竞争法的半抑制率IC50 的计算方法，以及用IC50 来评估是否需要抽提；

一. 半抑制浓度（或称半抑制率），即IC50
1.1 名词解释：半抑制率IC50
半抑制浓度（或称半抑制率），即IC50。在间接竞争ELISA标准曲线中是一个非常重要的数据，标准曲线是一个S型曲线。
ICELISA中，不添加抑制物质的对照孔的OD值为B0， 添加了抑制物质的孔的OD值为B，B/B0% 就叫做结合率， 在结合率为50%时所对应的抑制物质的浓度就叫做IC50。
一般IC50的数值越小，说明你的抗体的特异性能越强！
最小检测限为试剂盒标准曲线的最小的浓度，小于最小浓度或大于最大浓度，即不在曲线范围内的都不准确，大于最大浓度可以稀释后测。
IC50为50%抑制浓度即B/B0=50%时所对应的浓度，半数抑制是用来衡量抗体灵敏度，半数抑制越低，说明抗体的灵敏度越高。
通常来说，试剂盒最大和最小量程应该是该抗体用来检测标准品的检测限，检测限分最大和最小检测限。
检测下限一般为理论值，是根据标准曲线算出来的理论上可以检测到的最小的值；
定量限为实际检测时可以定量的最低的值；而最大检测限就是实际操作中抑制率达到100%时的药物浓度。
检测下限（LOD）对应的OD值：OD(LOD) =B0－3SD
定量限（LOQ）对应的OD值：OD（LOQ） = B0－10SD


1.2   半抑制率 IC 50的计算方法：
抑制法用四参数进行拟合，所得的C值就是IC50；
penisula 试剂盒提供了IC50的数据，根据样本进行不同稀释的结果，选择适当的稀释倍数，让样本的大多数位于IC50的附近。
注意，如果样本OD 值y = d or y > a or y < d，则不能计算出结果，或者得到阴性结果；
原文：
Plot a standard curve on a semi-log scale. Use the y axis for the average of the OD readings (minus the blank average) and the x axis
for the standard concentrations in ng/ml.
Use the equation shown below to calculate the values on the "FIT" column and plot a smooth line of FIT values versus standard
concentrations. Then change the parameters a (max), b (slope), c (IC50), and d (min), until you are satisfied that fit is good.
y = (a - d)/( 1+ (x/c)b)+ d
Next calculate the average of your sample readings and subtract the blank average (see arrows starting from A3 and A4, and the arrows leading to "Average").
Finally, you may use the "reverse" of the equation above to calculate the concentrations in ng/ml for all your samples.

Caution: when you calculate sample concentrations using the "reverse" equation if y = d or y > a or y < d, the reading is out of range
and the calculation will yield an error or a meaningless negative concentration。


二、需要提取流程的penisula的ELISA 试剂盒(ELABELA 以及其他)；如何让实验做的更准确，当您的样本是血清或者血浆时，penisula 的EIA 试剂盒可能需要进行是否提取的评估：
 部分类似需提取步骤的ELISA试剂盒可以参考评估方式，逻辑上是可以通用或者借鉴的：

提取流程；
提取流程需要购买提取试剂盒（或者自配试剂），并进行样本的预处理；
样本预处理需要客户自己进行，
其中需要一个离心浓缩仪和4度的离心机（大容量）；具体流程和样本取样量（2-6ml 全血）请参照下面的方式

2.1、评估是否需要提取流程，可以用以下的方法：
我们提供了过量的标准，您可以使用它来确定是否需要提取。例如，如果您使用血清，您可以在血清中加入已知量的标准品，并检查这些标准品是否通过提取和不提取的检测方法进行了准确测定。提取消除了潜在的干扰物质，如白蛋白。提取也可能是将样品浓缩到测量范围内所必需的。与任何纯化技术一样，所需物质的回收可能不完全。因此，建议对提取程序进行优化和量化，以实现更准确的测定。虽然我们无法为您提供提取优化和定量方案，但如果您愿意，我们已经在试剂盒中加入了足够的标准。

原文：We have provided an excess amount of standard that you may use to determine if extraction is required. For example, if you are working with serum, you may spike it with known amounts of standard and check if they are accurately determined by the assay with and without extraction. Extraction eliminates potentially interfering substances, such as albumin. Extraction may also be necessary to concentrate the sample to within the measuring range. As with any purification technique, recovery of the desired substance is likely to be incomplete. Therefore, both optimization and quantification of the extraction procedure are recommended for more accurate determinations. While we cannot provide you with extraction optimization and quantification protocols, we have included enough standard in the kit should you wish to use it for this purpose.

三、提取需要的材料，样本的准备和trouble shooting

3.1提取所需材料
含有2SEP-COLUMN containing 200 mg of C18 (Cat. No. Y-1000)
 缓冲液A (BUFF-A): 1%三氟乙酸(TFA，高效液相色谱等级)。(酸化血浆样品以去除干扰蛋白，例如
白蛋白) (Cat. No. Y-1040)，缓冲液B (BUFF-B): 60%乙腈(高效液相色谱等级)、1% TFA和39%蒸馏水。(从柱中洗脱肽) (Cat. No. Y-1045) 
您也可以考虑购买Extraction kits (Cat. No. S- 5000)，包括分离柱和缓冲液；

