Fv Antibody Fragment Research Articles

Efficient in vivo targeting of malignant melanoma by single-chain Fv antibody fragments.

Single-chain antibody fragments (scFvs) have superior pharmacokinetic and biodistribution characteristics compared with larger antibody fragments when used for radioimaging of human tumours. An scFv specific for malignant melanoma (B4 scFv) was tested in a mouse xenograft model for human melanoma and compared with the monoclonal antibody (MAb) LHM2. LHM2 is specific for the high molecular weight proteoglycan melanoma-associated antigen (HMW-MAA), while B4 scFv binds a novel melanoma-specific antigen. The B4 scFv was cleared rapidly from the circulation (t(1/2)alpha = 7 min) compared with LHM2 MAb (t(1/2)alpha = 37 min). However, the B4 scFv had an unusually long t(1/2)beta (437 min compared with 384 min for LHM2 MAb). Biodistribution studies showed that B4 accumulated specifically in the tumour grafts. Although non-specific accumulation in the liver, lung, spleen and blood was lower than with LHM2, non-specific accumulation of B4 in the kidney was higher. Despite this, the overall targeting efficiency in this study, as determined by tumour:normal tissue ratios, showed that B4 was superior to whole antibody. Immunoscintigraphy images showed good correlation with the biodistribution data for B4 and LHM2. This study represents the first use of an anti-melanoma scFv in vivo.

Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria

The equilibrium denaturation and unfolding kinetics of the domains V L and V H have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimeric assembly even further. Domain dissociation and domain unfolding are coupled processes, giving rise to a highly cooperative unfolding transition. For the Fv fragment, cooperative unfolding is only observed in the presence of antigen. At low protein concentrations and in the absence of antigen, the Fv fragment is significantly destabilized, leading to quantitative domain dissociation before significant domain unfolding takes place. The kinetic unfolding of V H, V L and the scFv fragment is monophasic. Unfolding of the scFv fragment is much slower, when extrapolated to zero denaturant, than either of the isolated domains, suggesting that the higher thermodynamic stability of the scFv fragment is at least partially due to a high-energy transition state for unfolding. These studies emphasize the enormous importance of mutual domain stabilization in engineering stable antibodies.

Crystal Structure of Anti-Hen Egg White Lysozyme Antibody (HyHEL-10) Fv-Antigen Complex: LOCAL STRUCTURAL CHANGES IN THE PROTEIN ANTIGEN AND WATER-MEDIATED INTERACTIONS OF FV-ANTIGEN AND LIGHT CHAIN-HEAVY CHAIN INTERFACES

In order to address the recognition mechanism of the fragments of antibody variable regions, termed Fv, toward their target antigen, an x-ray crystal structure of an anti-hen egg white lysozyme antibody (HyHEL-10) Fv fragment complexed with its cognate antigen, hen egg white lysozyme (HEL), was solved at 2.3 A. The overall structure of the complex is similar to that reported in a previous article dealing with the Fab fragment-HEL complex (PDB ID code,). However, the areas of Fv covered by HEL upon complex formation increased by about 100 A(2) in comparison with the Fab-HEL complex, and two local structural differences were observed in the heavy chain of the variable region (VH). In addition, small but significant local structural changes were observed in the antigen, HEL. The x-ray data permitted the identification of two water molecules between the VH and HEL and six water molecules retained in the interface between the antigen and the light chain complementarity determining regions (CDRs) 2 and 3 (CDR-L2 and CDR-L3). These water molecules bridge the antigen-antibody interface through hydrogen bond formation in the VL-HEL interface. Eleven water molecules were found to complete the imperfect VH-VL interface, suggesting that solvent molecules mediate the stabilization of interaction between variable regions. These results suggest that the unfavorable effect of deletion of constant regions on the antigen-antibody interaction is compensated by an increase in favorable interactions, including structural changes in the antigen-antibody interface and solvent-mediated hydrogen bond formation upon complex formation, which may lead to a minimum decreased affinity of the antibody Fv fragment toward its antigen.

Selection of phage antibodies to surface epitopes of Phytophthora infestans

Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments. The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by BstNI restriction enzyme digest patterns. Antibodies were subcloned as fusions at the C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in Escherichia coli. These antibody fusion proteins bound to P. infestans germlings and to mycelial homogenates from various Phytophthora species. The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower. Several phage-displayed scFvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings. The combination of procedures developed here with Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions.

