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 IgG(1), the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 10(11) affibody molecules. Four rounds of selection using three different mouse IgG(1) mAbs as alternating targets resulted in the identification of binders with broad mIgG(1) 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 mIgG(1) by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG(1) 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 IgG(1) had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG(1) nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Z(mab25). This selective binding profile towards murine IgG(1) was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.

Affibody molecules and VHH antibody fragments are two classes of affinity proteins, both characterized by a small size and a single subunit domain structure. Here, we report the selection and characterization of affinity proteins from both classes against the special AT rich sequence binding protein SATB1, a suggested marker protein for aggressive and metastasizing breast cancer. The selected VHH antibody fragments originate from a nonimmune phage display library, while affibody molecules were selected from a library constructed in vitro and displayed on ribosomes. It was observed that selected molecules recognizing one of three conserved DNA-binding domains of SATB1, also recognized its close homologue SATB2 while several of the selected molecules from both classes binding to other regions selectively recognized SATB1. Two of these SATB1 selective molecules, VHH clone 2D2 and affibody molecule clone Z_SATB1:2, performed well in differentimmunotechnology applications including ELISA, WB, IF and pull-out experiments and gave a selective staining of endogenous SATB1 in Jurkat T cells. These molecules may thus become useful tools, either alone or in combination, for the selective detection of SATB1 in breast tumor specimens. Due to their small size in comparison to immunoglobulins, such single domain binding proteins may be suitable for high resolution microscopy techniques such as Stimulated Emission Depletion (STED) microscopy, where the resolution may get constrained by the size of the affinity reagent.

Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed

The use of in vitro protein library technologies for the generation of high affinity binding proteins often includes an affinity maturation step, involving the construction of secondary libraries from which second generation variants with improved affinities are selected. We describe a novel approach for the affinity maturation of affibody molecules based on stepwise in vitro molecular evolution, involving cycles of whole gene error-prone PCR amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display for selection. Starting with a human Raf-1 binding affibody molecule of an initial 2 μM dissociation constant (KD), the in vitro evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor based monitoring of the collective target binding ability of expressed pools obtained after each selection cycle. After a first cycle of diversification and selection, no increase in the hRaf-1 target binding of the pool was observed, whereas after two cycles, a significant increase in the binding response was seen. DNA sequencing showed that an alanine to valine substitution in an earlier variegated position had been effectively enriched, and was present in 11% and 83% of all clones after cycle one and two, respectively, either alone or in combination with other substitutions. Further studies on a subset of individual variants isolated after cycle two showed that the observed A27V substitution alone accounted for a 13-fold increase in affinity, predominantly through increased on-rate kinetics. Additional substitutions in framework or non-framework positions typically resulted in a further two-fold increase in affinity. Interestingly, thermal melting point (Tm) analyses showed that an increased affinity could be associated with either slightly higher or lower Tm values, compared to the parental variant. All investigated variants showed excellent refolding properties, and the selectivity of the affinity matured hRaf-1 binders had not been compromised by the substitutions, as analyzed using a multiplexed bead-based binding assay involving 77 recombinant human control protein fragments.