Production and characterization of a monoclonal antibody for Pefloxacin and mechanism study of antibody recognition.

In this report, an artificial antigen (PFLX-BSA: Pefloxacin connected bovine serum albumin) was successfully prepared. The monoclonal antibody against pefloxacin was produced and characterized using a direct competitive ELISA. The linear range of detection was 0.115-6.564 µg/L. The limit of detection defined as IC15 was 0.170 ± 0.05 µg/L and the IC50 was 0.902 ± 0.03 µg/L. The antibody variable region genes were amplified, assembled, and sequenced. A three-dimensional structural model of the variable region was constructed to study the mechanism of antibody recognition using molecular docking analysis. Three predicted essential amino acids, Thr53, Arg97 of heavy chain and Thr52 of light chain, were mutated to verify the theoretical model. Three mutants lost binding activity significantly against pefloxacin as predicted. These may provide useful insights for studying antigen-antibody interaction mechanisms to improve antibody affinity maturation in vitro.

Ligation-anchored PCR unveils immune repertoire of TCR-beta from whole blood.

BACKGROUND: As one of the genetic mechanisms for adaptive immunity, V(D)J recombination generates an enormous repertoire of T-cell receptors (TCRs). With the development of high-throughput sequencing techniques, systematic exploration of V(D)J recombination becomes possible. Multiplex PCR has been previously developed to assay immune repertoire; however, the use of primer pools leads to inherent biases in target amplification. In our study, we developed a "single-primer" ligation-anchored PCR method that may amplify the repertoire with much less biases. RESULTS: By utilizing a universal primer paired with a single primer targeting the conserved constant region, we amplified TCR-beta (TRB) variable regions from total RNA extracted from blood. Next-generation sequencing libraries were then prepared for Illumina HiSeq 2500 sequencer, which generates 151-bp read length to cover the entire V(D)J recombination region. We evaluated this approach on blood samples from healthy donors and from patients with malignant and benign meningiomas. Mapping of sequencing data showed that 64% to 96% of mapped TCRV-containing reads belong to TRB subtype. An increased usage of specific V segments and V-J pairing were observed in malignant meningiomas samples. The CDR3 sequences of the expanded V-J pairs were distinct in each malignant individual, even for pairing of TRBV7-3 with TRBJ2-2 that showed increased usage in both cases. CONCLUSIONS: We demonstrated the technical feasibility and effectiveness of ligation-anchored PCR approach in capturing the TCR-beta landscapes. Further development of this technology may enable a comprehensive delineation of immune repertoire, including other forms of TCRs as well as immunoglobulins.

Fast track antibody V-gene rescue, recombinant expression in plants and characterization of a PfMSP4-specific antibody.

BACKGROUND: Monoclonal antibodies (mAbs) are essential tools in biological research, diagnosis and therapy, and are conventionally produced in murine hybridoma cell lines. Professional applications of mAbs depend on the steady supply of material. Because hybridoma cultures can stop producing the antibody or even die, preservation of the unique epitope specificity of mAbs by rescue of the sequences encoding the antibody variable domains (V regions) is important. The availability of these sequences enables not only the recombinant expression of the original antibody for further applications, but opens the road for antibody engineering towards innovative diagnostic or therapeutic applications. A time- and cost-efficient production system enabling the detailed analysis of the antibodies is an essential requirement in this context. METHODS: Sequences were rescued from three hybridoma cell lines, subjected to sequence analysis, subcloned into binary expression vectors and recombinantly expressed as chimeric mAb (constant regions of human IgG1:k1) in Nicotiana benthamiana plants. The properties of the recombinant and the murine mAbs were compared using competition enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. The recognition of native PfMSP4 by the recombinant mAb was analysed by immunofluorescence staining of Pf 3D7A schizonts and by western blot analysis of merozoite extract. RESULTS: The rescued sequences of all three hybridoma cell lines were identical. The recombinant mAb was successfully expressed as IgG in plants at moderate levels (45 mg/kg fresh leaf weight). Preservation of the original epitope was demonstrated in a competition ELISA, using recombinant mAb and the three murine mAbs. EGF_PfMSP4-specific affinities were determined by SPR spectroscopy to 8 nM and 10 nM for the murine or recombinant mAb, respectively. Binding to parasite PfMSP4 was confirmed in an immunofluorescence assay showing a characteristic staining pattern and by western blot analysis using merozoite extract. CONCLUSIONS: As demonstrated by the example of an EGF_PfMSP4-specific antibody, the described combination of a simple and efficient hybridoma antibody cloning approach with the flexible, robust and cost-efficient transient expression system suitable to rapidly produce mg-amounts of functional recombinant antibodies provides an attractive method for the generation of mAbs and their derivatives as research tool, novel therapeutics or diagnostics.

Expression, characterization, and evaluation of a RANK-binding single chain fraction variable: an osteoclast targeting drug delivery strategy.