Recombinant single-chain Fv antibody fragment–alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization

Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv). The anti-AAF scFv-PhoAv hybrid protein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further purification, for one-step immunodetection in dot-blot hybridization. The detection limit was identical and the test was quicker than the conventional two-step procedure with the purified anti-AAF MAb revealed with a secondary enzyme-labeled antibody. To assess the value of this new reagent for the immunodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv–PhoAv conjugate in a Southern-blot hybridization experiment. The present study shows that the genetic fusion with PhoAv provides a new tool for immunodetection which presents easier and quicker production and use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv–PhoAv conjugate is a promising alternative reagent for applications involving the immunodetection of specific DNA or RNA sequences, such as the detection and characterization of microorganisms.

ScFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies.

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the VH and VL domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody). Direct ligation of VH and VL domains further restricts association and forces three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructing a series of scFvs from anti-neuraminidase antibody NC10 in which the linker varied from one to four glycine residues. NC10 scFv molecules containing linkers of three and four residues showed a strong preference for dimer formation (diabodies), whereas a linker length of one or two glycine residues prevented the formation of diabodies and directed scFv association into trimers (triabodies). The data suggest a relatively strict transition from dimer (diabody) to trimer (triabody) upon reduction of the linker length from three to two glycine residues. Modelling studies are consistent with three residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multimers and an anti-idiotype Fab' showed that the dimer was bivalent for antigen binding and the trimer was trivalent.

Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding

Single chain variable fragments (scFv) of the murine monoclonal antibody HD37 specific to human B-cell antigen CD19 were constructed by joining the V H and V L domains with linkers of 18, 10, 1 and 0 residues. ScFv-18 formed monomers, dimers and small amounts of tetramers; scFv-10 formed dimers and small amounts of tetramers; scFv-1 formed exclusively tetramers; scFv-0 formed exclusively trimers. The affinities of the scFv-10 (diabody) and scFv-1 (tetrabody) were approximately 1.5- and 2.5-fold higher, respectively, than that of the scFv-0 (triabody). The tetrabody displayed a significantly prolonged association with cell-bound antigen ( t 1/2 cell surface retention at 37°C of 26.6 min) compared to both the diabody (13.3 min) and triabody (6.7 min). This increase in avidity of the tetrabody combined with its larger size could prove to be particularly advantageous for imaging and the immunotherapy of B-cell malignancies.

A bacterial single-chain Fv antibody fragment that inhibits binding of its parental anti- E-Selectin monoclonal antibody to activated human endothelial cells

Using the polymerase chain reaction, we cloned, modified, and linked antibody variable (V) region coding genes from a mouse hybridoma, and produced a bacterial single-chain Fv (scFv) antibody fragment specific for E-Selectin. A vector of pBR322 origin, bearing the tryptophan promoter and the ompA bacterial signal peptide, was used to direct scFv expression to periplasm. The vector included a six-histidine coding sequence 5′ to the scFv for the purification of the expressed protein using immobilized metal affinity chromatography (IMAC). We found that the V H-Linker-V L 32–33 kDa scFv remained insoluble after cellular fractionation, and transmission electron microscopy showed the new protein to be present in the periplasm as inclusion bodies. The scFv was solubilized using urea, purified using IMAC, and renatured to its active form. In a competitive enzyme-linked immunosorbent assay with activated human vein endothelial cells in the solid phase, the scFv competed for binding with the original monoclonal antibody.

High cytoplasmic expression in E. coli, purification, and in vitro refolding of a single chain Fv antibody fragment against the hepatitis B surface antigen

A single-chain Fv (scFv) antibody fragment against the hepatitis B surface antigen (HBsAg) was expressed in Escherichia coli in the form of two independent fusion proteins, with either 60 (‘long’) or 27 (‘short’) amino acid N-terminal encoding sequences related to human interleukin-2. Both fusion proteins were expressed insolubly and at high levels in the bacterial cytoplasm (approximately 30% of total bacterial protein in MM294 cells at a laboratory scale). When recombinant cells were cultured in 5-l fermentors, expression and optical density increased 2- and 4-fold, respectively, compared to a previous periplasmic insoluble version of the same anti HBsAg scFv. After extraction and solubilization in urea, the cytoplasmic scFvs were purified using immobilized metal ion affinity chromatography, followed by DTT treatment, and refolding by dialysis against a basic pH buffer containing EDTA. The refolded scFvs recognized the recombinant HBsAg in ELISA. Results of an ELISA where antigen affinity chromatography repurified scFvs were used as standards, indicated that refolding efficiencies were high: 56.2% for the ‘short’ fusion scFv, and 50.6% for the ‘long’ fusion scFv. Corrected final yields of active scFv were 30.3 and 27.3 mg l −1, respectively, for the aforementioned fusion proteins, 5–6 times better than those reported for the periplasmic scFv variant.

Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors.

The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.

Crystal structure of a ternary complex between human chorionic gonadotropin (hCG) and two Fv fragments specific for the α and β-subunits

Human chorionic gonadotropin (hCG), is a placental hormone which exerts its major effect by stimulating progesterone production, crucially sustaining the early weeks of pregnancy. Detection of hCG with specific monoclonal antibodies (mAbs) has become the chosen means for pregnancy diagnosis. We have used antibody Fv fragments derived from two high-affinity mAbs, one against the α and the other against the β-hCG subunit to enable the crystallisation of intact or desialylated hCG. Crystals of a ternary complex composed of Fv anti-α/hCG/Fv anti-β were found to diffract to 3.5 Å resolution, and the structure was solved by molecular replacement. In the crystal, the two Fvs keep hCG as in a molecular cage, providing good protein-protein contacts and leaving enough space for the saccharides to be accommodated in the cell solvent. The two Fvs were found not to interact directly through their complementary-determining regions with the hCG saccharides, but only with the protein. The hCG structure in the ternary complex was very close to that of the HF partially deglycosylated hormone, thus indicating that neither the saccharides nor the Fvs had any substantial influence on hormone structure.

Folding and assembly of an antibody Fv fragment, a heterodimer stabilized by antigen

The folding and assembly of the Fv fragment of the phosphorylcholine binding antibody McPC603, a non-covalent heterodimer of the variable domains VH and VL, was investigated. Since both domains, each engineered for stability and folding efficiency, could now be obtained in native and soluble form by themselves, fluorescence spectra of VH and VL in unfolded, folded and associated states can be reported. VH and VL only associate when they are native, and the stability of the heterodimer is strongly increased in the presence of antigen. VH rapidly folds into an hyperfluorescent intermediate, and the native state is reached in two parallel, proline-independent reactions. VL displays two fast refolding reactions, which are followed by two slower phases, limited by proline cis/trans-isomerization. The rate-limiting step for both the Fv and the scFv (single-chain Fv) fragment is the formation of the native VH-VL interface, which depends on ProL95 being in cis. The folding of the Fv fragment is fast after short-term denaturation or in the presence of proline cis/trans-isomerase catalysis, but the scFv fragment falls into a kinetic trap, observed by the persistence of the slow phases under all conditions. Furthermore, the scFv fragment, but not the Fv fragment, gives rise to premature interface formation, indicated by the fluorescence spectra and a much higher transient binding of 8-anilino-1-naphthalene sulfonate. The analysis of the folding pathway of the domains VH and VL in isolation and in non-covalent and covalent assemblies should provide helpful insights into the folding of multimeric proteins in general, and for the further engineering of stable and well-folding antibody fragments in particular.

High affinity single-chain Fv antibody fragments protecting the human nicotinic acetylcholine receptor

Univalent antibody fragments directed against the main immunogenic region (MIR) of the human acetylcholine receptor (AChR) are capable of protecting the AChR against loss induced by antibodies from myasthenia gravis (MG) patients. Our aim was to construct single-chain Fv (scFv) antibody fragments as a first step towards the production of therapeutic protecting molecules, from two high-affinity anti-MIR monoclonal antibodies (mAb 192 and mAb 195). During the construction of scFv192 fragment, two light chains co-secreted from the hybridoma mAb192 were identified. N-terminal amino acid and cDNA sequence analysis showed that one of the two light chains corresponded to the antigen binding molecule while the other originated from the non-secreting myeloma S194/5.XXO.BU.1 which was used in the production of the hybridoma. Functional scFv 192 and 195 fragments were constructed, expressed in Escherichia coli and affinity purified. The binding affinities of scFv192 and scFv195 ( K D=0.6 and 0.8 nM for human AChR) were two orders of magnitude higher than that of the earlier constructed scFv198. The scFv192 almost completely protected human AChR against binding of intact anti-MIR mAbs. Human AChR was also very efficiently protected (74–85%) by the scFv192 against binding of autoantibodies from MG sera with high anti-α subunit antibody fractions. These scFvs are good candidates for protection of MG patients after appropriate genetic modifications.

Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor

The single chain Fv fragment of mAb198 (scFv198) directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR), can efficiently protect the AChR in muscle cell cultures against the destructive activity of human myasthenic autoantibodies. Humanization of the scFv198 antibody fragment should prove useful for therapeutic application by reducing its immunogenicity. Framework sequences from human immunoglobulins homologous to the rat scFv198 sequences were selected and a totally synthetic humanized scFv198 antibody fragment was constructed in vitro. Humanized VH and VL domains were synthesized using two overlapping sets of 225 bases long oligonucleotides overlap extension and polymerase chain reaction (PCR), then assembled into a full-length gene by overlap extension of single-stranded DNA (ssDNA) fragments and PCR. The initial humanized antibody fragment had a very low affinity for the AChR. Molecular modeling was then performed and four residues from the framework regions (FR) of the humanized VH domain were selected to be replaced by the corresponding amino acid from the rat sequence. Three mutants were constructed by overlap extension, using PCR. The humanized variant containing replacements at VH residues 27, 29, 30 and 71 showed very good recovery of AChR binding activity; its binding affinities for Torpedo or human AChR ( K D: 8.5 or 323 nM, respectively) being only four times lower than those of the parental scFv198 ( K D: 2 or 80 nM, respectively). This variant was able to protect the human AChR against the binding of anti-MIR mAb and anti-α autoantibodies from a myasthenic patient. It was also able to protect AChR against antigenic modulation induced by the anti-MIR mAb198.

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-alpha.

Tumor necrosis factor (TNF)-alpha has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-alpha. Among the cell lines tested those resistant to TNF-alpha were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin beta1 resulted in a significant increase in the number of cells surviving TNF-alpha treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-alpha. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-alpha retained TNF-alpha activity. This scFv(14E1)-TNF-alpha molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-kappaB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays.

Strain specific recombinant antibodies to potato virus Y potyvirus

Single chain Fv antibody fragments have been selected from a synthetic phage-antibody library following three and four rounds of affinity selection with purified potato virus Y, common strain (PVY O). The selected fragments were highly specific for PVY and detected seven out of nine isolates of PVY O whilst failing to detect three isolates of PVY N and 12 isolates of PVY NTN. Nucleotide sequence of the scFv genes showed the variable heavy fragments belonged to the human VH4 family, whilst the variable light fragments belonged to the Vλ1 family. The fragments were used in ELISA to detect virus at concentrations of 50 ng/ml in plant sap and in comparisons with commercially available PVY monoclonal antibodies were shown to have similar limits of detection. This is the first report of the selection of a scFv specific for a member of the potyviridae, and its use in detecting and differentiating strains of PVY in infected plant sap. The results highlight the potential of the technology for the selection of strain specific antibodies with an avidity equivalent to traditional monoclonal antibodies raised against viral pathogens and their use for viral diagnosis.

Contributions of a Highly Conserved V H/V L Hydrogen Bonding Interaction to scFv Folding Stability and Refolding Efficiency

The assembly of single-chain Fv (scFv) antibody fragments, consisting of an interconnected variable heavy chain (V H) and variable light chain (V L), is a cooperative process that requires coupled folding and domain association. We report here an initial investigation of V H/V L domain-domain assembly with a site-directed mutagenesis study that probes a highly conserved V H/V L hydrogen bonding interaction. Gln 168 of the S5 scFv (Kabat V H 39) is absolutely conserved in 95% of all V H, and Gln 44 (Kabat V L 38) is found in 94% of all κ V L (Glx in 95% of all λ V L). These side chains form two hydrogen bonds in head-to-tail alignment across the V H/V L interface. Double mutant cycles at Gln 168 and Gln 44 were constructed to first investigate their contributions to thermodynamic folding stability, second to investigate whether stability can be improved, and third to determine whether refolding efficiencies are affected by mutations at these positions. The results demonstrate that the Gln 168-Gln 44 interaction is not a key determinant of S5 scFv folding stability, as sequential modification to alanine has no significant effect on the free energy of folding. Several mutations that alter the glutamines to methionine or charged amino acids significantly increase the thermodynamic stability by increasing the m g associated with the unfolding isotherm. These effects are hypothesized to arise largely from an increase in the V H/V L association free energy that leads to tighter coupling between domain-domain association and folding. All of the mutants also display a reduced antigen binding affinity. Single and double methionine mutants also displayed significant increases in refolding efficiency of 2.4- to 3-fold over the native scFv, whereas the double alanine/methionine mutants displayed moderate 1.9- to 2.4-fold enhancement. The results suggest that reengineering the V H/V L interface could be useful in improving the stability of single-chain antibodies, as Ala/Met mutations at these conserved positions increase the free energy of folding by 46% while minimally perturbing binding affinity. They also could be useful in improving scFv recovery from inclusion bodies as the mutations increase the refolding efficiency by more than twofold.

