Development and identification of a fully human single-chain variable fragment 29 against TSLP.

Thymic stromal lymphopoietin is a key initiator for inducing Th2-type inflammation and a potential therapeutic target for allergic disease. In the present study, the naive human antibody library was enriched using human thymic stromal lymphopoietin (hTSLP) as an antigen by phage display. Single clones were randomly picked from the enriched antibody library after three rounds of selection, and these were expressed for enzyme-linked immunosorbent assay (ELISA). The positive single-chain fragment variables (scFvs) determined by ELISA were further identified by Western blot, Biacore, and flow cytometry. After three rounds of phage display, 35% of the scFv clones were positive by ELISA and could bind well with hTSLP. Further identification revealed that scFv29 had satisfactory characteristics. The scFv29 was specific to hTSLP, and had no cross-reaction with hIL-33, hIL-4, and hIL-13. The scFv29 could bind to hTSLP in competition with the TSLP receptor and could also bind to mouse TSLP. Cellular experiments revealed that mTSLP could stimulate myeloid dendritic cell (DC) to mature, and scFv29 blocking could reduce the maturation rate of DC. These findings suggest that scFv29 could be used as a neutralizing antibody to block the signaling of TSLP, and this work provides the foundation for further study of the therapeutic roles of TSLP in allergic inflammation diseases.

Development of an immunoassay for the detection of carbaryl in cereals based on a camelid variable heavy-chain antibody domain.

BACKGROUND: The variable domain of camelid heavy-chain antibodies (VHH) is increasingly being adapted to detect small molecules in various matrices. The insecticide carbaryl is widely used in agriculture while its residues have posed a threat to food safety and human health. RESULTS: VHHs specific for carbaryl were generated from an alpaca immunized with the hapten CBR1 coupled to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) based on the VHH C1 and the coating antigen CBR2-BSA was developed for the detection of carbaryl in cereals. This assay, using an optimized assay buffer (pH 6.5) containing 10% methanol and 0.8% NaCl, has a half-maximum signal inhibition concentration of 5.4 ng mL-1 and a limit of detection (LOD) of 0.3 ng mL-1 for carbaryl, and shows low cross reactivity (≤0.8%) with other tested carbamates. The LOD of carbaryl using the VHH-based ELISA was 36 ng g-1 in rice and maize and 72 ng g-1 in wheat. Recoveries of carbaryl in spiked rice, maize and wheat samples were in the range of 81-106%, 96-106% and 83-113%, respectively. Relative standard deviations of repeatability and intra-laboratory reproducibility were in the range of 0.8-9.2% and 2.9-9.7%, respectively. CONCLUSION: The VHH-based ELISA was highly effective in detecting carbaryl in cereal samples after simple sample extraction and dilution. © 2019 Society of Chemical Industry.

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged ß2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

Single Chain Antibodies as Tools to Study transforming growth factor-ß-Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens.

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-ß signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-ß signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-ß signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

Survival of Escherichia coli harboring nucleic acid-hydrolyzing 3D8 scFv during RNA virus infection.

Previously, Escherichia coli harboring the codon-optimized 3D8scFv gene (E. coli 3D8scFv) was developed as a feed additive for use in preventing norovirus infection. Here, we evaluated whether the 3D8scFv gene affects the colonization of E coli when E. coli 3D8scFv passes through the mouse gastrointestinal tract. To determine the colonization ability of E. coli 3D8scFv, E. coli cells with or without the 3D8scFv gene were fed to mice. Total DNA was extracted from the animals' stools, stomach, small intestine and colon. All samples were amplified using 3D8scFv gene-specific primer sets. E. coli 3D8scFv begins to be excreted 1 h after feeding and that all E. coli 3D8scFv cells were excreted between 12 and 24 h after the last feeding of the cells. The previously measured gastrointestinal transit time of the mice was between 8 h and 22 h. The results of this study therefore show that E. coli 3D8scFv cannot colonize the gastrointestinal tracts of mice. In addition, if the purified 3D8 scFv protein is used as a feed additive, any associated E. coli 3D8scFv bacteria will not colonize the gastrointestinal tracts of the livestock. Thus, this feed additive meets the safety assessment criteria for the commercial use of bacteria.