A single chain Fraction variable (scFv) employs antibody-like target recognition specificity. Osteoclasts, responsible for bone resorption, express Receptor Activator of Nuclear factor Kappa B (RANK) receptors. This study aimed to express, characterize, and evaluate scFv against RANK receptors that may serve as a platform to target osteoclasts. Using phage display technology, scFv against RANK receptor was expressed and characterized by DNA sequencing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption-ionization time-of-flight (MALDI TOF), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunocytochemistry. The potential for cytotoxicity was evaluated using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, and its cross reactivity was evaluated using ELISA. Osteoclast-like cells were generated from RAW 264.7 cells, and the osteoclast targeting ability of scFv was evaluated using immunocytochemistry. ScFv's antiresorptive efficacy was studied using a tartrate-resistant acid phosphatase (TRAP) assay and resorption assay. Anti-RANK scFv was successfully expressed and characterized. No cross reactivity with other tumor necrosis factor receptor (TNFR) members and no cytotoxic effect on a non-RANK bearing cell line were observed. It showed specificity toward a RANK receptor and an inhibitory effect on osteoclast activity. With the increase in development trends for biologics as therapeutics and growing knowledge on the importance of osteoclast targeted therapy, this study may provide a drug delivery strategy to target osteoclasts, thereby leading to a promising therapy for resorptive bone diseases.

A mechanism for TCR sharing between T cell subsets and individuals revealed by pyrosequencing.

The human naive T cell repertoire is the repository of a vast array of TCRs. However, the factors that shape their hierarchical distribution and relationship with the memory repertoire remain poorly understood. In this study, we used polychromatic flow cytometry to isolate highly pure memory and naive CD8(+) T cells, stringently defined with multiple phenotypic markers, and used deep sequencing to characterize corresponding portions of their respective TCR repertoires from four individuals. The extent of interindividual TCR sharing and the overlap between the memory and naive compartments within individuals were determined by TCR clonotype frequencies, such that higher-frequency clonotypes were more commonly shared between compartments and individuals. TCR clonotype frequencies were, in turn, predicted by the efficiency of their production during V(D)J recombination. Thus, convergent recombination shapes the TCR repertoire of the memory and naive T cell pools, as well as their interrelationship within and between individuals.

Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner.

Halophilic ß-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649-658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The "Brevibacillus in vivo cloning" method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.

Human anti-EGFL7 recombinant full-length antibodies selected from a mammalian cell-based antibody display library.

Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.

Functional expression of recombinant anti-BNP scFv in methylotrophic yeast Pichia pastoris and application as a recognition molecule in electrochemical sensors.

Recent studies have revealed the potential of B-type natriuretic peptide (BNP) as a good prognostic marker for patients with heart failure. Antibodies against BNP are expected to be usefully employed in the diagnosis of heart failures. We established a more efficient method to produce functional anti-BNP, single chain variable fragment (scFv) using a eukaryotic expression system of Pichia pastoris. Although analysis of the N-terminal (NT) sequence of the expressed anti-BNP scFv indicated that the two Ste13 sites of the secreted anti-BNP scFv were not cleaved, the specificity of anti-BNP scFv was not affected significantly. The binding activity of anti-BNP scFv against other antigens, against four other antigens, NT probrain peptide (NT-pro BNP), atrial natriuretic peptide (ANP), carcinoembryonic antigen (CEA) and human serum albumin (HSA), was only one thousandth that of the original BNP antigen, which clearly demonstrated the specific binding of recombinant scFv toward BNP. The anti-BNP, scFv-based, electrochemical immunoassay exhibited excellent analytical performance with a detection limit of 1 fg/ml and a wide linear detection range from 1 to 10,000 fg/ml. The optimum culture conditions to obtain the maximum concentration of recombinant scFv were a pH range of 5.0-7.0 and an incubation temperature of 20°C. This anti-BNP scFv expressed in P. pastoris has the potential for promising applications in the diagnosis of heart failure.

Development and characterization of functional antibodies targeting NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.

Patterns of receptor revision in the immunoglobulin heavy chains of a teleost fish.

H chain cDNA libraries were constructed from the RNA derived from seven different organs and tissues from the same individual catfish. Sequence analysis of >300 randomly selected clones identified clonal set members within the same or different tissues, and some of these represented mosaic or hybrid sequences. These hybrids expressed V(H) members of the same or different V(H) families within different regions of the same clone. Within some clonal sets multiple hybrids were identified, and some of these represented the products of sequential V(H) replacement events. Different experimental methods confirmed that hybrid clones identified in the cDNA library from one tissue could be reisolated in the cDNA pool or from the total RNA derived from the same or a different tissue, indicating that these hybrids likely represented the products of in vivo receptor revision events. Murine statistical recombination models were used to evaluate cryptic recombination signal sequences (cRSS), and significant cRSS pairs in the predicted V(H) donor and recipient were identified. These models supported the hypothesis that seamless revisions may have occurred via hybrid joint formation. The heptamers of the cRSS pairs were located at different locations within the coding region, and different events resulted in the replacement of one or both CDR as well as events that replaced the upstream untranslated region and the leader region. These studies provide phylogenetic evidence that receptor revision may occur in clonally expanded B cell lineages, which supports the hypothesis that additional levels of somatic H chain diversification may exist.

