The Effect of Antibody Fragments on CD25 Targeted Regulatory T Cell Near-Infrared Photoimmunotherapy.

Regulatory T (Treg) cells play a major role in immune suppression permitting tumors to evade immune surveillance. Depletion of intratumoral Treg cells can result in tumor regression. However, systemic depletion of Tregs may also induce autoimmune adverse events. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cell-specific cancer therapy that locally kills specific cells in the tumor. Antibody-photoabsorber (IRDye700DX) conjugates (APC) are injected and bind to the tumor, and subsequent administration of NIR light to the tumor results in rapid cell death only in targeted cells. CD25-targeted NIR-PIT has been shown to induce spatially selective depletion of tumor-associated Treg cells. In this study, we compared the efficacy of an antibody fragment, anti-CD25-F(ab')2, and a full antibody, anti-CD25-IgG, as agents for NIR-PIT. Tumor-bearing mice were divided into four groups: (1) no treatment; (2) anti-CD25-IgG-IR700 i.v. only; (3) anti-CD25-F(ab')2-IR700 i.v. with NIR light exposure; and (4) anti-CD25-IgG-IR700 i.v. with NIR light exposure. Although both CD25-targeted NIR-PITs resulted in significant tumor growth inhibition, the anti-CD25-F(ab')2-IR700 based NIR-PIT was superior to the anti-CD25-IgG-IR700 NIR-PIT. The anti-CD25-F(ab')2-IR700 demonstrated faster clearance from the body than the anti-CD25-IgG-IR700. Sustained circulation of anti-CD25-IgG-IR700 may block IL-2 binding on the activated effector T-cells decreasing immune response. In conclusion, anti-CD25-F(ab')2 based NIR-PIT was more effective in reducing tumor growth than anti-CD25-IgG based NIR-PIT. Absence of the Fc portion of the APC leads to faster clearance and therefore promotes a superior activated T cell response in tumors.

Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using an IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex.

Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g., of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive [GD2(+)] tumors. For this purpose, an IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with ß-particle-emitting radiometals such as (177)Lu and (90)Y. A three-step regimen, including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with (177)Lu (as (177)Lu-DOTA-Bn; t1/2 = 6.71 days), was optimized in immunocompromised mice carrying subcutaneous human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were approximately 85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indices (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5/group; tumor volume, 240 ± 160 mm(3)) with three successive PRIT cycles (total (177)Lu: ∼33 MBq; tumor dose ∼3,400 cGy), revealed complete tumor response in 5 of 5 animals, with no recurrence up to 28 days after treatment. Tumor ablation was confirmed histologically in 4 of 5 mice, and normal organs showed minimal overall toxicities. All nontreated mice required sacrifice within 12 days (>1.0-cm(3) tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate subcutaneous GD2(+)-NB in mice while sparing kidney and bone marrow.

Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes.

Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins.

Immunological aspects of scorpion toxins: current status and perspectives.

Significant progress has been made in immunological studies of scorpion toxins and several formats of antibodies directed against scorpion toxins have been reported. Some of these are commonly used in a specific treatment against envenoming; others are primarily used for immuno-biochemical characterizations. The preparation protocol of the antibody or its fragments can be substantially different from one laboratory to another, which complicates a direct comparison of the potency of the antivenom. The use of immune sera, the total immunoglobulin fraction or Fab and Fab'2 fragments as the therapeutic agent is widespread. A number of monoclonal antibodies have also been reported and used for engineering of Fv, ScFv or Fab fragments. Recently, a novel antibody format - known as nanobodies - derived from HCAbs of camelids and selected after phage display shows great potential to provide a more efficient therapy against scorpion envenoming. Subsequent bispecific derivatives have been designed and their pharmacokinetics have been studied. Distinct advantages and disadvantages have been attributed to these equine, murine or camelid antibodies and their derived fragments. Some fragments are easily amenable into next generation therapeutics after proper manufacturing and provide an ensured availability of the product to the medical community. Through examples, we will show how the comparison of the serotherapeutic effectiveness is compromised due to the absence of standardization, on the preparation of immunogens, production processes and / or nature of the products. We will report on recent advances in the field.

High affinity anti-inorganic material antibody generation by integrating graft and evolution technologies: potential of antibodies as biointerface molecules.

