MIP-2 ELISA detailed questions - (Apr/25/2008 )

I'm doing MIP-2 ELISA for a cell line using a kit from Biosource (now part of Invitrogen). Reproducibility is bad in that the equations obtained from the standard curves and the values obtained between different experiments are quite different. The big intercepts (big blank readings) caused big variations in calculated MIP-2 values.
Could anybody give me general suggestions on how to get better results, including tricks to deal with 96-# plate?

Detailed questions:
1. Could ELISA kits of different batches make significant differences?
2. Which company provides the best ELISA kits, or MIP-2 kits in particular? Or does the company matter?
3. If you always do ELISA the same way with little variations in incubation times and such, is it true that you always get quite similar equations from standards (slope and intercept)?
4. I found that for my MIP-2 ELISA, the standard curve is quite linear (0-250 pg/ml) with an R^2 of 0.98-0.99. Is a linear curve the usual case for ELISA in general?
5. In obtaining the equation from the standards, is the blank value used to get standard curve or we deduct the blank value directly from all OD readings? as we know, as long as R^2 is <1, usually the intercept doesn't equal the blank value.
Sorry for so many Qs but I cannot help myself out any time soon.
Thanks.

The company is well known so the kit should not be a problem. The variation in your standard curve is troubling. Are you running dups and you should run controls between assays (frozen aliquots of large sample) to confirm any differences between kits. Determine if you have a precision problem, accuracy problem or both.

Sounds like you are washing or pipetting may be a culprit. When filling wells make sure you vigorously blot between each wash step. Also, you can allow the wash fluid to sit in the wells for 15 seconds before decanting/blotting.

Do your samples fall within the analytical range of the standard curve and/or do you have to make several dilutions (or also multiple dilutions of the standards)? This can magnify the variation you see.

Thanks a lot! I didn't dilute my samples since the values are within the range of the standards.
I diluted standards with blocking solution, as suggested in the kit. However, my samples were in cell culture medium containing 1% FBS. So I once pondered if it could be better to dilute standards using 1% FBS.
With quite limited experience, I actually worry if blotting too vigorously could blot off the molecules of interest! How vigorous can it be?
There are 12 columns, even with a 8-channel pipet, it still take some time to load the plate with either PBS or another reagent. Could this be a problem, since the last column could be slightly drier than the 1st? I even wonder if I should load to the columns in random sequence, and reverse the direction of pipet so that for some columns, staff in the 1st tip is loaded into the 8th well.