A possible hydrolysis mechanism of beta-naphthyl acetate catalyzed by antibodies.

The mechanism of ester hydrolysis has been extensively studied; however, the precise function of active-site residues in promoting catalysis is unclear. We describe here the structural models for the complex of a catalytic antibody Fv fragment with a phosphonate transition-state analogue, constructed by using gene cloning, sequencing and molecular modeling, mainly based on a known X-ray structure of a catalytic antibody. Hydrophobic and electrostatic analyses of the Fv/analog and Fv/substrate interaction suggest the hydrolysis mechanism: Tyr L91 and Tyr H97 play important roles to stabilize the beta-naphthyl group of hapten through pi-stack; His H35 donates a pair of free electrons at the atom NE2 to an active water and let it to be a partial hydroxide, which attacks the carbon atom of the carbonyl group of the substrate. Both His H35 and Arg L96 can form hydrogen bonds and stabilize the anionic tetrahedral intermediate formed during turnover. This mechanism emphasizes that an active water bridge may be formed during hydrolysis process.

Thermodynamics and kinetics of the reaction of a single-chain antibody fragment (scFv) with the leucine zipper domain of transcription factor GCN4.

Single-chain Fv (scFv) fragments of antibodies have become important analytical and therapeutic tools in biology and medicine. The reaction of scFv fragments has not been well-characterized with respect to the energetics and kinetics of antigen binding. This paper describes the thermodynamic and kinetic behavior of the high-affinity scFv fragment SW1 directed against the dimeric leucine zipper domain of the yeast transcription factor GCN4. The scFv fragment was selected by the phage display technique from the immune repertoire of a mouse that had been immunized with the leucine zipper domain of GCN4. The scFv fragment was produced in high yield in Escherichia coli inclusion bodies and refolded from the denatured state. Differential scanning calorimetry showed that SW1 was stable up to about 50 degreesC, but the subsequent thermal denaturation was irreversible (Tm approximately 68 degreesC). The scFv fragment specifically recognized the dimeric leucine zipper conformation. Two scFv fragments bound to the GCN4 dimer to form the complex (scFv)2-GCN4. Because of its repetitive structure, the rod-shaped GCN4 leucine zipper may present two similar epitopes for the scFv fragment. Surprisingly, the binding reaction was highly cooperative, that is, the species (scFv)2-GCN4 dominated over scFv-GCN4 even in the presence of a large excess of the antigen GCN4. It is speculated that cooperativity resulted from direct interaction between the two GCN4-bound scFv fragments. At 25 degreesC, the average binding enthalpy for a scFv fragment was favorable (-61 kJ mol-1), the entropy change was unfavorable, and the change in heat capacity was -1.27 +/- 0.14 kJ mol-1 K-1. As a result of enthalpy-entropy compensation, the free binding energy was virtually independent of temperature in the physiological temperature range. Antigen binding in solution could be described by a single-exponential reaction with an apparent rate constant of 1 x 10(6) M-1 s-1. Binding followed in a biosensor with the dimeric GCN4 coupled to the surface of the metal oxide sensor chip was 20 times slower.

Stability of a single-chain Fv antibody fragment when exposed to a high shear environment combined with air-liquid interfaces.

The effect of shear on the antigen binding activity of a recombinant scFv antibody fragment was investigated in the presence of air-liquid interfaces using a stirred vessel that was incompletely filled. Changes in binding activity of the scFv to its antigen were monitored using an optical biosensor which had been sensitized with hen egg lysozyme (the antigen). The biosensor response was used as a measure of scFv binding activity. In buffer solution (mean velocity gradient approximately 20,000 s-1), loss of binding activity followed a first-order model with a mean rate constant of 0.83 h-1. In unstirred buffer solution, no such loss was observed. Similarly, in sheared fermentation broth there was no loss of binding activity and protective effects were attributed to the antifoam PPG.