What would be the best design for spike-and-recovery and linearity-of-dilution validation experiments in one 96-well ELISA plate?
Background:  


There are a lot of recipes to extract protein from human tissues out there, but all of them boil down to one thing; preserving the tissue proteins as much as possible while obtaining a reasonable yield for downstream applications, using extraction buffer, extra techniques, and by adding protease inhibitors  
ELISA is one of the those downstream applications that you might be interested in, but one important question? is the sample matrix that you have already obtained valid for the ELISA assay? keep in mind, your sample is not serum, it is a mixture of tissue, extraction buffer, and protease inhibitors, and other things as well  
Two known assays are usually performed to address the above question. These are, spike-and-recovery (SAR), and linearity-of-dilution (LOD), and these two assays are specific to the analyte that you want to measure in your samples, e.g., cytokines, factors, Igs, etc...and specific also to the tissue, and assay kit as well  
Often times, you are interested in performing these two assays with minimum resources as much as possible, i.e., less time, less kit materials, and, most importantly, less sample material. This can be quite challenging and it depends on many issues that is out scope of this post to discuss them all  
If you are interested in performing these two assays using only one 96-well ELISA plate and examine validity of different sample matrices, sample buffer (lysis buffer or sometimes called extraction buffer) for a certain analyte, say X, then this scheme below is for you:


  

Technical Notes:


In this scheme, recovery is examined in 3 types of solutions; assay buffer, sample buffer and samples. These are different in their complexity. Typically, the assay buffer is usually optimized to detect the standard protein provided with the commercial kit  
The idea of spike-and-recovery is that you add a certain amount from standard stock solution into the wells containing the solution to be tested (e.g., sample buffer or samples) and measure them, to see whether you can recover that amount again, and how much you can recover from it in %. If, for any reason, you couldn't recover that amount in comparison with a control well, where that same amount was added into assay buffer, then this means that something in the test solution is not in favor of the assay. The specific amount you add into the wells is called spike, and it should be the same amount across all tested wells (try to be consistent). Make sure that the added amount when diluted in the well, its final concentration should still be measured by the assay and lie inside your standard curve. E.g., if your standard curve is from 4- 4500 pg/mL, and each well has 100µl of sample, if you want now to add spike into that sample well, you may add whatever amount you want that will still give you at the end a reading inside the standard curve, you may choose to add 10µl from the highest stock which is 500pg/mL, so you end up here with 1/10, that is 50pg/mL, which is fine as it lies inside the 4-500pg/mL range. Alternatively, you can add 50µl sample + 50µl spike (500pg/mL) and would end up with 1/2 dilution (which is the same in this scheme), that is 250pg/mL, which is also fine. So it is up to you to choose, keeping in mind the final concentration should lie inside your standard curve  
For the linearity-of-dilution, it is obvious that you need a high concentration solution that you can make two-fold (or whatever fold you like) serial dilution from and its reading should still lie inside the standard curve. The best candidate for this is the high spiked wells. The LOD would tell you about the effect of different dilutions on the precision of the assay  
For SAR and LOD you will get at the end an average percentage from 3 samples at least (calculation example will be provided later), I added a fourth sample, but this fourth can be a different sample type, say samples from tissue culture, so it is up to you to re-design this scheme and be innovative   
Choosing the best dilution is not an easy task, it is not without compromises sometimes. For example, if you have a harsh sample buffer, you may not have good recovery at 1/2, 1/4, 1/8 dilutions of the sample buffer in assay buffer, whereas almost good recovery is achieved at 1/16, then this dilution factor is most probably the one you should go for in your assay. On the other hand, 1/16 might not be detected by your ELISA assay, which is limited by its sensitivity. Here, the E wells can give you a clue whether you detect something in these unspiked samples or not, that's why I recommend you include at least one sample of high expected value of the analyte in question (this is not easy sometimes to predict) in this scheme, here this is sample 3. One more thing, if you get good recovery at 1/16 DO NOT go to 1/32 or 1/64, as it is obvious that with these higher dilutions you are reducing the chance of the analyte's detectability as it is limited by your assay's sensitivity  
In most of the cases, you will see that the dilution factor of good recovery of the sample buffer in columns 3 and 4, will coincide with that of the spiked samples in columns 5, 7, and 9. This will give you the assurance that you are in the right direction. If sample 4 is extracted by a different sample buffer, then it is clear that should not expect to end up with the same dilution factor (please, be more reasonable!)