IMMUNOANALYSIS OF HUMAN INSULIN USING MONOCLONAL-ANTIBODIES REVEALS ANTIGENICITY OF EVOLUTIONARILY CONSERVED RESIDUES

Seven anti-human insulin monoclonal antibodies (mAb) were produced acc ording to an efficient immunization protocol elaborated in our laborat ory. Their affinity constants for the binding to the insulin molecule ranged from 5.0 x 10(8) M(-1) to 1.0 x 10(10) M(-1) when insulin was i n solution and from 6.0 x 10(6) M(-1) to 2.5 x 10(8) M(-1) when insuli n was adsorbed onto the microtiter plate. The antigenic sites on the i nsulin molecule recognized by these mAbs were mapped using two approac hes. MAb pairs capable of binding simultaneously to human insulin in s olution (using a two-site ELISA) or adsorbed onto a microtiter plate ( using a competitive ELISA) were first sought. Three antigenic regions were defined on the surface of adsorbed human insulin and four on solu ble insulin. Two distinct antigenic regions common to both the adsorbe d and the soluble forms of insulin were defined by our mAbs. In a seco nd approach, the immunological cross-reactivities of these mAbs with s pecies variants of insulin, chemically modified insulin of known struc ture and a panel of 78 overlapping nonapeptides covering the entire se quence of human proinsulin were assessed. Evidence was obtained that t he epitopes recognized by the mAbs included residues conserved during evolution in the insulin molecule. The epitopic specificity of one gro up of mAbs (group I) was precisely defined. This group recognized a hi ghly conserved region of the insulin molecule including residues 10-17 of the A chain.