| 摘要: | 本研究目的以霍亂毒素（cholera toxin, CT）免疫小鼠來生產及純化霍亂毒素多株與單株抗體，應用霍亂毒素多株抗體建立霍亂毒素酵素連結免疫分析法（enzyme-linked immunosorbent assay, ELISA），並開發霍亂毒素單株抗體於免疫分析試片（immunoassay strip）檢測上。傳統檢測霍亂毒素以逆相被動乳膠凝集法（reverse passive latex agglutination, RPLA）檢測毒素蛋白質和利用聚合酶鏈鎖反應法（polymerase chain reaction, PCR）檢測毒素基因為主。RPLA雖能檢測出1-2 ng/mL CT，但易受檢體中其他腸內毒素而影響其準確性，且需費時20-24小時才能判讀結果。而PCR檢測霍亂毒素基因雖較免疫分析法靈敏且快速，但易造成誤判不具分泌霍亂毒素能力之非產毒型霍亂弧菌。
本研究首先進行霍亂毒素多株及單株抗體之生產與純化。小鼠腹水抗體使用Hitrap™ rProtein A Column純化出霍亂毒素多株抗體，經間接型ELISA免疫分析可有效檢測CT濃度為0.05 µg/mL，且具低交叉反應性。實驗成� 篩選出五株高效價之單株抗體細胞株，分別為CT-3A、CT-4C、CT-11D、CT-11H及CT-9A，經鑑定其單株抗體同種型（isotyping）均屬於IgM重鏈，λ輕鏈。其中CT-3A與CT-9A單株抗體經大量生產以Hitrap™ IgM Purification Column純化後，經間接型ELISA免疫分析抗體效價在1：15,625。第二部分進行膠體金（colloidal gold）合成工作，並成� 將霍亂毒素單株抗體與20 nm膠體金進行抗體染色，進一步應用於霍亂毒素免疫分析試片之開發上。實驗結果此霍亂毒素免疫分析試片在40-100 µg/mL CT間呈一線性關係且無交叉反應出現，並可應用於實際樣品檢測上，檢測所需時間在15-20分鐘。
The objective of this study is to develop an immunoassay strip (immunochromatographic strip) for cholera toxin (CT) using polyclonal and monoclonal antibodies against CT produced. In this study, the enzyme-linked immunosorbent assay (ELISA) for CT was set up using anti-cholera toxin polyclonal antibodies (anti-CT pAb), and the CT immunoassay strip using anti-cholera toxin monoclonal antibodies (anti-CT mAb) was developed. Conventional methods for determining this CT is to use reverse passive latex agglutination (RPLA) to determine CT protein and use polymerase chain reaction (PCR) to determine CT gene. Although RPLA can determine 1-2 ng/mL of CT protein, it can effect its accuracy by other enterotoxin of samples and took 20~24 hours. PCR has more sensitive and rapid than other immunoassays, but it is easy to mistake the non-toxin gene in other V. cholerae.
Firstly, this study was to produce and purify polyclonal and monoclonal antibodies against CT. The experiment result was that mouse anti-CT pAb from mouse ascites purified by Hitrap⢠rProtein A Column. This anti-CT pAb could determine 0.05 µg/mL of CT and low cross-reaction. The five high-titer anti-CT mAb-producing hybridoma cell lines selected and designated CT-3A, CT-4C, CT-11D, CT-11H, and CT-9A. The isotypes of the five anti-CT mAbs were identified as IgM heavy chain and λ light chain. For the purification of anti-CT mAb, Hitrap⢠IgM Purification Column was used. The highest titer of anti-CT mAb (CT-3A and CT-9A) determined by indirect ELISA was 1:15,625. Secondly, we made the colloidal gold from the chloroauric acid (HAuCl4) and labeled anti-CT mAb with the 20 nm colloidal gold. This anti-CT mAb immunoassay strip set up could determine in 40-100 µg/mL CT and without cross-reaction. Moreover, this strip could be applied to test samples in fact, and detection time was approximately 15-20 minutes. |
