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Elisa酶聯檢測


技術原理:

     酶聯免疫吸附試驗(又稱酵素免疫分析法,Enzyme-linked immunosorbent assay,簡稱 ELISA)利用抗原抗體之間專一性鍵結之特性,對檢體進行檢測;由于結合于固體承載物(一般為塑膠孔盤)上之抗原或抗體仍可具有免疫活性,因此設計其鍵 結機制后,配合酵素呈色反應,即可顯示特定抗原或抗體是否存在,并可利用呈色之深淺進行定量分析。根據待測樣品與鍵結機制的不同,ELISA可設計出各種 不同類型的檢測方式,主要以三明治法(sandwich)、間接法(indirect)、以及競爭法(Competitive)三種為主,以下為各種方法 之介紹。

 



三明治法


三明治法常用于檢測大分子抗原,一般之操作步驟為:

  •  將具有專一性之抗體固著(coating)于塑膠孔盤上,完成后洗去多余抗體;
  •  加入待測檢體,檢體中若含有待測之抗原,則其會與塑膠孔盤上的抗體進行專一性鍵結;
  •  洗去多余待測檢體,加入另一種對抗原專一之一次抗體,與待測抗原進行鍵結;
  •  洗去多余未鍵結一次抗體,加入帶有酵素之二次抗體,與一次抗體鍵結;
  •  洗去多余未鍵結二次抗體,加入酵素受質使酵素呈色,以肉眼或儀器讀取呈色結果。

三明治法分別以兩種抗體對檢體中的抗原進行兩次專一性辨認,因此專一性相當高,但此待測抗原必須是多價抗原,如此才可獲得兩種以上的專一性抗體,以分別進行夾心;而且此法需要足夠的表位空間以進行抗原抗體的夾心,所以并不適用于半抗原或小分子抗原等分子量較小之標的。


間接法

間接法常用于檢測抗體,一般之操作步驟為:

  •  將已知之抗原固著于塑膠孔盤上,完成后洗去多余之抗原;
  •  加入待測檢體,檢體中若含有待測之一次抗體,則其會與塑膠孔盤上的抗原進行專一性鍵結;
  •  洗去多余待測檢體,加入帶有酵素之二次抗體,與待測之一次抗體鍵結;
  •  洗去多余未鍵結二次抗體,加入酵素受質使酵素呈色,藉儀器(ELISA reader)測定塑膠盤中的吸光值(OD值),以評估有色終產物的含量即可測量待測抗體的含。量。


競爭法

競爭法是一種較少用到的ELISA檢測機制,一般用于檢測小分子抗原,其操作步驟為:

  •  將具有專一性之抗體固著于塑膠孔盤上,完成后洗去多余抗體;
  •  加入待測檢體,使檢體中的待測抗原與塑膠孔盤上的抗體進行專一性鍵結;
  •  加入帶有酵素之抗原,此抗原也可與塑膠孔盤上的抗體進行專一性鍵結,由于塑膠孔盤上固著的抗體數量有限,因此當檢體中抗原的量越多,則帶有酵素之抗原可鍵。結的固著抗體就越少,亦即,兩種抗原皆競相與塑膠孔盤上抗體鍵結,即所謂競爭法之由來;
  •  洗去檢體與帶有酵素之抗原,加入酵素受質使酵素呈色,當檢體中抗原量越多,代表塑膠孔盤內留下之帶有酵素的抗原越少,顯色也就越淺;
  •  當需要偵測無法獲得兩種以上單一性抗體的抗原,或是不易得到足夠的純化抗體以固著于孔盤上時,一般會考慮使用競爭法ELISA。


結果判斷

定性測定

  定性測定的結果判斷是對受檢標本中是否含有待測抗原或抗體作出”有”或”無”的簡單回答,分別用”陽性”、”陰性”表示。” 陽性”表示該標本在該測定系統中有反應。”陰性”則為無反應。用定性判斷法也可得到半定量結果,即用滴度來表示反應的強度,其實質仍是一個定性試驗。在這 種半定量測定中,將標本作一系列稀釋后進行試驗,呈陽性反應的最高稀釋度即為滴度。根據滴度的高低,可以判斷標本反應性的強弱,這比觀察不稀釋標本呈色的 深淺判斷為強陽性、弱陽性更具定量意義。

在間接法和夾心法ELSIA中,陽性孔呈色深于陰性孔。在競爭法ELISA中則相反,陰性孔呈色深于陽性孔。

定量測定

        ELSIA操作步驟復雜,影響反應因素較多,特別是固相載體的包被難達到各個體之間的一致,因此在定量測定中,每批測試均須用一系列不同濃度的參考 標準品在相同的條件下制作標準曲線。測定大分子量物質的夾心法ELISA,標準曲線的范圍一般較寬,曲線最高點的吸光度可接近2.0,繪制時常用半對數 紙,以檢測物的濃度為橫坐標,以吸光度為縱坐標,將各濃度的值逐點連接,所得曲線一般呈S形,其頭、尾部曲線趨于平坦,中央較呈直線的部分是最理想的檢測 區域。

測定小分子量物質常用競爭法,其標準曲線中吸光度與受檢物質的濃度呈負相關。標準曲線的形狀因試劑盒所用模式的差別而略有不同。ELISA測定的標準曲線注意圖中橫坐標為對數關系,這更有利于測定系統的表達。


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  • (1)實驗數據;
  • (2)數據分析;
  • (3)實驗報告。
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