勾狀效應 
潘暉真 醫檢師譯
  為了確保ELISA有精確的檢測數據,檢測過程中必須有足夠的抗體可以和抗原及酵素-抗體結合(第二抗體),使分析物可被完全偵測。在一般免疫反應中,若抗體量固定,逐漸加入抗原後所獲得的沉澱量曲線為鐘型曲線(或高斯曲線)。鐘型曲線的產生是一開始加入抗原時,由於抗原可和抗體結合,因此可得到一個向上的線性曲線,此曲線可提供未知檢體精確的濃度。當檢體內的抗原和抗體結合量達成平衡時,曲線不再上升而呈現平原狀態;若之後陸續增加抗原量,卻會因為抗體量不足無法與抗原結合而呈現反常的下降的曲線,此種情形稱為【高濃度勾狀效應】。
  為避免有勾狀效應的問題,不同檢體需建立線性稀釋法對抗體的沉澱曲線圖,才可確保有足夠的抗體可結合所有所有的抗原,以避免造成檢測數據過低。勾狀效應在具有許多抗原決定位的HCP ELISA更為複雜。HCP(host cell protein)分析的線性曲線應該要考慮到每個抗原決定位會被不同濃度的抗體結合,因此HCP部份抗原檢測會有勾狀效應產生。實際上限制抗體的濃度可用在HCP ELISA分析,有些檢體中的HCPs擁有足夠的抗體可與抗原結合,因此不會發生勾狀效應,但這種情形並不常見。在某些案例中,未稀釋檢體的吸光值會低於使用KIT作出的最高標準曲線的數值,即使增加稀釋次數提高HCP抗原量還是很難去推定何為最佳的稀釋比例或線性曲線。若抗原量大於相對應的抗體量,缺少稀釋的檢體其線性曲線必定會有勾狀效應產生。勾狀效應很可能會使多數抗原在最初清洗的步驟就被洗掉,但是清洗步驟會留下較具特異性HCPs抗原,而且也會產生最後的呈色,因此還是必須有清洗步驟。
  線性稀釋方法的建立在HCP分析上的發展與確認是很重要的工作。線性稀釋的研究主要是建立一系列的稀釋,我們可得到各種檢體最小需要的稀釋量(minimum required dilution, MRD)。MRD是處理有問題的檢體所執行的第一步稀釋步驟,MRD隨各檢體有不同的稀釋量,而接下來所有的稀釋步驟都維持不變。對某些檢體來說,HCP經稀釋後呈現的數值若超過或等於MRD就可以出具報告。部份檢體的MRD一旦被建立起來,SOP上面必須寫明在分析前須做稀釋動作以降低偽陰性的結果。
Hook Effect: What is “High Dose Hook Effect”?
For any ELISA to give accurate results there must be an excess of antibodies, both capture and enzyme conjugated, relative to the analyte being detected. It is only under the conditions of antibody excess that the dose response curve is positively sloped and the assay provides accurate quantitation. As the concentration of analyte begins to exceed the amount of antibody the dose response curve will flatten (plateau) and with further increase may paradoxically become negatively sloped in a phenomenon termed “High Dose Hook Effect”. Because the possibility exists that some samples may have analyte concentrations in excess of the antibody it is necessary to validate all sample types by dilutional linearity analysis to establish if they are on the valid, positively sloped region of the curve or on the negatively sloped hook region of the curve. Failure to validate the potential for Hook Effect can result in severe under-estimation of true contaminant concentrations! The issue of hook effect in multiple antigen assays such as HCP ELISA can be more complex. The dose response curve for an HCP assay should be thought of as the cumulative dose responses of all HCPs individually with each HCP having its own hook region determined by the concentration of antibody to that particular HCP. We are practically and fundamentally limited in the amount of antibody that can be used in an HCP ELISA. It is not uncommon in HCP assays for some samples to have certain HCPs in concentrations exceeding the amount of antibody for that particular HCP. In such cases the absorbance of the undiluted sample may be lower than the highest standard in the kit however these samples will fail to show acceptable dilutional recovery/linearity as evidenced by an apparent and significant increase in HCP concentration with increasing dilution. This lack of dilutional linearity is actually the result of the hook effect for the subset of analytes in excess over their respective antibodies. Poor dilutional linearity (Hook Effect) is most likely to be encountered in samples early in the purification process. However if the purification process is selective for certain HCPs, it may also be seen in downstream and final product samples. Thus the establishment of dilutional linearity is a most critical experiment in the development and validation of HCP assays. Dilutional linearity studies are performed at a series of dilutions to establish what we term the “minimum required dilution” (MRD) for a given sample type. The MRD is the first dilution at which the dilution adjusted value for the sample in question and all subsequent dilutions remains essentially constant. The HCP value to be reported for such samples is the dilution corrected value at or greater than the established MRD. Once an MRD is established for a particular sample type, your SOP should reflect that this sample requires this dilution prior to assay.
  Hook效應就是指當待測物的濃度過高,高到超過了固相上抗體所能夠接合的數目,因此反映在結果上就是超過了偵測的範圍,因此必須要將檢體先作稀釋(通常是以10倍開始),由於稀釋過後所計算出來的結果會高於未稀釋的結果,因此一般來說會作10倍、20倍以及40倍的稀釋,一直到兩次的稀釋結果都很接近為止,總之當(B)抗原過多的情形發生時,就必須小心hook效應,以免低估了待測抗原的的濃度。不過呢基本上這種hook效應在實際的操作上並不會出現,因為當檢體中存在過多抗原時,在目前已經自動化的操作過程中,機器的內部會根據不同檢驗的項目而有一個內設極限值,就是說在不稀釋檢體的情況下,檢驗試劑的原廠就會對該檢驗項目設定一個警告值,而這個數值會在低於真正會發生hook效應的濃度(如圖),譬如說某項檢查的設定值為1000,那麼待測物的濃度至少要到1500以上才有可能發生hook效應,而且就算發生hook效應,所得到的數值也會超過1000,因此在實驗結束後,機器會告訴你結果是>1000,請稀釋後再做一次,這樣就不會發生hook效應了。事實上hook效應只有在真正有非常多抗原存在時才會發生,一開始是因為在做標準曲線時發現為什麼當待測物的濃度越來越濃時所得到的數據不會呈線性的增加,而且在超過固相的結合能力時,應該是呈現一個高原式的線性關係,但是卻發生了濃度超高時標準曲線的末端有下垂的現象,我找不到有關hook效應這個名詞的原始出處,不過hook是鉤子,有彎彎的意思,這可能是形容那一段曲線下垂的部分,所以才取這個名字的吧。