From the crude polyclonal the crossreactive antibodies were extracted by incubation with Sepharose-bound human IgA and IgM. Specific antibodies were absorpted by incubation with Sepharose-bound human IgG. Specific antibodies were eluted by acidic buffer at pH 2.5 followed by neutralisation and dialysis. After repeated binding with immobilized human IgG a minimum of 65% protein bound.
The purified polyclonal was conjugated to fluorescein isothiocyanate isomer I followed by gel-filtation and ion-exchange chromatography to clear unbound conjugate.
Low background fluorescence of normal tissue in contrast to cases of autoimmune desease, depostited immunoglobulin IgG.