Reducing the concentration required for the linear range of the assay - (Mar/08/2012 )
We are using a kit to detect a range of cytokines using ELISA. The linear range for these cytokines is significantly higher than the concentration of the protein in our samples.
We would consider using more protein, with less dilution; however, our sample protein is a very limited resource, and this is impossible.
Is it possible to reduce the concentration of antigen at which the linear range shows itself--that is, shift the curve to the left--using some manipulation of reading equipment? Or is this only possible by changing antibody concentrations (i.e. not using a kit anddeveloping the assay in-house) or incubation times? Are there any other potential workarounds for this problem?
Increase incubation time, increase incubation temperature, inclusion of Peg 8K or 20K in sample buffer, concentrating sample before testing, mixing plate during incubations, pH shocking protein (briefly) then adding to wells...
Increase incubation time and try to agitate the plate during uncubation