Preparation of monoclonal antibody-ferritin conjugates of high specific activity.
125I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 1:1.4, containing over 90% ferritin-IgG "monomers"; 70-90% of the 125I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.
D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D005293:Ferritins; D005976:Glutaral; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D051379:Mice; D008854:Microscopy, Electron
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D005293:Ferritins; D005976:Glutaral