I was wondering if someone could analyze my current Direct ELISA protocol and perhaps tell me what am I doing wrong or what could be improved to give me a better signal intensity.
I start off with adding my tissue samples (5 microliters) to (2 milliliters of blocking buffer). My blocking buffer consists of PBS, BSA & Tween. It comes from an original stock I made that is 50 mL PBS, ~.5g of BSA and ~5 microliters of Tween.
Next I make sure to vortex these gently and add 100 microliters to each well (usually in triplicate) for my ALS tissue, Non-Neurological Control tissue, and background (Blocking Buffer) or no tissue as it is commonly referred to.
I then incubate at 37 degrees celsius for 25 minutes on a moderate shake.
After this I wash with PBS-Tween 300 microliters/well with each wash staying for one minute. I do this twice and then proceed to add 300 microliters of blocking buffer to each well. I incubate my plate at 37 degrees celsius but this time for 45 minutes. Now I prepare my "preincubation buffer" which also goes in the same incubator for 45 minutes. I prepare this by adding 10 mL of incubation buffer to a vial from my original stock of incubation buffer. The original stock of incubation buffer is 50 mL of PBS, ~.5g of BSA and ~5 uL of Tween. After I added the incubation buffer, I add 1 uL of AVIDIN, then vortex gently. Next I add 10 mL of my detection antibody to the preincubation buffer and gently vortex again. At this point I wait for both my preincubation buffer and plate to complete the 45 minute cycle.
I do my washes again, twice.
I add my preincubation buffer (100uL/well) and incubate at 37 degrees celsius for 25 minutes at moderate shake.
I do 4 washes this time but on the last wash I keep it until I have prepared my FEMTO which is what I use to analyze the plate. After I prepare the FEMTO I remove my last wash, then add 100 uL of FEMTO to the plate. This plus, a 3 minute incubation where the plate is left in complete darkness is the final step before I analyze the plate.
Do you have any suggestions or remarks concerning this direct ELISA method and why I am seeing low signal?
Do not add the sample to the blocking buffer. That will prevent the antigen from binding to the plate. Instead, dilute the sample into binding buffer (sodium carbonate/bicarbonate) and add it to the wells of the ELISA plate, which should be a high-binding surface plate. After incubating that, then wash the plate and treat the plate with the blocking buffer.
Adam B Shapiro thank you so much! Do you think the rest of my protocol is fine as is?
What is your "preincubation buffer"?
In my experience, the steps in a direct ELISA are as follows:
I'm sorry but I think what you just described is an indirect ELISA if I am not mistaken.
You're right. For a direct ELISA, the detection enzyme is attached to the primary antibody, so there are fewer steps, but less amplification, than an indirect ELISA.
I see. I even tried doing Sandwich ELISA and I got similar results. I strongly believe this is indicative of poor biotinylation and could be due to using expired desalting columns.
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