Hi researchers,
I want to ask a question about ELISA dilution factor with my data.
For the same sample, the first time I used 1:2 dilution (1 plasma + 1 diluent) and the OD value was 2.482. The second time I used 1:5 dilution (1 plasma + 4 diluent) and the OD value was 1.861.
My question is how to decide the dilution factor for this sample? I mean, can I assume that if I use 1:10 dilution (1 plasma + 9 diluent), the OD value for the same sample will become around 0.93(half of 1.861)?
I know this may not be correct because if 1:2 dilution made the OD 2.482, then 1:5 dilution would make the OD less than 1.241(half of 2.482). But actually it was 1.861. The 1:2 and 1:5 dilution were done in two different plates. How do you estimate the dilution factor, according to your existing data?
Thanks!
The dose-dependence absorbance curve in ELISA is not linear, that's why you did not see the drop in absorbance 5-fold when your diluted your sample 5-fold. If you have your protein purified, use it for the calibration curve which should be used in each of your ELISA plate. If you don't have your protein purified, use 10-fold dilutions of your sample like: neat, 1 : 10, 1 : 100, 1 : 1,000, 1 : 10,000, 1 : 100,000, 1 : 1,000,000. When you get this curve of absorbance, use only the middle 60% of your curve, ignoring the top 20% and the bottom 20% of the curve which also include the plateaus. In that case you will get the information about relative titer of your protein with the particular antibody used.
Is your ELISA a yes/no one or do you want to measure the levels of an analyte in your samples?
In the first case, I would choose a dilution which gives high values for several positive samples and totally negative (blank level) values for several negative samples. You could try with a small set of positive and negative controls if you have them. In that case the most important thing is the difference between positive and negative samples.
In the second case, you should have a standard curve. Then I would choose a dilution (or several dilutions if possible) that produce OD values within the range of the standard curve. You could try different dilutions with several samples of low, medium and high analyte content. If you know a priori the expected range of concentrations in the samples you could select the dilutions after calculating how many times you have to dilute to be within the standard curve range.
Of course, you cannot directly compare OD values from different plates.
The dose-dependence absorbance curve in ELISA is not linear, that's why you did not see the drop in absorbance 5-fold when your diluted your sample 5-fold. If you have your protein purified, use it for the calibration curve which should be used in each of your ELISA plate. If you don't have your protein purified, use 10-fold dilutions of your sample like: neat, 1 : 10, 1 : 100, 1 : 1,000, 1 : 10,000, 1 : 100,000, 1 : 1,000,000. When you get this curve of absorbance, use only the middle 60% of your curve, ignoring the top 20% and the bottom 20% of the curve which also include the plateaus. In that case you will get the information about relative titer of your protein with the particular antibody used.
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