Isoelectric focusing of human antibodies directed against a high molecular weight antigen.

A simple technique has been developed to assess the spectrotypes of antigen-specific human autoantibodies. Following isoelectric focusing in 3 M urea at 1200 V for 2 h, immunoglobulins were fixed in polyacrylamide gels by precipitation with sheep anti-human immunoglobulin in the presence of sodium sulphate. Specific anti-thyroglobulin antibody bands were labelled by reaction with [125I]thyroglobulin, an antigen with a molecular weight of 660,000 daltons which is too large to enter the gel. The advantages of this method include increased sensitivity and reproducibility, and the ability to produce sharper bands and less non-specific binding than other methods.