In my lab, I am using Biorad Galactomannan ELISA kit. During ELISA process, I washed the plate manually and using Ebra LisaScan reader for getting absorbance at 450/630 nm. But during validation my cut off and negative control are out of range. Please any one suggest how I can over come this problem. Here no automatic washer is available.
What is your blocking solution and percentage of BSA? You can increase the BSA concentration in blocking solution as well as sample diluent solution. This would reduce signal in the negative controls as well as positive controls. If you are getting saturated and near-saturated signal in positive controls and high background in negative controls, there is a lot of scope to increase the BSA concentration in blocking and sample diluent solutions.
Manual washing is as good or even better than using a machine, as you'll get out all the liquid when beating the plate against a stack of paper. Make sure you're filling your plate completely with washing buffer every time.
For a better troubleshooting, please post more details and your protocol. Are there any differences in the composition of the neg control and the cut-off and the samples?
What is the source of this unusually higher background is what you should try to find out. I would suggest to provide more details around the kit reagents, controls, your samples etc.
Chandru Subramani Dear Chandru, I am not using any blocking solution, here I attached the kit details and procedure which may help me to get your suggestion.
Thanking you.
Wolfgang Schechinger Dear Wolfgang, thank you for assuring me that manual washing is better than machine. Yes, I filled the strip of the plate completely.
Thanking you.
Chandra K Dixit Dear Chandra, here I attached the kit details. Please suggest.
Thanking you.