Single-chain Fv Antibody Fragment Research Articles

Selection Procedures for Nonmatured Phage Antibodies: A Quantitative Comparison and Optimization Strategies

Libraries of peptides and proteins can be displayed on the surface of filamentous bacteriophage. The efficient capturing of phage recognizing a defined target molecule remains a serious obstacle, in particular when the phage are present at a low frequency or have a reduced affinity like nonmatured phage antibodies and when the availability of target molecules is limited. We present theoretical considerations and experimental data which allowed us to substantially improve microselection under these conditions. We used a model phage displaying an anti-(2-phenyl-5-oxazolone) single-chain Fv antibody fragment. Following standard protocols and aiming at a low nonspecific binding, only 3.6 × 10 −3% of the phage input could be recovered from a single round of selection performed in the wells of a microtiter plate. Our results explain why this often employed panning in wells is not efficient, especially with high-molecular-weight target molecules. We devised a procedure which increased the probability of microselection by a factor of 34. An alternative capturing method using immunotubes with a new protocol decreased the amount of required work by a factor of 30. In the case of a nonlimited supply of target molecules, column-affinity chromatography is recommended.

The expression of recombinant proteins on the external surface of Escherichia coli. Biotechnological applications.

The expression of recombinant proteins on the external surface of Gram-negative bacteria is expected to open the way for a number of significant biotechnological applications, including the development of live bacterial vaccines, the production of whole cell adsorbents, the preparation of whole cell catalysts, and the display and selection of peptide and antibody libraries. We have developed a fusion protein system for the production of active recombinant proteins on the surface of Escherichia coli. Using this system we have expressed beta-lactamase, the Cellulomonas fimi exoglucanase Cex as well as its cellulose binding domain, and an antidigoxin single chain Fv antibody fragment on the cell surface. Recently we have begun to explore some of the potential applications for cell-surface expression.

Characterization of human blood group scFv antibodies derived from a V gene phage‐display library

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

Recombinant single-chain antibody peptide conjugates expressed in Escherichia coli for the rapid diagnosis of HIV

Recombinant single chain Fv (scFv) antibody fragments can form the basis of a rapid, whole-blood diagnostic assay. The scFv described in this study is derived from a monoclonal antibody which has a high affinity for glycophorin A, an abundant glycoprotein on the human red blood cell membrane surface. The prototype reagent built around the scFv was designed to detect, in whole blood samples, the presence of antibodies that have arisen through infection with a foreign organism such as human immunodeficiency virus. The scFv was composed of the antibody heavy-chain variable domain (V h) joined by a 15 residue linker-(GGGGS) 3- to the light-chain variable domain (V 1) terminated by either a C-terminal octapeptide tail (FLAG) or a 35 amino acid segment from the gp41 surface glycoprotein of HIV-1. Constructs were cloned into a Escherichia coli expression vector, pHFA, and expressed in a soluble form into culture supernatant. The product retained anti-glycophorin activity which could be detected directly in culture supernatants by ELISA. Furthermore, the scFv-epitope fusion functioned efficiently in the whole blood agglutination assay and was able to distinguish between HIV-1 positive and negative sera.

Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface.

We have expressed a single chain Fv (scFv) antibody fragment, consisting of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escherichia coli by fusing it to an Lpp-OmpA hybrid previously shown to direct heterologous proteins to the cell surface. This scFv fusion was expressed at a high level and was shown to bind the hapten with high affinity and specificity. Whole cell ELISAs, fluorescence microscopy, protease sensitivity, and flow cytometry all confirmed that the scFv was anchored on the outer membrane and was accessible on the surface. Utilizing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 10(5)-fold excess of control cells in only two steps. The expression of antibody fragments on the surface of E. coli is being evaluated as an attractive method for the in vitro production and selection of useful antibody fragments.

Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein-single-chain antibody fusion protein.

In order to achieve a stable and functional immobilization of antibodies, we investigated the possibility of adding hydrophobic membrane anchors to antibody fragments expressed in Escherichia coli. The DNA sequence encoding the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of E.coli was fused to the sequence of an anti-2-phenyloxazolone single-chain Fv antibody fragment [Takkinen et al. (1991) Protein Engng, 4,837-841]. The expression of the fusion construct in E.coli resulted in specific accumulation of an immunoreactive 28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment, the fusion protein was cell-associated, labelled by [3H]palmitate which is indicative of the presence of N-terminal lipid modification, partitioned into the detergent phase upon Triton X-114 phase separation and was localized predominantly in the bacterial outer membrane. The fusion antibody displayed specific 2-phenyloxazolone-binding activity in the membrane-bound form and after solubilization with non-ionic detergents. Furthermore, upon removal of detergent the fusion antibody was incorporated into proteoliposomes which displayed specific hapten-binding activity. Our results show that antibodies can be converted to membrane-bound proteins with retention of antigen-binding properties by introduction of lipid anchors during biosynthesis. This approach may prove useful in the design of immunoliposomes and immunosensors.

Engineered Proteins for Biomaterials

AbstractA molecular adaptor for interfacing environmentally sensitive, soluble polymers and antibody molecules has been developed. The gene coding for the minimally sized, 55 amino acid IgG binding domain from protein G has been constructed by total gene synthesis. This domain is thermally stable, exhibits a highly reversible folding and unfolding equilibrium, and recognizes IgG and Fab molecules with high affinity. These properties make the protein G domain a potentially useful adaptor for non-covalent immobilization of antibodies to soluble polymers and hydrogels. Engineered single-chain Fv antibody fragments have also been constructed and a method for expanding the usefulness of the protein G adaptor to these molecules is proposed. The engineered antibodies also provide a model system for developing general immobilization strategies aimed at maximizing binding affinities and therapeutic responses. The overall goal is to develop optimized engineering designs for functionally optimized antibody-material hybrids.