Hello everyone,
I have generated a monoclonal antibody and now I am screening it for its reactivity. I have read somewhere that I should also do immunoabsorption assay to check the specificity of the Ab. Can anyone suggest me the procedure for immunoabsorption?
You can measure the reactivity by ELISA. Involves all steps performed in ELISA.
Coat the plate with antigen. Incubate plate overnight at 4°C. Further steps involves blocking, washing and adding antibody solutions, dispensing test solution into each well, incubating and adding anti IgG- conjugates, washing and reading at 405nm.
I think what you are specifically looking for is Ouchterlony's double diffusion method.
http://www.austincc.edu/mlt/ser/OuchterlonyGelDiffuseionFall2009Edvotek.pdf
https://www.gbiosciences.com/image/pdfs/protocol/BE-501_protocol.pdf
You can do Sandwich ELISA. This a standard protocol for Sandwich ELISA https://www.abcam.com/ps/pdf/protocols/Sandwich_ELISA.pdf
For specificity determination of your antibody to protein of interest you can also do western blot.This a standard protocol for western blot http://docs.abcam.com/pdf/protocols/general-western-blot-protocol.pdf
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