Fab Format Research Articles

Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv.

Rabbit-derived recombinant antibodies have traditionally been viewed as intractable molecules due to the presence of a cysteine in position 80 of the VL domain that becomes rendered 'aberrant' when present in the 'unpaired' context of a single chain Fv (scFv) and chimeric Fab formats. This aberrant Cys80 can severely impinge on the achievable expression levels when rabbit recombinant antibodies are produced in prokaryote systems. The unpaired Cys residue also renders purification problematic. Consequently, researchers often disregard rabbit antibody libraries due to perceived limitations in accessible repertoire diversity. We have shown that by switching the orientation of the VH and VL domains in an aberrant-Cys-containing rabbit scFv isolated in a bona fide screening campaign, it was possible to substantially increase the expression and purification yields of this clone. Furthermore, by incorporating a novel rabbit C-kappa constant fusion domain, we were able to potentiate a further increase in expression level and purify this antibody to a high degree of homogeneity, hitherto impossible to achieve using the aberrant-Cys-containing wild-type scFv. Cumulatively, these findings demonstrate that facile re-formatting can help make the rabbit antibody repertoire, a very valuable resource, more accessible to researchers in the field.

An Improved Single-Chain Fab Platform for Efficient Display and Recombinant Expression

Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1–3mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (Tm of 81°C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.

Polyaniline Binder for Functionalized Acetylene Black: A Hybrid Material for Supercapacitor

Polyaniline-doped with functionalyzed acetylene black material has been successfully synthesized and used as an electrode material in supercapacitor, wherein, this material contributes both pseudocapacitative (PC) and electrical double layer capacitative (EDLC) behaviour. Functionalization of acetylene black (AB) has been carried out and used as dopant for THF soluble polyaniline base (PANI) to prepare PANI-functionalized acetylene black (PANI-FAB). Formation of FAB and PANI-FAB was confirmed with the help of FT-IR and XPS study. Functionalization of AB results in agglomerated FAB and doping with PANI results in agglomerated fibrous PANI-FAB, as compared to fibrous AB. High thermal stability of AB decreases on functionalization and again slightly increases for PANI-FAB. Functionalization of AB also results good bonding effect with PANI, where PANI acts as both PC and binder, instead of the higher amounts of binder (PVdF > 30 wt%) that are normally needed to hold AB. Electrochemical studies of FAB and PANI-FAB materials were carried out by cyclic voltammetric and electrochemical impedance spectroscopic methods. PANI-FAB supercapacitor cell shows capacitance of 132 Fg1 for 0.3 Ag1 current density with very low charge-transfer resistance and low time constant.

Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

Thermodynamic Analysis of the Binding of 2F5 (Fab and Immunoglobulin G Forms) to Its gp41 Epitope Reveals a Strong Influence of the Immunoglobulin Fc Region on Affinity

Immunotherapies and vaccines based on the induction of broadly neutralizing monoclonal antibodies (bNAbs) have become outstanding strategies against HIV-1. Diverse bNAbs recognizing different regions of the HIV-1 envelope have been identified and extensively studied. However, there is little information about the thermodynamics of binding of these bNAbs and their epitopes. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We found that these interactions are enthalpically driven and opposed by a negative entropy change. The highest affinity was found for 2F5 IgG for its functional epitope, indicating that additional interactions involving residues flanking the core epitope contribute strongly to higher affinity. In addition, the strong influence of the Fc region on the binding affinity suggests long-range allosteric effects within IgG. Our results provide useful information for developing new therapeutics against HIV-1 and, in a broader scope, contribute to a better understanding of antigen-antibody recognition.

Constant Domain of Fab Fragment Affects Antigen Binding of Antibodies: Molecular Dynamics Study

