Help me help the cows...

October 24, 2008 5:28 PM

Can I dilute my samples and keep them in the fridge for a couple of days without affecting my results?

I am using an ELISA to test for Babesia bigemina IgG antibodies. If I dilute my sera samples using PBS-Tween and then store them in the fridge for a couple of days, will this affect the results (as opposed to diluting them and running the ELISA right away)? I couldn't think of a good reason why it would, but I want to make sure. Thanks in advance.

posted by bolognius maximus to Science & Nature (3 answers total)

Depending on how dilute you're talking, you might have issues where a significant amount of your protein is sticking to the sides of your container. In a more concentrated solution you'd still have this effect, but if one µg of protein is going to stick to your tube (just pulling a number out of the air) that only represents a small fraction of the protein in neat serum. If you dilute the sample, it might be most (or even all) of the protein present.

If you have a known standard, spike a bit of one of your samples at say 10x your quantitation limit, dilute a portion of that now and more the day you're actually going to run and see if you see any difference it makes in your final result.

If this is something that you might want to do regularly, maybe add a little BSA to your diluent so that if something is non-specifically binding out of solution it's mostly BSA. Of course you'll have to confirm that doesn't interfere too.
posted by Kid Charlemagne at 5:47 PM on October 24, 2008

The Kid is right. Commercial ELISA kits usually use a diluent that's PBS + 0.1% Tween-20 + 1 mg/ml BSA (or thereabouts), to prevent loss of protein by adsorption to the test tube walls.

Do as the Kid says, with the additional suggestion to use a decent amount of BSA (somewhere in the 1 - 5 mg/ml range). If BSA interferes with your assay, you can try casein as an alternative blocker. There are also commercial blocking solutions available from Pierce. Good luck!
posted by Quietgal at 10:35 PM on October 24, 2008

Response by poster: Thanks for the suggestions! Back to the bench...
posted by bolognius maximus at 6:34 AM on October 25, 2008