ELISA——重復性保證

6.Reproducibility 重復性

       Ultimately, you are aiming for accuracy ａnd reproducibility to create usable ａnd valuable data. To achieve consistency, optimized performance ａnd accurate results, be fully cognizant of the protocol throughout. Remain consistent ａnd be systematic in your approach.

        *終，您的目標是獲得準確性和可重復性，以創(chuàng)建可用且有價值的數據。為了實現一致性，優(yōu)化性能和準確結果，請始終充分理解實驗方案，并在您的實驗中保持一致的操作體系。

—Before running the assay, allow about 30 minutes for the kit reagents to reach room temperature or the temperature stated in the information provided. Frozen samples should also be allowed to thaw completely before being used ａnd repeated freeze-thaw cycles should be minimized to less than three times.

在檢測前，提前將試劑盒試劑放置室溫（或按試劑盒說明書所建議的溫度）約30分鐘。冷凍樣品在使用前也應該完全融化，避免樣本多次反復凍融，凍融次數建議少于三次。

—Keep environmental conditions, e.g. temperature ａnd humidity constant throughout the assay ａnd between assays.

保證實驗的環(huán)境條件，例如整個測定和測定之間的溫度和濕度恒定。

—Ensure all equipment is calibrated including pipettes, plate washers ａnd readers.

確保所有設備都已校準，包括移液器，洗板機和讀取器。

—Use sufficient quantities of antibody.

使用足量的抗體。

—Substrate solutions should be freshly prepared, not stored for hours ahead of use.

底物溶液應該是新鮮制備的，而不是在使用前數小時的儲存溶液。

—Handle samples consistently throughout testing ａnd follow the same procedures each time.

在整個測試過程中持續(xù)處理樣品，并且每次都遵循相同的步驟。

—Throughout the process, visually inspect the tips ａnd wells to check aspiration, addition of reagents ａnd withdrawal. Levels should be equal.

在整個實驗過程中，目視檢查吸頭和孔的吸出物、添加和取出的試劑應與方案數值一致，不應該因為操作不當出現偏差。

—Don’t be tempted to mix lots between kits to get more out of your reagent. ELISA kits are each prepared so that the contents work together. Mixing lots between assays could adversely affect performance.

不要試圖將不同種類的試劑盒混用以獲得更多試劑。每種功能ELISA試劑盒都要準備好，在測定之間批次混合使用可能會對結果產生不利影響。

—Once a reagent has left the bottle, it should not be returned.

吸取試劑時，一旦試劑離瓶，不應該再放回原試劑瓶，防止交叉污染。

—Don’t allow the wells to dry out once testing has begun, keep the tray sealed to prevent drying.

一旦測試開始，不要讓孔干燥，保持板子密封以防止干燥。

7. Washing 洗滌

       Careful washing is necessary to reduce background signal caused by unbound, conjugated antibody. It will therefore increase the assay’s signal-to-noise ratio. Washing at each step will help to maintain purely the specific binding events. Ensure wash volume is high enough to remove all traces of antigen or antibody from the wells ａnd hence, reduce unwanted background signal. Maintain the recommended distance between the bottom of the well ａnd the wash tips to reduce residual antibody/antigen containing fluid being created that will affect your signal. Floating heads are more flexible ａnd easier to obtain a complete wash. Repeat wash cycles enough to ensure unwanted antigen ａnd antibody are removed but the bound antibody remains in place. Follow the guidelines but Biorbyt recommends three washes after each incubation. This will need to be altered depending on whether your plate is manufacturer coated or if you have coated it. You may need to increase the number of wash cycles if you have coated your own plate.

        實驗中，恰當的洗滌步驟對于減少由于未結合的偶聯(lián)抗體引起的背景信號是必要的，所以正確的洗滌步驟有助于增加分析的信噪比和保證單一特異性結合。確保洗滌液體積足夠多，以除去孔中游離的抗原或抗體的痕跡，從而減少不需要的背景信號。保持孔底和洗滌端之間的建議距離，以減少殘留的抗體/抗原液體，從而影響信號。浮動頭更靈活，更容易獲得完整的清洗。重復循環(huán)洗滌以確保除去不需要的抗原和抗體，但已結合的抗體會保留在結合原位。Biorbyt建議在每次孵育后進行三次洗滌。根據您所使用的板子是出廠包被還是您自己包被的，可以對洗滌方式進行調整，如果是您自己包被的板子，您可能需要增加清洗次數。

8. Buffers 緩沖液

         Use the buffers ａnd diluents provided within the kit or those that are specified in the protocol. Buffers can be single or multi-function. They are used throughout the assay for coating, blocking, washing ａnd for dilution. Coating is the process of adding a buffer to stabilize the antigen or antibody then incubating overnight to cause adsorption of the diluted antigen or antibody to the surface of the well. Biorbyt also provide multi-purpose, universal ELISA buffers that could potentially save time ａnd energy, speeding the process up without compromising function.

        使用試劑盒內提供的緩沖液和稀釋液，或實驗方案中建議的緩沖液和稀釋液。緩沖區(qū)可以是單個或多個功能，它們在整個分析過程中用于包被，封閉，洗滌和稀釋。包被是加入緩沖液以穩(wěn)定抗原或抗體，然后孵育過夜以使稀釋的抗原或抗體吸附到孔表面的過程。Biorbyt還提供多種類的通用ELISA緩沖液，可以節(jié)省時間和精力，加速實驗過程而不影響效果。

9.Blocking 封閉

      There are many blocking buffers available, some offering additional benefits such as increased stability, enhanced blocking, reduced cross-reactivity ａnd reduced non-specific binding. A blocking buffer containing an unrelated protein can be used to prevent non-specific binding of the detection antibodies to the plate. Blocking buffers usually contain BSA or milk proteins dissolved in PBS. Biorbyt’s range of Blocking buffers will include the perfect solutions for your assay.

