title: Ribosome display selection of a murine IgG₁ Fab binding affibody molecule allowing species selective recovery of monoclonal antibodies.
abstract: Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG₁, the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 10¹¹ affibody molecules. Four rounds of selection using three different mouse IgG₁ mAbs as alternating targets resulted in the identification of binders with broad mIgG₁ recognition and dissociation constants (K(D)) in the low nanomolar to low micromolar range. For one of the binders, denoted Z(mab25), competition in binding to full length mIgG₁ by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG₁ Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH₁ domain of mouse IgG₁ had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG₁ nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Z(mab25). This selective binding profile towards murine IgG₁ was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.
authors.affiliation: Division of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), Roslagstullsbacken 21, 10691 Stockholm, Sweden.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Grimm
authors.forename: Feifan
authors.initials: F
authors.lastname: Yu
authors.forename: Per-Åke
authors.initials: PÅ
authors.lastname: Nygren
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D015153:Blotting, Western; D002417:Cattle; D002846:Chromatography, Affinity; D020650:Combinatorial Chemistry Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D007163:Immunosorbent Techniques; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010206:Papain; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D013045:Species Specificity
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D010206:Papain