原文：Required Materials
? SEP-COLUMN containing 200 mg of C18 (Cat. No. Y-1000) 
? Buffer A (BUFF-A): 1% trifluoroacetic acid (TFA, HPLC Grade). (Acidifies plasma sample to remove interfering proteins such as albumin) (Cat. No. Y-1040)
? Buffer B (BUFF-B): 60% acetonitrile (HPLC Grade), 1% TFA, and 39% distilled water. (Elutes peptide from column) (Cat. No. Y-1045) 
? You may also consider purchasing Extraction kits (Cat. No. S- 5000), which include SEP-columns and buffers


3.2 血浆的提取和制备
将血样(2 - 6毫升)收集到预冷的注射器中，并转移到聚丙烯试管中，试管中含有作为抗凝剂的EDTA (1 mg/ml of blood)和作为蛋白酶抑制剂的Aprotinin (500 KIU/ml of blood)。不要使用肝素化试管，因为它们可能会干扰检测。
带有EDTA的Vacutainers是可以接受的。
将血液在4℃下以1,600xg离心15分钟
收集顶层(血浆)。可能能收取到1-3毫升血浆。
立即进行提取或在-70℃下冷冻以备后用。

原文：Withdrawal and Preparation of Plasma
? Collect blood samples (2 - 6 ml) into a chilled syringe and transfer into a polypropylene tube containing EDTA (1 mg/ml of blood) as an anticoagulant and Aprotinin (500 KIU/ml of blood) as a protease inhibitor at 4°C. Do not use heparinized tubes as they may interfere with the assay. Vacutainers with EDTA are acceptable.
? Centrifuge blood at 1,600xg for 15 minutes at 4°C.
? Collect the top (plasma) layer.
? Proceed to extraction immediately or freeze at -70°C for later use.


3.3 提取程序
向血浆中加入等量的缓冲液A。
在6,000xg to 17,000xg下离心20分钟，温度为4℃;
将上清液转移到新试管中，丢弃任何可能存在的沉淀;
用1毫升缓冲液B和3次3毫升缓冲液A洗涤SEP-COLUMN分离柱，使分离柱达到平衡;
将血浆溶液装载到平衡的SEP柱上;
用缓冲液A (3毫升，两次)缓慢清洗色谱柱，并丢弃清洗液。可以对色谱柱施加轻微真空(10秒/滴)。
用缓冲液B (3毫升，一次)缓慢洗脱肽，并将洗脱液收集在聚丙烯试管中。可以像前面的步骤那样施加轻微的真空。
使用干冰/甲醇浴将洗脱液冷冻干燥，以冷冻样品，并使用离心浓缩器将其蒸发；
将残留物溶解在适当体积的EIA buffer中，使目标物质的浓度接近IC50(在测量范围内)；

原文：
Extraction Procedure
? Add an equal amount of Buffer A to the plasma.
? Centrifuge at 6,000xg to 17,000xg for 20 minutes at 4°C.
? Transfer supernatant to a new tube discarding any pellet that may be present.
? Equilibrate a SEP-COLUMN by washing with 1 ml Buffer B followed by 3 X 3 ml Buffer A.
? Load the plasma solution onto the equilibrated SEP-Column.
? Slowly wash the column with Buffer A (3 ml, twice) and discard the wash. A light vacuum (10 sec/drop) may be applied to the column.
? Elute the peptide slowly with Buffer B (3 ml, once) and collect eluant in a polypropylene tube. A light vacuum may be applied as in previous step.
? Freeze-dry eluant to dryness using a dry ice/methanol bath to freeze the sample and a centrifugal concentrator to evaporate it；
? Dissolve the residue in a suitable volume of EIA buffer such that the concentration of the substance of interest will fall close to the IC50 (within the measuring range)；


3.4   试剂盒简要TROUBLESHOOTING：
3.4.1  如果你等得太久了，曲线会在顶部变平。如果您不熟悉本试剂盒，我们建议您在信号仍在增加的时候多读板几次。
对于EIAH和EIAS吸光度分析，显示蓝色(吸收650 nm)将不如终止反应黄色吸收强烈（450 nm ),但是数据仍然是好的，这样你就不会冒失去范围下限的风险。您可以先650nm 读数。

原文：
1.    If you wait too long to read, the curve will be flattened at the top. If you are not familiar with the kit we recommend you read the plate several times while the signal is still developing. 
For EIAH and EIAS absorbance assays the developing blue color (absorb. 650 nm) will be less intense compared to that of the terminated reactions (yellow - absorb. 450 nm) but the data are still good and this way you won't risk losing the lower end of the range.


3. 4.2   IC50并不像预期的那样
o请注意，我们每种产品报告的IC50都是基于添加到分析溶液之前制备的标准品的浓度。
o两个或三个因子的差异对于某些试剂盒来说可能是正常的，并且可能是由示踪剂和标准物的结合达到平衡所需的时间引起的。对于预培养方案来说尤其如此。如果可能的话，您应该始终包括您自己的可靠标准，其浓度接近预期的IC50，以检查试剂盒的准确性。
o在标准曲线几乎是直线的情况下，无法计算精确的IC50值。
o使用过量的抗血清或示踪剂，或使用降解的标准可能会提高IC50


原文：The IC50 is not as expected
o Note that the IC50 reported with each of our products is based on the concentration of the prepared standards before they are added to the assay solution.
o A difference by a factor of two or three may be normal for some kits and may be caused by the time it takes to equilibrate the binding of the tracer and the standard. This will be especially true for pre-incubation protocols. If possible you should always include your own reliable standard at a concentration close to the expected IC50 to check the accuracy of the kit.
o In cases where the standard curves are almost rectilinear, accurate IC50 values cannot be calculated.
o Using excessive amounts of antiserum or tracer, or using a degraded standard may elevate the IC50.