Chromatofocusing combined with the ELISA technique. A sensitive method for the analysis of immune complexes.

A sensitive method which permits analysis of IgG containing circulating immune complexes without detailed knowledge of the nature of the antigens and the specificity of the antibodies involved is described. Soluble BSA: anti-BSA were used as model immune complexes and isolated from serum. The procedure involves the use of gel chromatography for the separation of the high molecular weight fraction containing the immune complexes as measured by binding to 125I-labeled Clq, followed by absorption of the immune complex fraction to immobilized protein A-Sepharose CL-4B. After desorption from protein A-Sepharose the complexes were dissociated and separated into free antigen and antibody by chromatofocusing in the presence of urea. The isolated free antigen and antibody retained their immunological activity as shown by immunodiffusion, binding after their recombination to 125I-labeled Clq, and by recombining antigen and antibody with much enhanced sensitivity using a microplate ELISA system. By means of the ELISA recombination technique it is possible to analyze less than 1 microgram of BSA:anti-BSA model complexes. Application of this technique may provide more information about the nature of immune complex like material associated with diseases.