The fragment antigen binding (Fab) is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain (Fv), which contain the heavy (H) and the light (L) chains. Antibodies bind antigens via contacts with amino acids in complementarity-determining regions (CDRs) in Fv domain. It may seem to be plausible that there is no difference of the binding structure and affinity between Fv and Fab format if the both format have same CDRs. Recently, however, numerous studies have compared the binding affinity of the single chain Fv (scFv) and Fab variants of the same antibody and have found that the changing the format to a Fab resulted in equal or higher binding affinity. However, the affects of the structural format on the antibody binding affinity remains poorly understood. In order to investigate the difference of physical properties between Fab and Fv fragments, from a structural and thermodynamical point of view, we have carried out 1 μs molecular dynamics (MD) simulations for each format. We have used the antibodies of epiregulin (EPR) as a simulation model, which is a member of the epidermal growth factor (EGF) family and a factor affecting pancreatic cancer, exhibiting high biological activities.We have found that constant region of Fab fragment of the antibody play an important role in the Fv domain stability, which reduce a fluctuation between H chain and L chain, and reduce the surface area of basic and hydrophobic amino acid exposed to solvent. And also find that binding free energy of Fab fragments and EPR is lower than that of Fv fragments. This indicates that the presence of the constant region of Fab fragment affects the binding structure of EPR and antibody.

Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-β1-adrenergic (β1AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at β1AR. Immunization with the β1AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at β1AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through β-arrestin. In vitro characterization also verified different antibody-receptor interactions reflecting the different epitopes on the extracellular surface of β1AR to which the mAbs bind. The anti-β1AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with β1AR agonism.

Polyaniline binder for functionalized acetylene black: A hybrid material for supercapacitor

Polyaniline-doped with functionalized acetylene black material has been successfully synthesized and used as an electrode material in supercapacitor, wherein, this material contributes both pseudocapacitive (PC) and electrical double layer capacitive (EDLC) behavior. Functionalization of acetylene black (AB) has been carried out and used as dopant for THF soluble polyaniline base (PANI) to prepare PANI-functionalized acetylene black (PANI-FAB). Formation of FAB and PANI-FAB was confirmed with the help of FT-IR and XPS study. Functionalization of AB results in agglomerated FAB and doping with PANI results in agglomerated fibrous PANI-FAB, as compared to fibrous AB. High thermal stability of AB decreases on functionalization and again slightly increases for PANI-FAB. Functionalization of AB also results good bonding effect with PANI, where PANI acts as both PC and binder, instead of the higher amounts of binder (PVdF>30wt%) that are normally needed to hold AB. Electrochemical studies of FAB and PANI-FAB materials were carried out by cyclic voltammetric and electrochemical impedance spectroscopic methods. PANI-FAB supercapacitor cell shows capacitance of 132Fg−1 for 0.3Ag−1 current density with very low charge-transfer resistance and low time constant.

Thermodynamic Stability and Flexibility Characteristics of Antibody Fragment Complexes

Free energy landscapes, backbone flexibility and residue-residue couplings for being co-rigid or co-flexible are calculated from the minimal Distance Constraint Model (mDCM) on an exploratory dataset consisting of VL, scFv and Fab antibody fragments. Experimental heat capacity curves are reproduced markedly well, and an analysis of quantitative stability/flexibility relationships (QSFR) is applied to a representative VL domain and several complexes in the scFv and Fab forms. Global flexibility in the denatured ensemble typically decreases in the larger complexes due to domain-domain interfaces. Slight decreases in global flexibility also occur in the native state of the larger fragments, but with a concurrent large increase in correlated flexibility. Typically, a VL fragment has more co-rigid residue pairs when isolated compared to the scFv and Fab forms, where correlated flexibility appears upon complex formation. This context dependence on residue- residue couplings in the VL domain across length scales of a complex is consistent with the evolutionary hypothesis of antibody maturation. In comparing two scFv mutants with similar thermodynamic stability, local and long-ranged changes in backbone flexibility are observed. In the case of anti-p24 HIV-1 Fab, a variety of QSFR metrics were found to be atypical, which includes comparatively greater co-flexibility in the VH domain and less co-flexibility in the VL domain. Interestingly, this fragment is the only example of a polyspecific antibody in our dataset. Finally, the mDCM method is extended to cases where thermodynamic data is incomplete, enabling high throughput QSFR studies on large numbers of antibody fragments and their complexes.

