title: Evaluation of the mode of binding of immunoglobulin to activated agarose.
abstract: Determining the orientation of the immobilization of proteins to solid-phase matrices is of critical importance in the development of systems that employ immobilized proteins. Among these are enzyme-linked immunoassays, immobilized enzymes and affinity chromatography matrices. To determine the orientation of immunoglobulin G (IgG) on activated agaroses, we coupled the immunoglobulin covalently to various activated matrices. The IgG was then cleaved with papain and the liberated fragments collected and analyzed using high-performance liquid chromatography. Only Fab fragments could be detected regardless of the activation method used. This implies that IgG binds to these matrices predominantly via the Fc domain. In order to develop a quantitative method of measuring the Fab and Fc fragments, we compared the binding of IgG and its papain cleavage fragments to S-Zephyr columns and Mono-S columns. Comparison between these columns showed that IgG is bound more tightly to the S-Zephyr column and, in contrast, its retention on Q-Zephyr is less than on a comparable Mono-Q column. The resolution of IgG and its fragments was better in all cases on S-Zephyr than on Mono-S under the conditions employed.
authors.affiliation: Department of Chemistry, Baylor University, Waco, TX 76798.
authors.forename: W H
authors.initials: WH
authors.lastname: Scouten
authors.forename: C M
authors.initials: CM
authors.lastname: Adler
authors.forename: R
authors.initials: R
authors.lastname: Rongen
authors.forename: L
authors.initials: L
authors.lastname: Mallee
mesh_terms: D002021:Buffers; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000737:chemistry; D007136:Immunoglobulins / Q000737:chemistry*; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry*
chemical_list: D002021:Buffers; D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D007136:Immunoglobulins; D012685:Sepharose