以抗NS1單源抗體建立ELISA應用於檢測抗禽流感病毒之抗體

Abstract

禽流感病毒 (avian influenza virus, AIV)之非結構蛋白NS1，於病毒複製過程中才會產生，並不存在於病毒顆粒，因此藉由檢測血清中抗NS1抗體，可區別動物血清抗體來自於病毒感染或死毒疫苗免疫，而目前尚未有可應用的抗NS1抗體檢測工具。故本研究以H6N1 AIVs免疫BALB/c小鼠，進行融合製備抗NS1單源抗體 (αNS1 MAb)，以間接免疫螢光染色法 (indirect immunofluorescence assay, IFA)、真核表現系統 (eukaryotic expression system, EES)、西方墨點法 (western blot, WB)及間接型酵素連結免疫吸附法 (indirect ELISA, iELISA)等方式進行特性鑑定。另一方面，以E. coli表現系統及桿狀病毒表現系統製備之A/chicken/Taiwan/2838V/00 (H6N1)NS1重組蛋白 (rNS1與brNS1)，搭配αNS1 MAbs分別做為docking Ab與tracer Ab，篩選出最佳抗體組合 (MAb pair)，最終建立以rNS1為抗原之sandwich blocking ELISA (rNS1 bELISA)；此外也建立以rNS1抗原搭配αNS1 MAb做為docking Ab之sandwich ELISA (rNS1 sELISA)及直接coating rNS1之indirect ELISA (rNS1 iELISA)。以真核表現系統之IFA結果作為判定雞血清是否含抗NS1抗體之黃金標準，以三種ELISA檢測115個雞血清樣品，rNS1 bELISA的臨界值為10%，敏感性為77.8% (35/45)，特異性為74.3% (52/70)；rNS1 sELISA的臨界值為0.59，敏感性為93.3% (42/45)，特異性為92.9% (65/70)；rNS1 iELISA的臨界值為0.76，敏感性為91.1% (41/45)，特異性為97.1% (68/70)。rNS1 sELISA與rNS1 iELISA具有較高之敏感性及特異性，有潛力應用於檢測雞血清中抗NS1抗體，以助於禽流感之監測與防疫。

The non-structural protein 1 (NS1) of AIV is an important indictor Ag of AIV infection because it's not essential for formation of the viral particle but produced during viral replication. Detection of anti-NS1 antibodies in the serum can differentiate AIV infection from vaccination. Therefore, we intended to establish a blocking ELISA which enable to detect antibodies against avian influenza virus. In this study, BALB/c mice were immunized with H6N1 AIVs for generating anti-NS1 monoclonal antibodies (αNS1 MAbs). αNS1 MAbs were screened by immunofluorescence assay (IFA) and characterized by IFA, eukaryotic expression system (EES), western blot (WB), and indirect ELISA (iELISA) to identify the specificity to NS1. On the other hand, recombinant NS1 proteins (rNS1 and brNS1) from A/chicken/Taiwan/2838V/00 (H6N1) were produced by E. coli and baculovirus-insect cell expression system respectively. After optimizing the condition, we established three ELISA systems with αNS1 MAbs and rNS1, they are rNS1 blocking ELISA, rNS1 sandwich ELISA and rNS1 indirect ELISA. The IFA test of EES were taken as the golden standard method of detecting anti-NS1 antibodies in the chicken sera. 115 chicken sera were tested by three ELISA systems. The cut-off value, sensitivity and specificity of rNS1 bELISA were 10%, 77.8% (35/45) and 74.3% (52/70). The cut-off value, sensitivity and specificity of rNS1 sELISA were 0.59, 93.3% (42/45) and 92.9% (65/70). The cut-off value, sensitivity and specificity of rNS1 iELISA were 0.76, 91.1% (41/45) and 97.1% (68/70). These results show that rNS1 sELISA and rNS1 iELISA have potential to be applied on detecting serum anti-NS1 antibodies.