title: Nature of immobilization surface affects antibody specificity to placental alkaline phosphatase.
abstract: Retention of native conformation of immobilized protein is essential for various applications including selection and detection of specific recombinant antibodies (scFvs). Placental alkaline phosphatase (PAP), an onco-fetal antigen expressed on the surface of several tumors, was immobilized on supermagnetic particles for selection of recombinant antibodies from a human phage display antibody library. The isolated antibodies were found to be cross-reactive to either of the isozymes of alkaline phosphatase, i.e., bone alkaline phosphatase (BAP) or intestinal alkaline phosphatase (IAP) and could not be used for tumor targeting. A specific anti-PAP monoclonal antibody H17E2 was tested for retention of specificity under these conditions. Binding of the antibody to magnetic beads conjugated IAP and BAP along with PAP and the ability of the two isozymes to inhibit its binding to PAP depicted the loss of isozyme specificity of the antibody. However, the antibody retained its specificity to PAP immobilized on polyvinyl chloride (PVC) surface. Enzyme activity was observed on both surfaces. This demonstrates that nature of immobilization may affect antigen-antibody binding in subtle ways, resulting in alteration of conformation of the epitopes. This may have consequences for determining the specificity of antibody binding for proteins that share a high degree of homology.
authors.affiliation: a National Brain Research Centre , Manesar , Haryana , India.
authors.forename: Mukesh
authors.initials: M
authors.lastname: Kumar
authors.forename: Imran
authors.initials: I
authors.lastname: Khan
authors.forename: Subrata
authors.initials: S
authors.lastname: Sinha
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000276:immunology*; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology*; D011143:Polyvinyl Chloride / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D004800:Enzymes, Immobilized; D058851:GPI-Linked Proteins; D007527:Isoenzymes; D011143:Polyvinyl Chloride; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental
keywords: ELISA; antibody specificity; immobilization surface; placental alkaline phosphatase; protein conformation stability