Cellular effects and orientation of immobilized immunoglobulin are correlated to the charge-mediated influence of the antibody variable region.

Ligand binding to a cell receptor often insufficiently triggers cellular immune responses. Receptor clustering through cross-linking occurs when a ligand binds to two or more receptors, amplifying cellular responses. This is required in certain monoclonal antibodies (mAbs), including effector mechanism activation [binding to fragment crystallizable receptors (FcRs)] or acting as agonists for therapeutic signaling. Therefore, immobilized immunoglobulin immunoassays were developed for efficient diagnostic and therapeutic approaches. The immobilized mAb density and orientation influence the sensitivity and accuracy of these assays. Limited evidence shows that different epitope motifs with the same target mAbs affect immobilized density and orientation in the solid-phase state. Here, we developed a series of fully humanized antidendritic cell immunoreceptor (DCIR) mAbs with different epitopes but the same Fc region. Immobilized anti-DCIR mAbs trigger the effector response from FcR through the Fc region and induce inhibitory pathways from the DCIR intracellular immunoreceptor tyrosine-based inhibitory motif through the fragment variable (Fv) region. In the immobilized immunoglobulin immunoassay, the isoelectric points (pI) of the DCIR mAb Fv region, not the total pI, significantly correlate to the surface density and orientation of immobilized mAbs on negatively charged plates. Cytokine production and protein phosphorylation in human monocytes were affected by vary binding abilities of immobilized mAbs to the plate. Methods, such as increasing hydrophobicity or ionic interactions, have improved the surface density and consistent orientation of immobilized anti-DCIR mAbs. Our study highlights the critical relationship between the net charge of the antibody Fv region and its immobilization potential in the solid-phase state.