Measurement of high affinity antibodies on antigen-immunoblots.
We describe a semi-quantitative method for measuring the relative affinity of antigen-specific oligoclonal IgG bands separated by isoelectric focusing followed by blotting onto antigen-coated membrane and incubation with sodium thiocyanate. When the developed blot is digitised in greyscale, densitograms can be made and peak areas calculated using ImageJ freeware. By expressing peak area as a percentage of the total area under the curve we have shown that there is a statistically significant rise in percentage of peak area for a given band which persists with increasing molarities of sodium thiocyanate.
Chapman
Miles D
MD
Department of Neuroimmunology, Institute of Neurology, Queen Square, London, UK. m.chapman@ion.ac.uk
Keir
Geoffrey
G
Petzold
Axel
A
Thompson
Edward J
EJ
D019540:Area Under Curve; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting / Q000379:methods*; D007136:Immunoglobulins / Q000032:analysis*; D007525:Isoelectric Focusing; D012709:Serum Albumin / Q000737:chemistry*; D013861:Thiocyanates / Q000737:chemistry*
D007136:Immunoglobulins; D012709:Serum Albumin; D013861:Thiocyanates; C024553:sodium thiocyanate