Recent advances in molecular evolution technology enabled us to identify peptides and antibodies with affinity for inorganic materials. In the field of nanotechnology, the use of the functional peptides and antibodies should aid the construction of interface molecules designed to spontaneously link different nanomaterials; however, few material-binding antibodies, which have much higher affinity than short peptides, have been identified. Here, we generated high affinity antibodies from material-binding peptides by integrating peptide-grafting and phage-display techniques. A material-binding peptide sequence was first grafted into an appropriate loop of the complementarity determining region (CDR) of a camel-type single variable antibody fragment to create a low affinity material-binding antibody. Application of a combinatorial library approach to another CDR loop in the low affinity antibody then clearly and steadily promoted affinity for a specific material surface. Thermodynamic analysis demonstrated that the enthalpy synergistic effect from grafted and selected CDR loops drastically increased the affinity for material surface, indicating the potential of antibody scaffold for creating high affinity small interface units. We show the availability of the construction of antibodies by integrating graft and evolution technology for various inorganic materials and the potential of high affinity material-binding antibodies in biointerface applications.

The development of nanobodies for therapeutic applications.

Evolution has been continuously honing the design of antibodies to function as specific molecular markers that are able to alert the immune system to the presence of pathogenic antigens, and to recruit complement- and Fc receptor-bearing effector cells. During the past 25 years, the versatility of antibodies has been applied to several therapeutic applications. The development of new technologies, combined with data obtained using a new generation of antibody reagents, have allowed the adaptation of the design of antibodies to better match drug development requirements. Nanobodies are therapeutic proteins derived from the heavy-chain variable (VHH) domains that occur naturally in heavy-chain-only Ig molecules in camelidae. These VHH domains are the smallest known antigen-binding antibody fragments. Nanobodies can be easily produced in prokaryotic or eukaryotic host organisms, and their unique biophysical and pharmacological characteristics render these molecules ideal candidates for drug development. This review describes the structural properties of nanobodies and focuses on their unique features, which distinguishes these molecules from other antibody formats and small-molecule drugs. Possible therapeutic applications of nanobodies are discussed and data from phase I clinical trials of the novel 'first-in-class' anti-thrombotic agent ALX-0081 (Ablynx NV) are presented.

Therapeutic effect of llama derived VHH fragments against Streptococcus mutans on the development of dental caries.

Streptococcus mutans is the main cause of dental caries. We evaluated the therapeutic effect of variable regions of a llama heavy chain antibody fragments directed against S. mutans named S36-VHH (S for Streptococcus) alone or fused with glucose oxidase (GOx) from Aspergillus niger. Western blot analysis and ELISA revealed binding of the S36-VHH to the streptococcal antigen I/II adhesin molecule of S. mutans serotype C. In a rat-desalivated caries model, daily administration of S36-VHH significantly reduced the development of smooth surface caries. No additional therapeutic effect of GOx was observed. Our results suggest that llama VHH antibodies may be a potential benefit as prophylaxis against dental caries.

Construction and expression of a single chain Fv fragment against pharmacologically active paeoniflorin in Escherichia coli, and its potential use in an enzyme-linked immunosorbent assay.

A recombinant single chain variable-fragment (scFv) antibody against paeoniflorin (PF) was produced using the hybridoma cell line C31B9. Variable regions of heavy (V (H)) and light (V (L)) chain antibody genes were directly cloned from cDNA resources of hybridoma C31B9 and assembled using splicing by overlap extension (SOE)-PCR using a (Gly (4)Ser) (3) linker DNA. The constructed scFv genes were cloned into pET28a vectors for the generation of recombinant proteins in Escherichia coli. Most of the recombinant proteins were expressed in inclusion bodies. The yield of refolded and purified scFv was 1.89 mg per 100 mL of cell culture. The recombinant scFv displayed cross-reactivity as its mother monoclonal antibody (MAb) C31B9. Therefore, the newly expressed scFv protein was applied to quantitative ELISA to determine the total paeoniflorin (PF) and albiflorin (Alb) concentrations in peony root samples. Using PF as a standard compound, the full linear range of the assay was extended from 0.78 to 25 microg/mL. The results obtained by ELISA employing both the recombinant scFv and the original MAbC31B9 showed a reasonably good agreement with each other.

Molecular characterization of a human monoclonal antibody that interacts with a sulfated tyrosine-containing epitope of the GPIb receptor and inhibits platelet functions.

