值為0.92。經試驗顯示sigleplex Luminex與sandwich ELISA二者於感染後的豬隻最早可於第八天檢測到NSP-3ABC抗體。然而，以sigleplex Luminex方法檢測15頭經攻毒而仍出現典型水泡病變的免疫豬皆呈現NSP抗體陽轉反應，sandwich ELISA則只有11頭呈現陽轉反應。第三項研究以xMAP Luminex評估單一血清樣品可同時檢測到FMDV SP-VP1及NSP-3ABC等抗體。經試驗結果顯示，於661支來自感染、健康及免疫等豬隻血清樣品，評估FMDV-SP抗體之Dsn約為90.0-98.7%，及Dsp為93.0-96.5%。然而評估FMDV-NSP抗體之Dsn為90.0%，及Dsp為93.3-99.1%。以xMAP檢測SP及NSP抗體之最早出現時期分別在感染後之第4天及第8天，且可自免疫一次的15頭攻毒豬同時檢測出SP及NSP陽性抗體。以xMAP分別與病毒中和試驗 (VNT) 及一商品化3ABC polypeptide blocking ELISA等試驗檢測感染豬血清樣品，經比較後發現於定性檢測SP及NSP抗體有存在顯著的正相關，然於xMAP及VNT之定量比較後發現二者之相關性頗低，若以xMAP與blocking ELISA 檢測FMDV-NSP抗體之特異性分析則介於93.3 -94.9%。這些研究顯示各種方法之敏感性及特異性皆高於 90 % 以上，可作為區別診斷及免疫狀況等評估之參考，且不會與口蹄疫病毒以外的水疱性疾病如豬水疱病病毒及水疱性口炎病毒等所誘發的抗體產生交叉反應，因此即具有高度的試驗特異性。|
Foot-and-mouth disease (FMD), swine vesicular disease (SVD), vesicular stomatitis (VS), and vesicular exanthema (VE) are highly contagious vesicular diseases of animal but are not able to be differentiated clinically. For the purpose of instant detecting FMD and differentiating it from the other vesicular diseases, many methods have been developed and evaluated in recent years. The structural protein VP1 gene of FMDV O/TW/1997 and the non-structural protein 3ABC gene of FMDV O/TW/1999 were constructed, respectively, into expression vectors, which were based on an Escherichia coli expression system. Subsequently, monoclonal antibodies were generated by immunizing mice with the recombinant proteins, and they were employed to develop the complex interface measurement platforms for FMDV tests. Plate and microsphere formats were developed and evaluated for their abilities in antibody detection from serum samples against structural protein (SP-VP1) and non-structural protein (NSP-3ABC) of the FMDV. In those studies, the sandwich enzyme-linked immunosorbent assay (ELISA), singleplex Luminex and multipex Luminex (xMAP) were developed for rapid detection of the FMD antibodies. In the first study, the developed sandwich ELISA demonstrated a diagnostic sensitivity (Dsn) of 98.4 % and a diagnostic specificity (Dsp) of 100 % for naive and vaccinated pigs; the detection ability of the assay was comparable with those of PrioCHECK and UBI kits. There were 97.5, 93.4 and 66.6 % agreement between the results obtained from our sandwich ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics between our ELISA and the kits were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies to nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in sera of infected cattle. In the secondary study, sera from 64 infected, 307 vaccinated, and 280 naive pigs were tested for the FMDV-NSP antibody by the sigleplex Luminex assay. The Dsn of the assay was 100%. The Dsp of the assay was 98.7% in vaccinated pigs and 97.5% to 100% in naive pigs. Agreement between the results from the singleplex Luminex and those from a 3ABC polypeptide blocking ELISA was 96.3%, and kappa statistics gave a value of 0.92. The singleplex Luminex can detect the immune response to NSP-3ABC in swine as early as eight days post-infection as same as sandwich ELISA. Moreover, the NSP-3ABC antibody in all of the 15 vaccinated but unprotected pigs which presented vesicular lesions were detected by the singleplex Luminex assay, and the antibodies in 11 of the 15 pigs were detected this antibody by the sandwich ELISA. In the third study, an xMAP was optimized to detected antibodies to SP-VP1 and NSP-3ABC of the FMD virus simultaneously in a single serum sample. To detect SP antibodies in 661 sera from infected, naive pigs and vaccinated pigs, the DSn and DSp of the xMAP were 90.0-98.7% and 93.0-96.5%, respectively. To detect NSP antibodies, the DSn was 90% and the DSp ranged from 93.3% to 99.1%. The xMAP can detect the immune response to SP and NSP as early as 4 and 8 dpi, respectively, in the experimentally infected pigs Moreover, the SP and NSP antibodies in 15 vaccinated but unprotected pigs were detected by the xMAP. Comparing the abilities in detecting the SP and NSP antibodies in the sera of infected samples, the results from the xMAP had high positive correlation with those from the virus neutralization test (VNT) and commercially available 3ABC polypeptide blocking ELISA. However, the results of xMAP had no quantitative relationship with those of the VNT. Furthermore, the specificity was 93.3-94.9% with the blocking ELISA for detecting the FMDV-NSP antibody. These studies showed that the sensitivity and specificity of the methods developed are higher than 90%, which can be as references for diagnosis and assessment of the immune status. Furthermore, the specificities of these assays were also highlighted by the absence of cross-reactions generated by antibodies against the SVDV and VSV at different titers.
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