C-反應蛋白 ( C-reactive protein, CRP ) 是一種急性時期的血漿蛋白質，當身體受到細菌感染或組織細胞受到傷害時，其在血液中的濃度會很快的上升。傳統檢測CRP是以酵素連結免疫吸附分析法 ( enzyme-linked immunosorbent assay, ELISA ) 及螢光免疫連結分析法為主，但是這些方法在檢測上步驟繁雜、需標識 ( labeling ) 來產生訊號，且需昂貴儀器來進行分析。本研究之目的是以CRP 免疫小鼠來生產CRP多株與單株抗體，並利用所生產之CRP多株抗體建立ELISA檢測系統，同時應用其單株及多株抗體於免疫層析試片 ( immunochromatographic strip ) 之開發上。本研究所製備之可拋棄式免疫層析試片，不僅具有方便、快速、經濟、有效之優點，且不需要任何儀器之使用。
本研究首先進行CRP多株及單株抗體之生產與純化。小鼠血清抗體使用Hitrap™ rProtein A Fast Flow Column純化出CRP多株抗體，經間接型ELISA免疫分析可有效檢測CRP濃度為0.05 µg/mL，且具低交叉反應性。實驗成正z選出八株高效價之單株抗體融合瘤細胞株，分別為CRP-3C、CRP-5E、CRP-9G、CRP-11F、CRP-11G、CRP-11H、CRP-12C 及CRP-12F，經鑑定其單株抗體同種型 ( isotype ) 有五株屬於IgG2b重鏈，κ輕鏈，另有三株為IgA重鏈，κ輕鏈。其中CRP-9G單株抗體經大量生產後，以Hitrap™ rProtein A Fast Flow Column純化，經間接型ELISA分析抗體效價為1：15,625。第二部分進行膠體金粒子 ( colloidal gold particles ) 之合成工作，並成弗NCRP單株抗體與25 nm膠體金結合，成扒}發出CRP免疫層析試片。實驗結果顯示此CRP免疫層析試片在 5-100 µg/mL間呈線性關係且無交叉反應出現，並可應用於老鼠血清樣品檢測，檢測所需時間約5-10分鐘。 C-reactive protein (CRP) is a major acute-phase plasma protein displaying rapid and pronounced rise of its serum concentration in response to infection or tissue injury. Conventional methods for determining CRP were enzyme-linked immunosorbent assay (ELISA) and fluorescence immunoassay. These methods needed long procedure, labeling and specific machine for determination. The objective of this study is to develop an immunochromatographic strip for CRP using polyclonal and monoclonal antibodies against CRP produced. In this study, the ELISA for CRP was set up using anti-CRP polyclonal antibodies (anti-CRP pAb). The CRP immunochromatographic strip using anti-CRP monoclonal antibodies (anti-CRP mAb) and anti-CRP pAb was also developed. The self-assembled immunochromatographic strip was convenient, rapid, low price and no any machine needed in clinical diagnosis.
Firstly, this study was to produce and purify polyclonal and monoclonal antibodies against CRP. The anti-CRP pAbs obtained from ICR mouse ascites were purified by Hitrap™ rProtein A Fast Flow column. The anti-CRP pAb could determine 0.05 µg/mL CRP by ELISA and had low cross-reaction. The eight high-titer anti-CRP mAb-producing hybridoma cell lines were selected and designated CRP-3C、CRP-5E、CRP-9G、CRP-11F、CRP-11G、CRP-11H、CRP-12C and CRP-12F. The isotypes of the five anti-CRP mAbs were IgG2b heavy chain and κ light chain and those of three mAbs were IgA heavy chain and κ light chain. For the purification of anti-CRP mAb, Hitrap™ rProtein A Fast Flow column was also used. The highest titer of anti-CRP mAb (CRP-9G) determined by indirect ELISA was 1:15,625. Secondly, the colloidal gold was made from the chloroauric acid (HAuCl4) and labeled anti-CRP mAb with 25 nm of colloidal gold. This CRP immunochromatographic strip prepared could determine for 5-100 µg/mL CRP and no cross-reaction. Moreover, this strip could be applied to test mouse serum samples, and the detection time was approximately 5-10 minutes.
