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ELISAs [Nov. 1st, 2008|10:55 pm]
Lab Rats

lab_gripes

[bubbleteagirl02]
So, first off, 'ello all!

Second, if anyone works with mice, perhaps you can help me with this question.

Do you know of any company (not R&D or PBL) that makes a good Interferon-alpha ELISA for mouse serum that is really sensitive? I just ran the R&D ELISA, know I did everything perfectly, and it stunk, royally. Thus I am frustrated at the lack of good ELISA readings (the standard curve wasn't even graphable!) and wondering if there is another good ELISA out there or if I need to start looking for other readouts for Interferon-alpha.
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[User Picture]From: lyssabits
2008-11-04 01:04 am (UTC)
R&D's website is a bit irritating, as they do not have anything beyond the data sheet for the assay. A couple of questions/thoughts..

kevisannasdad's suggestion to try letting the color develop longer was a good one. If it's unusually cold that could be slowing your development. If all the readings for the stands tend to be low (like, say, if your highest standard isn't much above 1) then letting the color develop longer may help. I know I've screwed up ELISAs through no fault of my own when the heater in here was broken and it was about 90 degrees. I'd also try diluting the samples less. I know mouse serum is precious, if you need it for something else it can be tempting to try and stretch it.

How are you storing the serum? How many times has this serum been freeze/thawed? Serum's finicky that way, it's not recommended to freeze/thaw it more than 3 times.

When you say your standard curve isn't graphable, what do you mean? Most of the time these curves aren't completely linear, but look more like an S, so it's difficult to detect changes at the lower and upper ends of the curve. However if they're erratic, then that's more likely to indicate a dilution error.

This kit is optimized for media and you're using it for serum? How did you trouble shoot this? (I admit, I have no idea how you'd even begin to trouble shoot for this. ;) ) My first thought was that there was something in the serum inferring with the primary antibody. Do you have any idea what the circulating levels of INF-alpha should be? Some factors circulate at very low levels and the serum needs to be concentrated.

Also noticed that you said you were using a plate washer instead of manually washing.. that does change how many times you're "supposed" to wash. Generally you can wash fewer times with a plate washer than when you do it by hand.. so be careful you're not over-washing.
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