ELISA——重复性保证

6.Reproducibility 重复性

       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.

        *终，您的目标是获得准确性和可重复性，以创建可用且有价值的数据。为了实现一致性，优化性能和准确结果，请始终充分理解实验方案，并在您的实验中保持一致的操作体系。

—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.

在检测前，提前将试剂盒试剂放置室温（或按试剂盒说明书所建议的温度）约30分钟。冷冻样品在使用前也应该完全融化，避免样本多次反复冻融，冻融次数建议少于三次。

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

保证实验的环境条件，例如整个测定和测定之间的温度和湿度恒定。

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

确保所有设备都已校准，包括移液器，洗板机和读取器。

—Use sufficient quantities of antibody.

使用足量的抗体。

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

底物溶液应该是新鲜制备的，而不是在使用前数小时的储存溶液。

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

在整个测试过程中持续处理样品，并且每次都遵循相同的步骤。

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

在整个实验过程中，目视检查吸头和孔的吸出物、添加和取出的试剂应与方案数值一致，不应该因为操作不当出现偏差。

—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.

不要试图将不同种类的试剂盒混用以获得更多试剂。每种功能ELISA试剂盒都要准备好，在测定之间批次混合使用可能会对结果产生不利影响。

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

吸取试剂时，一旦试剂离瓶，不应该再放回原试剂瓶，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.

一旦测试开始，不要让孔干燥，保持板子密封以防止干燥。

7. Washing 洗涤

       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.

        实验中，恰当的洗涤步骤对于减少由于未结合的偶联抗体引起的背景信号是必要的，所以正确的洗涤步骤有助于增加分析的信噪比和保证单一特异性结合。确保洗涤液体积足够多，以除去孔中游离的抗原或抗体的痕迹，从而减少不需要的背景信号。保持孔底和洗涤端之间的建议距离，以减少残留的抗体/抗原液体，从而影响信号。浮动头更灵活，更容易获得完整的清洗。重复循环洗涤以确保除去不需要的抗原和抗体，但已结合的抗体会保留在结合原位。Biorbyt建议在每次孵育后进行三次洗涤。根据您所使用的板子是出厂包被还是您自己包被的，可以对洗涤方式进行调整，如果是您自己包被的板子，您可能需要增加清洗次数。

8. Buffers 缓冲液

         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.

        使用试剂盒内提供的缓冲液和稀释液，或实验方案中建议的缓冲液和稀释液。缓冲区可以是单个或多个功能，它们在整个分析过程中用于包被，封闭，洗涤和稀释。包被是加入缓冲液以稳定抗原或抗体，然后孵育过夜以使稀释的抗原或抗体吸附到孔表面的过程。Biorbyt还提供多种类的通用ELISA缓冲液，可以节省时间和精力，加速实验过程而不影响效果。

9.Blocking 封闭

      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.

        封闭缓冲液有许多种类可供选择，有助于增加的反应的稳定性，增强阻断性，减少的交叉反应性和减少的非特异性结合。含有不相关蛋白质的封闭缓冲液可用于防止检测抗体与板的非特异性结合。封闭缓冲液通常含有溶解于PBS中的BSA或牛奶酪蛋白。Biorbyt的阻断缓冲液系列将为您的分析提供完美的解决方案。

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.

        我们温馨提示，以干净和有序的方式工作是实验成功步。在开始分析之前，确定您需要使用的试剂量，准备适量的试剂并丢弃任何过量的试剂以防止交叉污染。通过更换每个样品、标准品或添加试剂之间的移液器吸头，避免样品或试剂的交叉污染。在液体清除和清洗过程中要格外小心。对于每次转移，请使用新的一次性试剂储存容器。

ELISA——精确度、获得可靠的标准曲线

11.Accuracy 精确度

         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. 

        保证整个试验过程的准确性将提高您对数据的信心。您的目标是以高精确度生成多条标准曲线。您需要至少以一个样本两次重复（是三个重复）测试您的样本。您需要每个重复之间维持*小的可变系数（％CV），保证重复结果的稳定性。如果数值明显异常，就需要分析可能导致结果异常的潜在原因。异常数值产生的一个可能原因是由于96孔板生产原因或由于外部孔引起的“边缘效应”。会影响实验CV值的因素还包括：样品不正确的储存或制备方式，孔中有气泡，洗板不完全，试剂混合不当，不恰当的温度控制以及移液操作技术。光密度（OD）高于或低于标准曲线线性范围的样品将分别导致目标浓度被低估或被高估。

12.Obtaining Reliable Standard Curves 获得可靠的标准曲线

     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.

       按照试剂盒制造商的建议，对试剂盒试剂进行恰当的保存，处理和移液操作至关重要。根据说明书和实验方案的推荐进行稀释剂，试剂和清洁剂的选择，并保持试剂盒使用的正确pH，温度和强度条件，检查小瓶/管试剂在（离心）旋转后充分溶解。每种测定方法不一定完全相同，但应该有可以接受的变化范围。应为每组样品制备标准曲线。在厂家建议的稀释范围内生成标准曲线，并确保您有足够的数据点。试图扩大延长标准曲线没有任何好处，因为抗体结合只能在一定范围内产生数据。如果您在结果中注意到“钩状效应”，可能的原因是分析物不足以与样品中抗原的量结合。这个问题也可以通过首先在一系列稀释度下进行预实验测试来解决。如果您发现结果灵敏度较低，则需要检查：

- 试剂盒保存是否正确

- 检测试剂是否充分发挥作用

- 您的样品类型和缓冲液是否兼容

- 靶蛋白和底物浓度/数量是否是足够的

- 读板器的吸附波长设置是否正确

- 记录时间是否足够长

- 您的ELISA试剂盒对您的测定是否具有正确的灵敏度。