Low OD's for ELISA - (Aug/28/2006 )

I have been doing ELISA since many days and generated good data many times. But now recently, I am having very low OD values and very slow development after adding the substrate. I repeated by coating fresh plates again and doing ELISA. But still the development was very slow. And another time, I got good standard and sample OD values and color development but the positive control (which I used a number of times so far) is very less. Can someone help me what the reason could be? (I am talking about a simple ELISA such as Rat IgG1) Any inputs are greatly appreciated. Thank you in advance.

May be something wrong with your substrate.
Try adding the substrate to the diluted secondary Ab in a eppendorf tubes...to see whether the development of color is normal.

I hope this may help.