Sandwish ELISA Problems and Troubleshooting

[proatpcr]

Hello, I have a big problem with my sandwich ELISAs.

I am using a mouse monoclonal antibody to coat the plate after which I block with PBS/BSA 1%.


I am using rabbit polyclonal serum against my protein of interest.
Maybe the serum polyclonal antibody contains antibodies against BSA?

The fact is that the lysis buffer has an value important and more interestingly my cells lysates without the protein of interest have the same value as 12500pg of the protein alone using the same buffer.

I do not know what i can change in the protocol. If someone can help thanks

[danfive]

Hi,
Here are some good online resources from BD biosciences:
Typical protocol: [Only registered users see links. ]
Troubleshooting videos: [Only registered users see links. ]
Look at the video for Troubleshooting: high background --> [Only registered users see links. ]

[premkumarkalpana]

Hello sir,
This is premi i have a problem in getting linearity in standard of IL8 , my optical density not rised so please help me to get higher Optical density.

[danfive]

Originally Posted by premkumarkalpana

Hello sir,
This is premi i have a problem in getting linearity in standard of IL8 , my optical density not rised so please help me to get higher Optical density.

OD not high enough--either extend the color development step or increase the detector (secondary Ab-conjugate) concentration.