Discovery of diverse and functional antibodies from large human repertoire antibody libraries

Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

Antibody-mediates inhibition of human C1s and the classical complement pathway

Disregulation of complement activation plays a critical role in numerous inflammatory diseases and therefore, inhibition of the complement pathway is of great therapeutic interest. In the classical complement pathway, immune complexes formed by IgM, IgG1, IgG2 and IgG3 antibodies result in the activation of the C1s protease that in turn cleaves C4 and then C4-bound-C2 yielding the proteolytic fragments C4b and C2a which associate to form a C3 convertase enzyme. We report here the engineering of a potent human antibody inhibitor of C1s protease activity. Phage panning of a very large synthetic (FAB) antibody fragment library using a truncated version of C1s, comprising the second CCP domain and serine protease domain (CCP2-SP) and expressed in insect cells, resulted in the isolation of a FAB that inhibited the catalytic activity of C1s. An affinity matured variant of the FAB format antibody displaying subnanomolar KD for C1s was shown to exhibit >80% inhibition of C2 processing at a 5:1 antibody:C1s molar ratio. We show that this engineered antibody, D.35, displays potent inhibition of complement deposition and lysis of Ramos cells by the anti-CD20 therapeutic antibody rituximab relative to the approved, but less-specific, human plasma-derived C1-inhibitor (CINRYZE). C1s inhibitory antibodies should be useful for delineating the role of the classical pathway in disease models and may hold promise as therapeutic agents.

Impact of Intrinsic Affinity on Functional Binding and Biological Activity of EGFR Antibodies

Aberrant expression and activation of EGF receptor (EGFR) has been implicated in the development and progression of many human cancers. As such, targeted therapeutic inhibition of EGFR, for example by antibodies, is a promising anticancer strategy. The overall efficacy of antibody therapies results from the complex interplay between affinity, valence, tumor penetration and retention, and signaling inhibition. To gain better insight into this relationship, we studied a panel of EGFR single-chain Fv (scFv) antibodies that recognize an identical epitope on EGFR but bind with intrinsic monovalent affinities varying by 280-fold. The scFv were converted to Fab and IgG formats, and investigated for their ability to bind EGFR, compete with EGF binding, and inhibit EGF-mediated downstream signaling and proliferation. We observed that the apparent EGFR-binding affinity for bivalent IgG plateaus at intermediate values of intrinsic affinity of the cognate Fab, leading to a biphasic curve describing the ratio of IgG to Fab affinity. Mathematical modeling of antibody-receptor binding indicated that the biphasic effect results from nonequilibrium assay limitations. This was confirmed by further observation that the potency of EGF competition for antibody binding to EGFR improved with both intrinsic affinity and antibody valence. Similarly, both higher intrinsic affinity and bivalent binding improved the potency of antibodies in blocking cellular signaling and proliferation. Overall, our work indicates that higher intrinsic affinity combined with bivalent binding can achieve avidity that leads to greater in vitro potency of antibodies, which may translate into greater therapeutic efficacy.

An Antidote for Acute Cocaine Toxicity

Not only has immunopharmacotherapy grown into a field that addresses the abuse of numerous illicit substances, but also the treatment methodologies within immunopharmacotherapy have expanded from traditional active vaccination to passive immunization with anti-drug monoclonal antibodies, optimized mAb formats, and catalytic drug-degrading antibodies. Many laboratories have focused on transitioning distinct immunopharmacotherapeutics to clinical evaluation, but with respect to the indication of cocaine abuse, only the active vaccine TA-CD, which is modeled after our original cocaine hapten GNC, has been carried through to human clinical trials. The successful application of murine mAb GNC92H2 to the reversal of cocaine overdose in a mouse model prompted investigations of human immunoglobulins with the clinical potential to serve as cocaine antidotes. We now report the therapeutic utility of a superior clone, human mAb GNCgzk (K(d) = 0.18 nM), which offers a 10-fold improvement in cocaine binding affinity. The GNCgzk manifold was engineered for rapid cocaine clearance, and administration of the F(ab')₂ and Fab formats even after the appearance of acute behavioral signs of cocaine toxicity granted nearly complete prevention of lethality. Thus, contrary to the immunopharmacotherapeutic treatment of drug self-administration, minimal antibody doses were shown to counteract the lethality of a molar excess of circulating cocaine. Passive vaccination with drug-specific antibodies represents a viable treatment strategy for the human condition of cocaine overdose.

