Detection and Quantitation of Human Ia-Type Antigens with Iodinated Protein A and Specific Purification of Antibodies Against Ia-Type Alloantigens

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Abstract
Antibodies directed against specific human Ia-type antigens can easily be detected and quantitated by an improved radioimmunoassay using iodinated protein A bound to a specific antibody-Ia-antigen complex on the surface of freshly drawn peripheral human leukocytes, cultured human cell lines, or lymphoid cells fixed with glutardialdehyde or formaldehyde. The same principle can also be used for the detection of Ia alloantigens on human lymphocytes when testing them with specific antisera known to contain antibodies against transplantation antigens. These anti-Ia-alloantigen antibodies had been purified by a two-step procedure involving ion-exchange chromatography on DEAE-cellulose at pH 6.3 and the specific absorption on formaldehyde-fixed Ia-alloantigen-carrying homozygous cell lines, followed by elution of these antibodies with isotonic citrate buffer at pH 3.0. In this way an about 90-fold purification could be achieved. After such a purification the highly enriched antibody fraction still reacted selectively with one specificity of the Ia antigen system.