Sample dilution depends upon the concentration of your analyte in urine samples and the sensitivity of the method. If the expected concentration at a given dilution of urine is above the detection limit of the assays, you can use such a dilution. Without prior information, you would have to test different dilutions and assess whether the results are logical. In general its good to use a diluting solution identical to he blocking solution you are using to preclude non-specific interactions. I normally use 4% milk/PBS, but yo can also add detergents like Tween 20.

In my experience milk is much better than BSA to eliminate non-specific background, unless you expect milk components to interefere with your paticular assay.

Hi Gertudis

Thank You Very Much for your response.

I found that diluting the sample will take the concentration of protein below the detection limit. So, I thought, after diluting, I will spike known amounts of recombinant protein to bring the concentration back to the detectable range.

Is standard addition a better method than dilution in my case where concentration of the protein is very low ?

I was using 1% BSA for blocking as well as for diluting the samples. I will try with milk and see the percentage recovery.

Thank you

You can analyze recovery in the way you said, but I guess you are doing that to use the assay with real samples later. So at the end you cannot dilute the samples in a way that their concentrations are going to be below the detection limit. I dont know how to remove matrix effects without diluting, but yo can try to modify your detection system to achieve higher sensitivities, and then higher dilutions will be useful.

Many thank for you Dear Gertrudis, I found your explanation very helpful