ELISA optimization problems - ELISA (Jan/16/2008 )

Hi
I'm having a problem developing an ELISA for GFP. I previously used the Peirce reactibind anti-GFP plates and had no problems. I used a goat polyclonal anti-GFP alkaline phosphatase secondary antibody. Unfortunately Peirce stopped making those plates. So i am now trying to reproduce those plates.

I tried using a mouse monoclonal anti-GFP capture antibody on Costar ELISA plates. I used 5% non-fat milk in TBST as a blocking reagent. I found out today that Pierce uses a Polyclonal goat antibody for the plate. I am getting a polyclonal antibody in this week to try that.

I incubated 100ul of primary antibody for 2hr at RT. I washed 4x with PBS / Tween 80 then added 300ul of my blocking reagent and let that sit for 1hr at RT. I washed again 4x with the PBS tween and added my recombinant GFP. I let that sit overnight at 4C. washed 4x with PBS tween, incubated with the same secondary antibody as before, developed with alk phos substrate and then read the plate.

My standard curves all came out very flat, no matter what concentration of primary antibody i used. the numbers were as follows:

1 to 6000
1000pg 0.903
100pg 0.86
10pg 1.109
1pg 0.605
0pg 0.083

1 to 12000
1000pg 0.588
100pg 0.588
10pg 0.573
1pg 0.412
0pg 0.086

1 to 24000
1000pg 0.439
100pg 0.575
10pg 0.529
1pg 0.264
0pg 0.153

1 to 36000
1000pg 0.341
100pg 0.328
10pg 0.279
1pg 0.165
0pg 0.114

1 to 48000
1000pg 0.293
100pg 0.324
10pg 0.261
1pg 0.196
0pg 0.072

1 to 60000
1000pg 0.24
100pg 0.194
10pg 0.156
1pg 0.123
0pg 0.083

I've also uploaded an excel file to make it easier to visualize. I'm getting great sensitivity, and it seems like there is a direct correlation between capture antibody concentration and amount of absorbance. so i think that what I am seeing is actually GFP. I need to get a standard curve. Any suggestions. I'm going to try blocking with 5% BSA next.
thanks in advance.

OK, your problems may be from a few sources.
1. The plates might be different. Try a different plate and see if you get better signal to noise. If you have any of the old Pierce plates, see what type they were.
2. I would try PBST with BSA for the blocking step and the washes.

For a great set of reagent conditions, look at the R&D Systems site http://www.rndsystems.com . The "ELISA/assay development Kits and Reagents" section of the Products menu has the conditions for all of the R&D assays. You should get some ideas of different buffers to try.

3. Because you don't know the coating concentration, you will probably need to optimize the secondary Ab concentration. What sort of dynamic range did you get with the Pierce kit?

I know it is a bit off topic, but there are so many ELISA topics, so ...

Has anyone tried or considered to freeze the standard samples of his ELISA kit in oder to use them again next time? Actually I froze my 2000, 1000, 500pg/mL and so on, and now I wonder whether I could use them again? I mean I don't know why I couldn't ...

I know it is a bit off topic, but there are so many ELISA topics, so ...

Has anyone tried or considered to freeze the standard samples of his ELISA kit in oder to use them again next time? Actually I froze my 2000, 1000, 500pg/mL and so on, and now I wonder whether I could use them again? I mean I don't know why I couldn't ...