A means to study details of complex antigen mixture neutralizations when limited to antibodies unresponsive to Fc region dependent technologies for immunoprecipitation. (144.6)

Abstract The Crofab™ antivenin, an ovine Fab IgG fragment product, thus lacking an Fc region for typical immunochemical physical separations, is the sole antivenin used in American for treatment of the thousands of life threatening Crotalid envenomations each year, but has unexplained clinical failures. We postulated that by removing the immune complexes and unreacted antibody from neutralization mixtures, thus leaving only the unreacted venom components, that high resolution chromatographic analysis of in vitro binding studies could be developed. So we evaluated biotinylation of the ovine Fab antivenin, although successful biotin conjugation of Crofab™ or any ovine Fab fragments has not been reported, and reduced activity of biotinylated ovine Fab2 had been reported. Our results show with varying coupling reaction mass ratios tested, up to a 75% coupling yield and a ratio of 2-3 biotin per Fab. By SE-HPLC analysis, the biotinylated antivenin did not lose binding activity. RP-HPLC showed differential neutralization of various venom components. This innovative method provides a means to conduct a much more detailed analysis of any ovine Fab antivenin neutralization or similar antibody-complex antigen mixtures, and suggests that Fab forms of other species of antivenin, or other species’ antibodies which cannot be immunoprecipitated with Fc region dependent technologies might be responsive to this approach, leading to detailed analyses of neutralizations.

Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs.

The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc2) but not to GPVI monomer (GPVIex) through a phage display method. Six Fabs were found: B-F, only reactive with GPVI-Fc2, and A, mainly reactive with GPVI-Fc2, with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc2 binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc2. Adding the anti-GPVI monoclonal antibody 204-11 increased the B(max) of m-Fab-F binding to GPVI-Fc2, suggesting that 204-11 binds to GPVI-Fc2 molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface.

A novel human monoclonal antibody that binds with high affinity to mesothelin-expressing cells and kills them by antibody-dependent cell-mediated cytotoxicity

Mesothelin is a potential new target for cancer immunotherapy because it is present at relatively low levels only in mesothelial cells of pleura, peritoneum, and pericardium of healthy people, but is significantly elevated in a number of tumors, including mesothelioma, ovarian, pancreatic, and lung cancers. However, all currently available antibodies against mesothelin are either murine or chimeric, which could limit their use because of increased likelihood of immunogenicity compared with fully human antibodies. Here, we report the identification and characterization of a novel fully human monoclonal antibody, m912, which was isolated from a human Fab library by panning against recombinant mesothelin. This antibody in scFv, Fab, and IgG1 formats bound specifically and with high affinity (equilibrium dissociation constant in the nmol/L range) to cell surface-associated human mesothelin and to recombinant mesothelin. It specifically lysed cancer cells engineered to express mesothelin in the presence of peripheral blood mononuclear cells isolated from healthy donors most likely by antibody-dependent cellular cytotoxicity. M912 is the first reported fully human monoclonal antibody to mesothelin, which has potential for cancer treatment and diagnosis.

Isolation of therapeutic human monoclonal antibodies for varicella‐zoster virus and the effect of light chains on the neutralizing activity

Therapeutic antibodies against varicella-zoster virus (VZV) were isolated from a combinatorial library of human antibodies using a phage-display system. Purified gH:gL was used to screen the library, and approximately 300 clones were isolated. Eight kinds of Fab-cp3-fused molecules (clones 10, 24, 36, 60, 94, 120, 192, and 431) neutralized viral infectivity. After conversion of Fab-cp3 antibodies to the Fab-protein A form, the concentrations of antibodies showing 50% inhibition of plaque formation ranged from 0.12 to 400 nM. Clones 10, 24, 94, 120 and 431 neutralized wild strains without showing strain specificity and were further converted to human IgG(1). Two clones (24 and 94) were confirmed to react with gH:gL and VZV-infected cells. IgG of clone 94 prevented spreading of infected cells. Thus these antibodies exhibited the typical phenotype of anti-gH antibody. Next the contribution of light (L) chains to neutralizing activity was analyzed by comparing the effect of L chain of clones 10, 120, and 192 with the identical heavy chain on their neutralizing activity. The L chain in the Fab form of clone 94 was replaced by L chains of clones 10, 24, 36, and 60 and the neutralizing activity of these replaced antibodies was weaker than that of the prototype clone 94. When the kappa-L chain of clone 94 was replaced by the lambda-L chain of clone 24, this antibody possessed neutralizing activity despite the kappa-lambda class change. Thus, human antibody library against VZV-gH has been established and characterized the role of the L chain in VZV-neutralizing activity to engineering further an antibody with stronger neutralizing activity.