Modification of tyrosine residues in extracellular proteins by a sulfate moity plays an important role in many ligand/receptors interactions. In the present work, we describe a unique human monoclonal antibody, termed Y1-scFv, that is specific for a sulfated epitope in the platelat receptor GPIb. The Y1-scFv single chain antibody (scFv) competes with von Willebrand factor (vWF) for binding to human platelets and thus effectively inhibits platelet aggregation. Limited proteolysis of GPIb molecule, using the endoproteases, mocarhagin and cathepsin G, revealed that a seven amino-acid epitope, Tyr-276 to Glu-282, contains the recognition site for Y1-scFv. This GPIb region contains three sulfated tyrosine residues. Binding studies of Y1-scFv to cells and to synthetic peptides in vitro indicated that of the seven residues comprising the epitope only sulfo-Tyr-276 and adjacent Asp-277 are critical for the interaction. To identify the reciprocal sequences in the antibody that recognize the sulfated epitope, we introduced mutations within the complementary-determining region of the heavy chain (CDR3H) of Y1-scFv (MRAPVI). Arginine residue in the second position was critical for the binding. Moreover, a mutant, containing two sequential arginine residues, in the second and third positions of the CDR3H (MRRPVI), showed a nine-fold increased binding to GPIb. This antibody mutant also demonstrated a significant increase in inhibition of vWF-dependent platelet aggregation and adhesion under flow. In conclusion, this unique antibody and mutants, that recognize a sulfated epitope in GP1b receptor, efficiently inhibited platelet adhesion and aggregation, making it a candidate for a new anti-thrombotic agent.

Recombinant human antibodies against aldehyde-modified apolipoprotein B-100 peptide sequences inhibit atherosclerosis.

BACKGROUND: Accumulation and oxidation of LDL are believed to be important initiating factors in atherosclerosis. Oxidized LDL is recognized by the immune system, and animal studies have suggested that these immune responses have a protective effect against atherosclerosis. Aldehyde-modified peptide sequences in apolipoprotein B-100 (apoB-100) are major targets for these immune responses. METHODS AND RESULTS: Human IgG1 antibodies against 2 malondialdehyde (MDA)-modified apoB-100 peptide sequences were produced through screening of a single-chain antibody-fragment library and subsequent cloning into a pcDNA3 vector. Three weekly doses of these antibodies were injected into male apoE-/- mice. Phosphate-buffered saline and human IgG1 antibodies against fluorescein isothiocyanate were used as controls. One of the IgG1 antibodies significantly and dose-dependently reduced the extent of atherosclerosis as well as the plaque content of oxidized LDL epitopes and macrophages. In cell culture studies, human monocytes were incubated with native LDL or oxidized LDL, in the presence of antibodies. The same antibody induced an increase in monocyte binding and uptake of oxidized LDL. CONCLUSIONS: These findings suggest that antibodies are important mediators of atheroprotective immune responses directed to oxidized LDL. Thus, passive immunization against MDA-modified apoB-100 peptide sequences may represent a novel therapeutic approach for prevention and treatment of cardiovascular disease.

A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions.

We describe a novel approach for high-throughput screening of recombinant antibodies, based on their immobilization on solid cellulose-based supports. We constructed a large human synthetic single-chain Fv antibody library where in vivo formed complementarity determining regions were shuffled combinatorially onto germline-derived human variable-region frameworks. The arraying of library-derived scFvs was facilitated by our unique display/expression system, where scFvs are expressed as fusion proteins with a cellulose-binding domain (CBD). Escherichia coli cells expressing library-derived scFv-CBDs are grown on a porous master filter on top of a second cellulose-based filter that captures the antibodies secreted by the bacteria. The cellulose filter is probed with labeled antigen allowing the identification of specific binders and the recovery of the original bacterial clones from the master filter. These filters may be simultaneously probed with a number of antigens allowing the isolation of a number of binding specificities and the validation of specificity of binders. We screened the library against a number of cancer-related peptides, proteins, and peptide-protein complexes and yielded antibody fragments exhibiting dissociation constants in the low nanomolar range. We expect our new antibody phage library to become a valuable source of antibodies to many different targets, and to play a vital role in facilitating high-throughput target discovery and validation in the area of functional cancer genomics.

Direct phage to intrabody screening (DPIS): demonstration by isolation of cytosolic intrabodies against the TES1 site of Epstein Barr virus latent membrane protein 1 (LMP1) that block NF-kappaB transactivation.

