Originally Posted by Sharpie
Thanks for the reply danfive,
Our starting material is cell culture supernatant, containing the antigen in the order of hundreds of ng/ml.
No, we do not have a hook effect as you describe. The greater the dilution, the less signal we have. But, within the dynamic range of the standard curve, we get divergent results for the different dilutions chosen.
With the same example of 12 molecules, in our hands we would get at dilution ½ a measurement of 8, so final concentration of 8 * 2 = 16 and at a dilution of 1/4 a measurement of 5, so final concentration of 5 *4 = 20. Our final calculated concentration increases as the dilution factor increases. But, no, it is not a hook effect.
We add our samples and standard curve on the plate, and then add 25µl of 2.5% Triton X-100 (final 0.5%). The purpose of this is to disrupt the lipididic envelope of the virus and release the p24 protein inside (our antigen). So yes we add a non-ionic detergent. I tried adding the detergent to the sample before making dilutions, but ended up with the same results.
We have never optimized for dilution factor nor dilution buffers. This did pique my curiosity and think it could help us. How do you achieve that ? We are currently using PBS 1X + 1%BSA both for diluting the standard and the samples.
Thanks for your help
I see what you mean about the no hook effect.
Triton X-100 is great.
Do you believe that you are preparing a homogenous mixture before adding to plate? I ask because incongrous mixing during dilution can create weird effects.
You may consider comparing the following and see which works best.
1. Hand mixing via pipette (5-8 times)---limitation-not 100% homogenous mix accomplished.
2. Vortexing (medium, 5-10 sec)--limitation-some proteins will clump, change structure. Usually not recommended, but ideal for some v. small proteins/molecules.
3. Microtiter plate shaking (5-600 rpm 1.5-3min)--depending on the solution tends to create excellent homogenous mixture with tight CV's; limitation-none really, just need to optimize for time and rpms.
4. Inversion of tubes (10-12 times)--gentle, thorough mixing with larger # inversions--limitation-tough to standardize for inter-day runs etc.
As to optimizing for dilution factor and dilution buffer:
Dilution factor is for diluting past the hook in the "hook effect"--you may not have this need. but normally you would run the assay with samples set up as dilution series (DF = 2, 4, 8, 16 etc).
You may benefit from optimizing for dilution buffer/detergent type and concentration.
You use PBS 1X + 1%BSA.
So you could try PBS vs TBS (round 1, on rare occassion the buffer salt combination makes a big difference).
Assume PBS is the better buffer for you application.
Then try (round 2):
PBS 1% BSA
PBS 0.5% Triton X100 (BSA optional)
PBS 1.0% Triton X100 (BSA optional)
Here if the percentages don't show any difference, I'd keep the lower one 0.5% standard, and try different detergents:
PBS 0.5% Triton X100
PBS 0.5% Tween 20
PBS 0.5% Igepal CA 630
PBS 0.5% Brij 35
You would run sample (3-5 to keep it simple) concurrently in all the above solutions and compare them head to head, to see which underlying buffer solution/mix is better.
I'm sure some things will need clarification. Post any Q's when the time comes.