        封閉緩沖液有許多種類可供選擇，有助于增加的反應的穩(wěn)定性，增強阻斷性，減少的交叉反應性和減少的非特異性結合。含有不相關蛋白質的封閉緩沖液可用于防止檢測抗體與板的非特異性結合。封閉緩沖液通常含有溶解于PBS中的BSA或牛奶酪蛋白。Biorbyt的阻斷緩沖液系列將為您的分析提供完美的解決方案。

10.Cross Contamination 交叉污染

      Forgive us for stating the obvious, but working in a clean ａnd organised manner is the first step. Before beginning the assay, establish the amount of reagents you will need to use. Prepare the right amount of reagent ａnd discard any excess. Avoid cross contamination of samples or reagents by changing pipette tips between each sample, standard or reagent addition. Be extra careful during liquid removal ａnd washing. For each transfer, use new, disposable reagent reservoirs.

        我們溫馨提示，以干凈和有序的方式工作是實驗成功步。在開始分析之前，確定您需要使用的試劑量，準備適量的試劑并丟棄任何過量的試劑以防止交叉污染。通過更換每個樣品、標準品或添加試劑之間的移液器吸頭，避免樣品或試劑的交叉污染。在液體清除和清洗過程中要格外小心。對于每次轉移，請使用新的一次性試劑儲存容器。

ELISA——精確度、獲得可靠的標準曲線

11.Accuracy 精確度

         Accuracy throughout the procedure will increase confidence in your data. You are aiming to generate more than one standard curve with a high degree of accuracy. You will need to test your samples in duplicate at minimum, ａnd preferably in triplicate. You need the smallest possible coefficient of variability (%CV) between each replicate. Outliers will be more obvious so can be investigated before being included in calculations ａnd affecting your results. One possible cause of outliers is ‘Edge Effect’ caused by issues with production of multiwell plates or with assay processes that affect the outer wells. Things that will affect your CV include: incorrect storage and/or preparation of samples, bubbles in wells, incomplete plate washing, poor mixing of reagent, temperatures not controlled ａnd poor pipetting technique. Samples with optical densities (OD) above or below the linear range of the standard curve will result in target concentrations being underestimated or overestimated, respectively. 

        保證整個試驗過程的準確性將提高您對數據的信心。您的目標是以高精確度生成多條標準曲線。您需要至少以一個樣本兩次重復（是三個重復）測試您的樣本。您需要每個重復之間維持*小的可變系數（％CV），保證重復結果的穩(wěn)定性。如果數值明顯異常，就需要分析可能導致結果異常的潛在原因。異常數值產生的一個可能原因是由于96孔板生產原因或由于外部孔引起的“邊緣效應”。會影響實驗CV值的因素還包括：樣品不正確的儲存或制備方式，孔中有氣泡，洗板不完全，試劑混合不當，不恰當的溫度控制以及移液操作技術。光密度（OD）高于或低于標準曲線線性范圍的樣品將分別導致目標濃度被低估或被高估。

12.Obtaining Reliable Standard Curves 獲得可靠的標準曲線

     It is crucial to follow the manufacturer’s recommendations for storage, handling ａnd pipetting. Always use the recommended diluents, reagents ａnd detergents, ａnd keep to the correct conditions of pH, temperature ａnd strengths for your kit. Check vials don’t contain any undissolved residue after spinning. Assays will not be identical, but there is a range of variation that is acceptable. A standard curve should be produced for each set of samples assayed. Generate each standard curve within the manufacturer’s recommended dilution range ａnd ensure that you have sufficient data points. There are no benefits to trying to extend the curve, the antibodies used for binding will only produce data within a set range. If you notice the ‘Hook Effect’ in your results the probable cause is not enough analyte to bind with the quantity of antigen in your sample. This issue is also resolved by testing first at a range of dilutions. If you are finding sensitivity is low, it is worth checking:

—That the kit was stored correctly

—Your detection reagent is fully functioning

—Your sample type ａnd buffers are compatible

—You had an adequate concentration/amount of target protein ａnd of substrate

—The adsorbance wavelength settings of your plate reader are correct

—Incubation times are long enough

—Your ELISA kit had the right level of sensitivity for your assay.

       按照試劑盒制造商的建議，對試劑盒試劑進行恰當的保存，處理和移液操作至關重要。根據說明書和實驗方案的推薦進行稀釋劑，試劑和清潔劑的選擇，并保持試劑盒使用的正確pH，溫度和強度條件，檢查小瓶/管試劑在（離心）旋轉后充分溶解。每種測定方法不一定完全相同，但應該有可以接受的變化范圍。應為每組樣品制備標準曲線。在廠家建議的稀釋范圍內生成標準曲線，并確保您有足夠的數據點。試圖擴大延長標準曲線沒有任何好處，因為抗體結合只能在一定范圍內產生數據。如果您在結果中注意到“鉤狀效應”，可能的原因是分析物不足以與樣品中抗原的量結合。這個問題也可以通過首先在一系列稀釋度下進行預實驗測試來解決。如果您發(fā)現結果靈敏度較低，則需要檢查：

- 試劑盒保存是否正確

- 檢測試劑是否充分發(fā)揮作用

- 您的樣品類型和緩沖液是否兼容

- 靶蛋白和底物濃度/數量是否是足夠的

- 讀板器的吸附波長設置是否正確

- 記錄時間是否足夠長

- 您的ELISA試劑盒對您的測定是否具有正確的靈敏度。