Question

why the concentration of rabbit IgG antibodies (ammonium sulphate followed by ion-exchange chromatography) in the lab experiment is lower than the published concentration of IgG in rabbit serum. Two reason?

Answer 1

1. confirm the saturation of Ammonium sulphate and slowly add the to the concentraion of 40-50 % to collect the IgG fraction and dialyse. Instead of ion-exchange chromatography, try affinity chromatography (protein A-column or protein G column)

2. For best amount of antiboy to be collected, serum has to be taken from animal at 5 days to 9 days every alternate day, pooled together for affinity purification/ion exchange chromatography. Another reason might be the lack of immunogenicity of the sample itself. Try to do a control experiment (protein ovalalbumin or Keyhole Limpet Hemocyanin (KLH) alone to isolate antibodies and check concentration)