ELISA - direct protein assay with ELISA (Jan/30/2002 )

I would like some advice with ELISA's. We are trying to measure protein concentrations in various cell lines and are having trouble with replicability. Even when we repeat the dilutions four times, we still get a large variability in OD405, and are not sure what to trust (no textbook results here!) -- any suggestions?
Nicola

find out whether your plate reader is calibrated and if so question each step in your handling technique and pipetting, also how you handle the wash. try reverse pipetting to ensure volume reproducibility ensure they are also calibrated.  It might still be difficult getting exactly 2 of same od readings, however , the difference between reading such not have any significance.
good luck
ps, the smallest error in handling can arise to alot of problems, try using a reagent or protein of known od to check the machine and reproducibility. The producers of the plate reader sometimes have a calibration reagent. ask them.