科研動態

Simoa技術:飛克級蛋白質高靈敏檢測服務——華盈生物

2019/03/27

        蛋白質是生命功能的最終執行者,也是臨床診斷、疾病分型、藥物篩選等生物醫學相關領域重要、應用廣的檢測對象。酶聯免疫吸附實驗(ELISA)技術一直以來在蛋白質檢測中占據著主導地位。ELISA的靈敏度一般只能達到pg/mL水平,對于超低豐度蛋白的檢測往往力不從心。然而,在疾病早期,精確檢測這些超低豐度蛋白又對預判疾病的發生與轉歸有著重大意義,伴隨著大健康產業和精準醫學的發展,傳統方法的技術瓶頸與巨大市場需求之間的矛盾日趨凸顯。

        解決矛盾就意味著獲得海量市場,各生物技術公司試圖從各個角度入手改進現有的蛋白檢測技術,以突破超低豐度蛋白質檢測的瓶頸問題。它們普遍分為兩個技術流派:

        1)以增強檢測信號強度為出發點,結合電化學發光而開發的技術,如MSD。

        2)以降低背景噪音為出發點,結合毛細管電泳而開發的技術,如Ella和Erenna等。

        然而這些技術只能稱為高靈敏,不能稱為超靈敏,它們依然無法滿足NfL、Tau等神經因子在外周血清中的檢測,也無法在疾病初期近健康狀態下檢測IL-1β這些低豐度蛋白,應用范圍仍然受到限制。

        Simoa(Single-molecule Array)技術靈敏度比ELISA高1000倍以上,它的出現將蛋白質檢測技術直接帶入到單分子、數字化檢測時代,成為fg級超低豐度蛋白質檢測領域的優勢技術

 

Simoa是如何解決超低豐度蛋白檢測問題的呢?

 

        Simoa核心技術環節有兩個:
        1. 超低的反應體系,在提高靈敏度的同時,指數級降低背景噪音和信號擴散;

        2. 數字化檢測設計與定量方法,實現單分子信號的獨立識別與計算,不放過任何一個檢測信號。


        Simoa檢測的生物學原理仍然是經典的免疫反應-雙抗夾心法,所不同的是,Simoa技術將約250,000個捕獲抗體包被在2.7 μm的小磁珠上,檢測時加入生物素標記的檢測抗體及親和素偶聯的酶和底物,通過一層油將單個磁珠分別封閉在238,000個4.5 μm的反應孔(Well)中進行反應。由于每個小孔的反應體系僅僅為50飛升,比傳統ELISA小20億倍,這時小孔中即使只有一個分子,其催化底物就可產生3000個熒光分子,通過CCD攝像頭即可捕獲到信號,利用泊松分布理論可計算出陽性熒光小孔(On Well)對應的蛋白濃度值,實現數字化單分子檢測的愿望。

 

對比.png

圖1 Simoa與ELISA對比


        ELISA檢測如同墨水滴入2000個鳥巢大小的泳池中,信號會無限地擴散和稀釋,只有樣品濃度達到一定閾值(比如pg/mL以上),才能被ELISA檢測到,再根據所有信號的平均值來計算濃度。而Simoa的檢測就好比將一滴墨水滴入礦泉水瓶中,很容易就被觀察到。之后通過對238,000個孔中每一個陽性信號孔(On Well)獨立納入濃度計算公式,即Simoa是將前沿的“數字化”檢測原理在蛋白檢測中進行運用的技術

David Yeung [1]等研究人員對比了目前世界上幾乎所有超敏蛋白質檢測技術對于IL-2、IL-6、IL-17A、TNF-α等低豐度炎癥因子的檢測情況,發現Sioma技術無論在靈敏度還是在數據重現性上,相比其他技術均具有顯著優勢(見表1)。

表1 Simoa與其它高敏蛋白檢測技術對比
表1.png

FEAD: Frequency of endogenous analyte detection

 圖2.png

 

        綜上,我們可以看出Simoa技術是低豐度蛋白檢測領域真正的王者,它的檢測能力超乎我們的想象,可以滿足其它所有技術無法完成的檢測需求。

 

        1. Simoa可以在血清/血漿中檢測NfL、Tau、pTau、Aβ40、Aβ42等超低豐度神經因子;

        2. Simoa可以通過加大稀釋倍數,檢測房水、玻璃體、眼淚等微量樣品中的炎癥因子;

        3. Simoa可以在單個細胞中定量蛋白,實現單個胚胎細胞培養上清中的蛋白檢測;

        4. Simoa可以在外泌體等稀少樣品類型中檢測PD1、PD-L1等蛋白;

        5. Simoa可以在阿爾茲海默癥初期(提前16年),在接近正常人的患者血清中檢測到蛋白標志物NfL[2]

 
        目前Simoa的商品化試劑盒已經廣泛覆蓋腫瘤、神經、心血管、免疫等,華盈生物可以為多種類型的樣品(如:血清、血漿、腦脊液、細胞培養上清、呼出冷凝氣、外泌體、房水、玻璃體、眼淚、汗液、尿液、唾液、微量細胞、單個囊胚培養基、血拓片、凝血塊等)提供這些指標的檢測服務。

檢測目錄
商業化試劑盒-01.png


Simoa實測靈敏度范圍

檢測下限.png



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