Macrophage recognition of immune complexes: development and application of novel cell surface labeling procedures

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Abstract
A fluorescein- and lactoperoxidase-conjugated ferritin-anti-ferritin immune complex was been prepared for murine cell surface labeling experiments on immune recognition and effector function. Lactoperoxidase (LPO) has been covalently coupled to affinity-purified anti-ferritin antibodies with p-benzoquinone by a modified version of the method of Ternynck and Avrameas. The conjugate is a heterodimer of MW 230,000 with linkages to either or both of the H and L chains of the antibody, as judged by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence and presence of 2-mercaptoethanol. The conjugate retains antibody-binding activity as measured by a quantitative precipitin assay. When incorporated into immune complexes, the modified antibody also retains Fc receptor recognition ability as determined by erythrocyte-antibody rosette inhibition assays. EM demonstrated that the antigen, ferritin, was monodisperse with complete apoprotein sheaths surrounding the core. Ferritin-anti-ferritin-LPO complexes were formed in 4-fold antigen excess. Complexes were verified by fluorescence and EM. Immune complexes were masked with cold I by use of the endogenous LPO activity. The complexes bound to cells at 4.degree. C as shown by EM and fluorescence video/intensification microscopy. The LPO delivered to the cell surface in this fashion can be utilized to iodinate the surface with 125I. Under saturation conditions, the labeling with local LPO delivery followed by SDS-PAGE and autoradiography is identical with labeling with free LPO. Labeling was also conducted under conditions of substrate deficit. Bovine serum albumin was employed to scavenge I+, which may diffuse from the local LPO delivery sites. These conditions reveal that, during immune complex recognition, a local polymerization reaction occurs that can be at least partially reversed with 2-mercaptoethanol.