Detection and purification of backbone-cyclized proteins using a bacterially expressed anti-myc-tag single chain antibody.

A myc-tag and of which recognition by an antibody 9E10 has long been used for the detection and purification of recombinant proteins. We have previously expanded the application of the tag to the specific detection and purification of backbone-cyclized proteins. Here we sought a more practical way to using the 9E10 antibody by expressing its single chain antibody (scAb) form in Escherichia coli. The combined use of a strong T7 promoter and auto-induction strategy rather than early to mid-log induction of a Lac promoter resulted in the soluble over-expression of 9E10 scAb. However, the co-expression of a chaperone, Skp, was absolutely necessary for the activity even when the protein was expressed in a soluble manner. We could purify about 4 mg of 9E10 scAb from 1 l of culture, and the resulting scAb could be used to detect and purify the backbone-cyclized protein as the parental full-length 9E10. Moreover, the immunoaffinity resin prepared using 9E10 scAb could be regenerated several times after the elution of bound proteins using an acid, which added more value to the ready preparation of the active antibody in bacteria.

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains.

BACKGROUND: Different strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv. RESULTS: Extracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L-1) and highest space-time-yield (8 mg L-1 h-1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac). CONCLUSIONS: The genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space-time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains.

Proteomic differences in recombinant CHO cells producing two similar antibody fragments.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. "Omics" studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label-free LC-MS proteomic analyses to investigate product-specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single-chain Fv-Fc homodimeric antibody fragments (scFv-Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase-mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label-free proteomic analysis. LC-MS-MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902-1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.

Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.

Selection of bisphenol A - single-chain antibodies from a non-immunized mouse library by ribosome display.

Developing reagents with high affinity and specificity are critical to detect the environmental hormones or toxicants. Ribosome display technology has been widely used in functional protein or peptide screening and in directed evolution of protein molecules in vitro. In this study, single-chain variable fragments (scFvs) against bisphenol A (BPA) were selected from a library constructed from splenocytes of non-immunized mice. After five rounds of selection, the selected scFvs bound to BPA with high affinity. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was introduced to screen the antibody affinity and specificity to BPA. The equilibrium dissociation constants (KDS) of one clone was 1.76µM as determined by surface plasmon resonance (SPR). This study indicated that ribosome display can isolate binders to small molecules from a non-immunized naive library without any in vivo steps and can generate recombinant antibodies efficiently and rapidly. In addition, this study provides a methodological framework for detection of small molecules using recombinant antibodies.

Cloning, expression and identification by immunohistochemistry of humanized single-chain variable fragment antibody against hepatitis C virus core protein.

Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library containing the gene for scFv antibody against Hepatitis C virus core protein was panned with core protein immobilized on microtiter plate wells. After five rounds of panning 60 phage clones specific to core protein were obtained and one selected clone was sequenced. It was found that the specifically detected antigen consists of 774bp and is capable of encoding 257 amino acids in the patients but not in healthy persons.

Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is type III secretion system (T3SS). PcrV is an important structural protein of the T3SS. Methods: In the current investigation, a recombinant single-chain fragment variable (scFv) mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results: : Increased efficiency was achieved by EnBase® compared to Luria­Bertani broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 µg/mL. Conclusion: : Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).

Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume.

