||[Nov. 1st, 2008|10:55 pm]
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.
1) My boss looked at my results and we basically came to the conclusion that the lower half of the standard curve stunk for whatever reason but a fair number of my duplicate sample wells were consistent, just lower than even the standard curve should have read, so we can't actually say anything about that. While I am totally willing to admit technique error, I was ridiculously meticulous about this ELISA since I had messed up my last one on IL-10.
2) A lab mate wanted me to use this ELISA to measure INF-a levels in mouse serum in his experiment. We had hoped that post transplant that there may have been a spike in the levels. We have never used this kit before but it is one of the only IFN-a kits on the market (unfortunately not designed for serum but we trouble shot that). Kit was not expired, came in that day.
3) The directions say nothing about poor results. I barely had enough directions to figure out how/of to dilute the samples even if I was using culture media and not mouse serum.
4) Did that the best I could. Everything was in duplicate and I even spiked extra serum samples to check for masking/depletion of a known amount of INF-a. Problem was that I couldn't get the curve and thus only have super low OD values.
The duplicates will tell you if your pipetting and your washes are good...
So the top half your your std curve, did you get good numbers from that? Could the issue be in the dilutions? What was the range of your curve that was attempted and what was the range that worked? Did you dilute your standards and your samples in the appropriate buffer?
If nothing else, call the company monday morning?
Sorry if I got defensive, I am really pissed at this ELISA.
Some of the duplicates were perfect, and some were weird. And it had no correlation to the samples groups or the standard, it was just... weird.
The top half of my standard curve would have gotten great readings if any of the samples had the same level as the top half of the curve. Unfortunately all my samples fell in the bottom bad half of my curve. And further, since the program wouldn't even make a standard curve, I have no idea the actual levels of IFN-a in my samples beyond my OD.
Don't know if I mentioned this, but it was a precoated plate sandwich ELISA.
The curves range should have been from 12.5 pg/mL to 500 pg/mL. Ranged that worked was the top three or four which I think was 500 to 100 pg/mL 50 if the fourth standard was good (which I can't remember if it was or not).
Samples and standard were diluted in the buffer provided by the kit. It was the first time I used a plate washer, but I did use the provided wash solution in the plate washer.
Sometimes ELISA is like that. The standards never actually make a straight line, it really is a curve. When you're at the low end the values can sometimes appear to be all over the place. If you're using a kit you tend to assume that they wouldn't tell you to do standards that are below the linear part of the curve because they'd be useless, but the thing is that especially when it depends on room temp for reactions the colour development isn't the same every time. So perhaps you needed to leave the development longer or something.
However, if that is the case it's basically irrelevant if your samples are at the same point in the curve. To get good results you would then need to dilute your samples less so they end up nearer the top half of the curve where it works nicely. That can be a total pain in the butt with mouse serum because you don't have of it (we need special mutant mice that have huge volumes of blood!). It can sometimes be helpful to half volumes and do a 50uL ELISA (assuming you usually use 100ul) so that you don't need as much of it. This has always worked well for me :)
...you don't have a lot of it, that is. I should start proof-reading _before_ posting ;)