Coupling of antibody-binding fragments to solid-phase supports: site-directed binding of F(ab')2 fragments.
A method to covalently bind antibody fragments, via their carboxyl termini to solid supports, is presented. The strategy involves: (1) reversibly blocking all the accessible carboxyl groups on the antibody molecule with phenylhydrazine, (2) exposing the carboxyl termini of the fragment by enzymatic digestion with pepsin and (3) subsequently coupling the fragment to an appropriate support. Experiments with an anti-bovine serum albumin monoclonal antibody and C-14 phenylhydrazine revealed that the blocking step was nearly completely reversible with a dilute solution of FeCl3. Radioiodinated blocked F(ab')2 fragments were then coupled to an amino-functionalized Sepharose 4B column, and characterized as to their coupling capacity (mass of protein coupled/ml of bead), and antigen-binding activity. The coupling capacity of the blocked fragments was found to be 12%, half the coupling efficiency of unmodified radioiodinated F(ab')2. The antigen-binding capacity (mol antigen bound per mol antibody coupled) for the blocked F(ab')2, on the other hand, was found to be 1.9, which was approx. 3.5-times greater than for the unmodified F(ab')2. Comparisons with other conventional coupling techniques were also made. These preliminary studies suggest that this technique can provide one with the means to obtain more uniform and active populations of immobilized antibody fragments.
Yarmush
M L
ML
Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ 08855.
Lu
X M
XM
Yarmush
D M
DM
D000911:Antibodies, Monoclonal / Q000737:chemistry; D001666:Binding Sites, Antibody; D002846:Chromatography, Affinity / Q000379:methods; D005240:Feasibility Studies; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007164:Immunosorbents / Q000737:chemistry*; D010434:Pepsin A / Q000378:metabolism; D010659:Phenylhydrazines / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000276:immunology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007164:Immunosorbents; D010659:Phenylhydrazines; C030299:phenylhydrazine; D012710:Serum Albumin, Bovine; D010434:Pepsin A