title: The physical and functional behavior of capture antibodies adsorbed on polystyrene.
abstract: Six monoclonal and two polyclonal antibodies to fluorescein (FLU) were affinity purified and immobilized on Immulon 2 polystyrene as capture antibodies (CAbs): (a) by passive adsorption at pH 9.6, (b) via a streptavidin bridge to a biotinylated carrier molecule, and (c) via an antiglobulin which had been previously adsorbed passively to the polystyrene. Data show that less than 3.0% of the binding sites of monoclonal CAbs and approximately 5-10% of those of polyclonal CAbs were capable of capturing antigen (FLU4.2-BSA) after passive adsorption. Immobilization of CAbs via an antiglobulin or a streptavidin bridge, resulted in the preservation of antibody binding sites to greater than 70% for some monoclonals although immobilization via the streptavidin bridge resulted in the highest number of functional sites/well. The data presented are consistent with studies on other adsorbed proteins which demonstrate that passive adsorption on polystyrene results in the loss of protein function. Furthermore, these data show that generally less than half of the binding sites of antibodies available in solution are available after solid-phase immobilization even when non-adsorptive methods are employed. Some polyclonal anti-FLU also have lower average avidity following passive adsorption compared with CAbs immobilization via a streptavidin bridge. Immunochemical studies revealed that adsorbed polyclonal-CAbs performed like monoclonals when tested with multivalent antigens (FLU10-IgA) but in an expected heterogeneous manner in Scatchard plots when tested using univalent FLU-insulin. This observation implied cross-linking of immobilized CAbs by the multivalent antigen. Because only 5-10% of adsorbed polyclonal CAbs are active, the survivors must be non-randomly distributed in clusters to explain the cross-linking. This was confirmed by scanning electron microscopy which gave rise to the hypothesis that antibodies which retain activity after adsorption, are those present in clusters, i.e., the functional adsorbed CAb is an antibody cluster. Data presented in this report on the behavior of adsorbed CAbs, and reviewed from the work of others for various adsorbed proteins, indicate that the method of passive adsorption at pH 9.6, which is widely used in popular microtiter ELISAs, and which has in many ways revolutionized immunoassay, is a method of protein denaturation. Assayists that utilize passive adsorption of proteins on hydrophobic supports as part of their research need to be cognizant of this phenomenon, while inventors of immunoassay should develop alternative methods of immobilization which do not destroy 90% of the functional activity of solid-phase reactant.
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City 52242.
authors.forename: J E
authors.initials: JE
authors.lastname: Butler
authors.forename: L
authors.initials: L
authors.lastname: Ni
authors.forename: R
authors.initials: R
authors.lastname: Nessler
authors.forename: K S
authors.initials: KS
authors.lastname: Joshi
authors.forename: M
authors.initials: M
authors.lastname: Suter
authors.forename: B
authors.initials: B
authors.lastname: Rosenberg
authors.forename: J
authors.initials: J
authors.lastname: Chang
authors.forename: W R
authors.initials: WR
authors.lastname: Brown
authors.forename: L A
authors.initials: LA
authors.lastname: Cantarero
mesh_terms: D000327:Adsorption; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D007118:Immunoassay / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D011137:Polystyrenes
chemical_list: D000906:Antibodies; D011137:Polystyrenes