The expression of intracellular antibodies (intrabodies) in eukaryotic cells has provided a powerful tool to manipulate microbial and cellular signaling pathways in a highly precise manner. However, there have been several technical issues that have restricted their more widespread use. In particular, single-chain antibodies (sFv) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use sFv-phage libraries as a source of intrabodies unless a pre-selection step to identify these rare sFvs from natural libraries or libraries of engineering sFvs that could fold properly in the absence of disulfide bonds were used. Here, we investigated whether target specific sFvs that are isolated from a 15 billion member non-immune human sFv-phage display library could be directly screened in pools as intrabodies without prior knowledge of their individual identity or purity within pools of antigen-specific sFvs. As the target, we used a synthetic transformation effector site 1 (TES1) polypeptide comprising the membrane-most proximal 34 amino acid residues of the carboxy-terminal cytoplasmic tail of the oncogenic latent membrane protein 1 (LMP1) of Epstein Barr virus, which serves as a docking site for adapter proteins of the tumor necrosis factor (TNF) receptor (TNFR)-associated factor (TRAF) family. Anti-TES1 sFvs, initially identified by phage ELISA screens, were grouped into pools according to the absorbance reading of the antigen-specific phage ELISA assays and then transferred as pools into eukaryotic expression vectors and expressed as cytoplasmic intrabodies. Using the pooling strategy, there was no loss of individual anti-TES1 sFvs in the transfer from prokaryotic to eukaryotic expression vectors. In addition, the initial assignments into sFv pools based on phage ELISA readings allowed the segregation of individual anti-TES1 sFvs into discrete or minimally overlapping intrabody pools. Further assessment of the biological activity of the anti-TES1 intrabody pools demonstrated that they were all able to selectively block F-LMP1-induced NFkappaB activity that was mediated through the TES1-site and to bind LMP1 protein with high efficiency. This direct phage to intrabody screening (DPIS) strategy should allow investigators to bypass much of the in vitro sFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic sFv phage libraries already in existence to identify intrabodies that are active in vivo.

Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning.

Several human monoclonal antibodies can neutralize a range of human immunodeficiency virus type 1 (HIV-1) primary isolates but their potency and related ability to suppress generation of HIV-1 escape mutants is significantly lower than the activity of antiretroviral drugs currently in clinical use. Recently, a human Fab, X5, was identified and found to neutralize primary isolates from different clades. Further improvement of the potency and breadth of HIV-1 neutralization by this antibody could be critical for its potential use in the treatment of HIV-1-infected patients. However, increasing potency of an antibody by selection from libraries may lead to a decrease in the breadth of neutralization. In an attempt to solve this problem, we subjected a random mutagenesis library of the scFv X5 to sequential rounds of selection on non-homologous HIV-1 envelope glycoproteins (Envs) dubbed sequential antigen panning (SAP). By using SAP, we identified two scFv antibodies, m6 and m9, that were tested with a panel of 33 diverse primary HIV-1 infectious isolates in an assay based on a reporter cell-line expressing high levels of CD4, CCR5 and CXCR4. The IC(50) was less than 50 microg/ml for 21 (m6) and 19 (m9) out of 29 isolates from group M (subtypes A-C, F, G and CRF-01AE) and one isolate from group N; three isolates from group O were not significantly inhibited at 50 microg/ml. The average IC(50) values for the two antibodies were significantly (p<0.001, n=29) lower compared to scFv X5. Their inhibitory activity does not appear to be related to the HIV-1 subtype, coreceptor usage or the disease stage. m9 inhibited infection of peripheral blood mononuclear cells by the primary isolates JRCSF, 89.6 and BR020 with IC(90) of 4, 6 and 25 microg/ml, respectively; for a single-round infection by pseudovirus, the IC(90) for JRSCF, 89.6, YU2 and HXBc2 was 15, 5, 15 and 5 microg/ml, respectively. In these two assays the IC(90) for m9 was, on average, two- to threefold lower than for scFv X5. These results demonstrate that both the potency and the breadth of HIV-1 neutralization of one of the few known potent broadly cross-reactive human monoclonal antibodies, scFv X5, could be improved significantly. However, only experiments in animal models and clinical trials in humans will show whether these new scFvs and the approach for their identification have potential in the development of prophylactics and therapeutics for HIV-1 infections.

Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants.

We constructed a recombinant antibody fragment--single chain fragment-variable (scFv) antibody--derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene. The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb). Up to 220 ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities. These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.

Immunomodulation of enzyme function in plants by single-domain antibody fragments.

Immunomodulation involves the use of antibodies to alter the function of molecules and is an emerging tool for manipulating both plant and animal systems. To realize the full potential of this technology, two major obstacles must be overcome. First, most antibodies do not function well intracellularly because critical disulfide bonds cannot form in the reducing environment of the cytoplasm or because of difficulties in targeting to subcellular organelles. Second, few antibodies bind to the active sites of enzymes and thus they generally do not neutralize enzyme function. Here we show that the unique properties of single-domain antibodies from camelids (camels and llamas) can circumvent both these obstacles. We demonstrate that these antibodies can be correctly targeted to subcellular organelles and inhibit enzyme function in plants more efficiently than antisense approaches. The use of these single-domain antibody fragments may greatly facilitate the successful immunomodulation of metabolic pathways in many organisms.

Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains.

To study the molecular structure and function of gene products in situ, we developed a molecular immunolabeling technology. Starting with cDNA from hybridomas producing monoclonal antibodies against biotin, catalase, and superoxide dismutase, we bioengineered recombinant single-chain variable fragment antibodies (scFv) and their derivatives containing metal-binding domains (scFv:MBD). As tested with surface plasmon resonance and enzyme-linked immunosorbent assay, affinity binding constants of the scFv (5.21 x 10(6) M(-1)) and scFv:MBD (4.17 x 10(6) M(-1)) were close to those of Fab proteolytic fragments (9.78 x 10(6) M(-1)) derived from the parental IgG antibodies. After saturation of MBD with nickel or cobalt, scFv:MBD was imaged with electron spectroscopic imaging at each element's specific energy loss, thus generating the element's map. Immunolabeling with scFv:MBD resulted in a significant improvement of the labeling fidelity over that obtained with Fab or IgG derivatives, as it produced a much heavier specific labeling and label-free background. As determined with radioimmunoassay, labeling effectiveness with scFv:MBD was nearly the same as with scFv, but much higher than with scFv conjugated to colloidal gold, Nanogold, or horseradish peroxidase. This technology opens possibilities for simultaneous imaging of multiple molecules labeled with scFv:MBD at the molecular resolution within the same sample with electron spectroscopic imaging. Moreover, the same scFv:MBD can also be imaged with fluorescence resonance energy transfer and lifetime imaging as well as positron emission tomography and magnetic resonance imaging. Therefore, this technology may serve as an integrative factor in life science endeavors.

[Cloning and expression of ScFv gene against alpha-toxin of Clostridium perfringens type A].

The VH and VL genes from a hybridoma cell line producing mouse McAb against alpha-toxin of Clostridium perfringens type A were amplified by RT-PCR. The VH and VL genes were connected thought a flexible linker (Gly4Ser)3 and the VH-linker-VL (ScFv) gene was cloned into a vector pGEM-T. The ScFv gene consists of 726 bp encoding 242 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region. According to kabat classed method, the VH and VL gene segments belong to mouse Ig heavy chain subgroup II (B) and kappa light chain subgroup III respectively. The ScFv gene was amplified inserted the expression vector pHOG21 and transformed into E coli XL1-BLUE. The ScFv protein was highly expressed in recombinant strain XL1-BLUE (pHOG-2E3) and the expression level of the ScFv was about 25% of total bacteria protein by SDS-PAGE. The neutralization assay showed that the expressed ScFv protein could neutralize the phospholipase C activities of alpha-toxin.

Receptor-mediated targeting of fluorescent probes in living cells.

A strategy was developed to label specified sites in living cells with a wide selection of fluorescent or other probes and applied to study pH regulation in Golgi. cDNA transfection was used to target a single-chain antibody to a specified site such as an organelle lumen. The targeted antibody functioned as a high affinity receptor to trap cell-permeable hapten-fluorophore conjugates. Synthesized conjugates of a hapten (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and fluorescent probes (Bodipy Fl, tetramethylrhodamine, fluorescein) were bound with high affinity (approximately 5 nM) and specific localization to the single-chain antibody expressed in the endoplasmic reticulum, Golgi, and plasma membrane of living Chinese hamster ovary cells. Using the pH-sensitive phOx-fluorescein conjugate and ratio imaging microscopy, pH was measured in the lumen of Golgi (pH 6.25 +/- 0.06). Measurements of pH-dependent vacuolar H+/ATPase pump activity and H+ leak in Golgi provided direct evidence that resting Golgi pH is determined by balanced leak-pump kinetics rather than the inability of the H+/ATPase to pump against an electrochemical gradient. Like expression of the green fluorescent protein, the receptor-mediated fluorophore targeting approach permits specific intracellular fluorescence labeling. A significant advantage of the new approach is the ability to target chemical probes with custom-designed spectral and indicator properties.

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 Vlambda1 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.

A scFv-alkaline phosphatase fusion protein which detects potato leafroll luteovirus in plant extracts by ELISA.

A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV). Nucleotide sequence analysis showed that the scFv belongs to the human V(H)3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants. Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.