Bradyrhizobium is an endophytic bacterium under investigation as an efficient biofertilizer for sustainable legume-rice rotational cropping system. Monitoring and bio-imaging of this nitrogen fixing bacterium is essential for the study of plant-microbe evolution, soil microbiome, as well as quality control in organic farming. While phage display antibody technology has been widely used to generate recombinant antibody for myriad medical purposes, so far, this technology has been minimally applied in the agricultural sector. In this study, single-chain variable fragments (scFv) against two Bradyrhizobium strains SUTN9-2 (yiN92-1e10) and DOA9 (yiDOA9-162) were isolated from a human phage display antibody library. Specific binding of scFv was demonstrated by ELISA and confocal-immunofluorescence imaging techniques. Bradyrhizobium localization in both endophytic and bacteroid forms could be observed inside rice tissue and plant nodule, respectively. Moreover, successful application of the recombinant antibody for the evaluation of nodule occupancy was also demonstrated in comparison with standard GUS-staining method. The results of this study showed for the first time the potential use of human phage display scFv antibody for imaging and monitoring of Bradyrhizobium biofertilizer and thus could be further applied for point-of-detection of bacterial inoculum in the legume-rice rotational crop system. IMPORTANCE Human scFv antibody generated from phage display technology was successfully used for the generation of specific recombinant antibodies: yiN92-1e10 and yiDOA9-162 for the detection of Bradyrhizobium strains SUTN9-2 and DOA9, respectively. These two recombinant scFv antibodies could be used for precise detection of the rhizobia both in symbiosis with legume and endophyte in rice tissue by ELISA and immunofluorescent staining, during legume-rice rotational cropping system in the field. This methodology can be further employed for the study of other plant-microbe interactions and monitoring of biofertilizer in diverse sustainable cropping systems as well as in precision agriculture.

Characterization of single chain antibody targets through yeast two hybrid.

BACKGROUND: Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. RESULTS: We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. CONCLUSIONS: Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.

A peptide-based fluorescent ratiometric sensor for quantitative detection of proteins.

A ratiometric fluorescent sensor was obtained by solid-phase synthesis of a peptide singly labeled at its N-terminus with a 3-hydroxychromone (3HC) derivative, an environmentally sensitive fluorophore with a two-band emission. The construct contains the binding site recognized by an antibody fragment, scFv1F4(Q34S), with nanomolar (nM) affinity. The dye only marginally affected the kinetic and equilibrium binding parameters of the scFv-peptide interaction, as measured by surface plasmon resonance. On interaction with the antibody fragment, the sensor showed up to 47% change in the ratio of its two emission bands, indicating an enhanced screening of the 3 HC fluorophore from bulk water. Competition with two unlabeled peptides of different lengths led to a dynamic displacement of the construct governed by the relative binding constants. Calibration showed that the response is proportional to the ratio of scFv1F4(Q34S) to labeled peptide. The detection limit of scFv1F4(Q34S) was 15 nM. In a more complex medium (100 microg/ml bovine serum albumin), the scFv could be detected in the 50- to 100-nM range. This work demonstrates that, with the perspective of further improvements of the dye spectroscopic properties, fluorescent ratiometric sensing based on small synthetic peptides represents a promising tool for quantitative target detection.

Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin.

A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly(4)Ser)(2) between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL(-1)) was improved to eightfold higher than that in conventional ELISA (0.2 microg mL(-1)), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.

Single chain variable fragment antibodies selected by phage display against the sporozoite surface antigen S16 of Cryptosporidium parvum.

Two human single chain variable fragment (scFv) libraries were used to select clones that bound to the surface glycoprotein S16 of Cryptosporidium parvum. Panning of the Tomlinson libraries I and J resulted in the isolation of nine distinct clones. Of the four clones which had full-length scFv, three contained stop codons. The remaining five clones were truncated, with four missing the heavy chain, and one missing most of the light chain. The full-length clones exhibited better binding to native C. parvum proteins and recombinant S16 than the truncated clones, with the exception of one truncated clone. None of the selected clones cross-reacted with Giardia lamblia, Escherichia coli, Streptococcus pyogenes, Listeria monocytogenes, Bacillus cereus or another immunogenic target of C. parvum, P23. Clones expressed as the soluble scFv-gIIIp construct were able to detect C. parvum native proteins and sporozoites. Panning from naïve libraries was an useful method for isolation and identification of recombinant antibodies that have the potential for use in pathogen detection and immunotherapy.

Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.

The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.