Binding specificity of a monoclonal antibody to human Hb A

HemoCardTM kits use monoclonal antibody-conjugated metal sol particles in an immuno-concentration assay. The HemoCard Hbs S, C, and E kits detect the three most common hemoglobin variants in both adult and newborn samples. The HemoCard Hb A kit can be used in conjunction with HemoCard Hbs S and C (but not HemoCard Hb E) to distinguish heterozygotes from homozygotes. We examined the antigen-binding domain of a monoclonal antibody to Hb A by testing samples from human and animal sources. We confirmed that the Hb A conjugate recognizes an epitope in which glutamic acid at beta 6 is necessary to provide the proper conformation for binding. This binding is not affected by substitutions at positions directly adjacent to beta 6. However, extensive substitutions in the primary sequence (e.g. in chicken, beta 6: glutamic acid, substitutions at beta 3, beta 5, beta 9, beta 10, beta 11) may produce conformational changes, resulting in diminished binding.