The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies

The antigen-binding fragment (Fab) has been considered a more functionally stable version of recombinant antibodies than single chain antibody fragments (scFvs), however this intuitive consideration has not been sufficiently proven in vivo. This communication shows that three out of four specific scFvs against a scorpion toxin, with different affinities and stabilities, become neutralizing in vivo when expressed as Fabs, despite the fact that they are not neutralizing in the scFv format. A scFv fragment previously obtained from a neutralizing mouse antibody (BCF2) was used to produce three derived scFvs by directed evolution. Only one of them was neutralizing, however when expressed as Fab, all of them became neutralizing fragments in vivo. The initial scFvBCF2 (earlier used for directed evolution) was not neutralizing in the scFv format. After expressing it as Fab did not become a neutralizing fragment, but did reduce the intoxication symptoms of experimental mice. The stability of the four Fabs derived from their respective scFvs was improved when tested in the presence of guanidinium chloride. The in vitro stability of the Fab format has been shown earlier, but the physiological consequences of this stability are shown in this communication. The present results indicate that improved functional stability conferred by the Fab format can replace additional maturation steps, when the affinity and stability are close to the minimum necessary to be neutralizing.

Optimizing recombinant antibody function in SPR immunosensing

Recombinant antibody fragments are valuable tools for SPR-based detection of small molecules such as illicit drugs. However, the multiple structural formats of recombinant antibody fragments are largely uncharacterised with respect to their respective performance in SPR sensing. We have expressed a model anti-M3G antibody in both scFv and chimeric Fab formats to examine its sensitivity and binding profiles in a microplate immunoassay format and Biacore. We have further examined the influence of scFv multimerisation, Fab constant region stability and SPR chip surface coating chemistry, on anti-hapten SPR assay development. Under optimised competition ELISA conditions, the anti-M3G scFv was found to have an IC(50) value of 30 ng/ml, while the most stable Fab construct exhibited an IC(50) value of 2.4 ng/ml. In SPR competition assay on an M3G-OVA-coated SPR chip surface, the two constructs again differed in sensitivity, with IC(50) values of 117 and 19 ng/ml for the scFv and Fab, respectively (the scFv also exhibiting poor linearity of response). However, when the SPR chip surface was directly coated with M3G, both antibody constructs exhibited good linearity of response, similar high sensitivity IC(50) values (scFv 30 ng/ml, Fab 14 ng/ml) and high reproducibility (50 effective regenerations for M3G-OVA, 200 for M3G direct). During SPR assay development it was noticed that scFv and Fab constructs gave differing off-rate profiles. Subsequent HPLC, ELISA and electrophoretic analyses then confirmed that a portion of the scFv population multimerises. Bivalent scFv was found to profoundly affect the dissociation curve for scFv in stringent SPR kinetic analyses, leading to a 40-fold difference in calculated off-rate values (Fab off rate 4.7 x 10(-3)S(-1), scFv off rate 1.03 x 10(-2)S(-1)). The structural format of recombinant antibody fragments and chip functionalisation methodology can both profoundly affect the function of anti-M3G SPR assay, with direct coating and Fab format proving to be optimal. The confirmation of scFv multimerisation and resulting changes in SPR kinetics profile, in comparison with a Fab, further suggest that caution must be taken in the interpretation of SPR sensorgrams, which are commonly used in the 'affinity ranking' of scFv panels in which the extent of dimerisation in each sample is unknown.

Display and selection of chicken IgA Fab fragments

Passive immune therapy is regaining interest to prevent and cure infectious diseases both in human and veterinary medicine. Therefore, systems are required that enable efficient targeted selection of antibodies originating from virtually any animal species. Here, a system for the selection of chicken IgA, using phage display, is described. A novel phagemid vector (pChick3) for the display and selection of chicken IgA antibodies in Fab format was developed. The functionality of pChick3 was demonstrated by construction of an immune antibody library using B cells from chickens infected with Eimeria acervulina. From this library, 10 different IgA fragments with specific binding to the E. acervulina antigen mix, the sporozoite or oocyst fractions were selected. These results demonstrate the efficiency and versatility of the pChick3 vector system that can readily be applied to construct libraries and subsequently select antibodies of the alpha isotype against a wide variety of pathogens and parasites.