Selection of complementary single-variable domains for building monoclonal antibodies to native proteins.

Antibodies are now indispensable tools for all areas of cell biology and biotechnology as well as for diagnosis and therapy. Antigen-specific single immunoglobulin variable domains that bind to native antigens can be isolated and manipulated using yeast intracellular antibody capture technology but converting these to whole monoclonal antibody requires that complementary variable domains (VH or VL) bind to the same antigenic site. We describe a simple approach (CatcherAb) for specific isolation of such complementary single domains allowing the constitution of functional Fv, forming the basis of antigen-specific whole immunoglobulin and thus antibody production. We illustrate this approach by developing high-affinity Fv from single variable domains binding to RAS and LMO2 oncogenic proteins.

Expression and purification of an anti-clenbuterol single chain Fv antibody in Escherichia coli.

Recombinant antibodies with desirable characteristics that can replace polyclonal or monoclonal antibodies are important for enzyme-linked immunosorbent assay (ELISA) of residues of clenbuterol (CBL), an illicit veterinary drug. Here, we report our work on expression and purification of a mouse-derived anti-CBL single chain Fv (scFv) antibody in Escherichia coli (E. coli). An expression plasmid pBV220-CBL was constructed and transformed into E. coli BL21 (DH3) strain cells. After induction by temperature, the 6x His-tagged anti-CBL scFv antibodies were expressed with the yield of 31%. The solubilized inclusion bodies were extracted, denatured and then purified by Ni-NTA column chromatography. The purified recombinant target protein was analyzed by high performance liquid chromatography, SDS-PAGE and Western blotting, respectively. The results showed the prepared anti-CBL scFv antibodies posed HRP-anti-His-tag antibody-recognized activity and their purity was up to 96%. Moreover, an indirect competitive ELISA based on the anti-CBL scFv antibodies revealed that the limit of detection for CBL was 0.5 ng/ml and the linear range was 1.5-10.6 ng/ml. Taken together, these findings suggest that the prepared recombinant antibody can be used for future immunoassay detection for CBL.

Purification and functional characterization of a Camelid-like single-domain antimycotic antibody by engineering in affinity tag.

Single-domain single-chain variable fragment (scFv) antibody is sometimes critical for purification using affinity tagging strategy. We failed in our initial effort to purify a prematurely developed Camelid-like E-tagged short scFv-K2 antibody that contained a complete variable region of the heavy chain and partial region of the light chain by using an anti-E-tag affinity column. To expedite the purification of this altered but interesting antimycotic agent, we replaced a long and large E-tag by a short and hydrophilic 6x-Histidine (His(6)) affinity tag by polymerase chain reaction. The short and compact His(6)-tag was placed on the previously constructed expression vector pCANTAB 5 E that contained the large affinity E-tag sequence (13 amino acids) by PCR-based mutagenesis and was expressed in Escherichia coli. The recombinant protein can then be purified by immobilized metal affinity chromatography (IMAC) and be used for biochemical and other functional characterization. This His(6)-tagged short scFv-K2 antibody (20 kDa) had strong cytocidal activity against Saccharomyces and Candida species with a IC(50) value of 0.44x10(-6)M and 1.10 x 10(-6)M, respectively. Tag replacement facilitates the purification of a Camelid-like single-domain scFv antibody and after that meets its different functional characteristics. The present study reflects that the V(H) domain of the scFv antibody is mainly responsible for its biological activity and single-domain scFv antibody may acts as a potent antimicrobial agent.

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris.

BACKGROUND: Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs. RESULTS: Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli. CONCLUSION: Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.

Improving GPX activity of selenium-containing human single-chain Fv antibody by site-directed mutation based on the structural analysis.

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se-scFv-B3 (selenium-containing single-chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv-B3 was carried out. A three-dimensional (3D) structure of scFv-B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer-aided docking and energy minimization (EM) calculations of the antibody-GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni(2+)-immobilized metal affinity chromatography (IMAC), then transformed to selenium-containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se-scFv-B3-A180S was significantly increased compared to the original Se-scFv-B3.

Expression of anti-K99 scFv in transgenic rice tissues and its functional characterization.

As a first step towards manufacturing functional anti-K99 single chain variable antibody fragment (scFv) in a plant system to prevent colibacillosis in neonatal calves, we investigated the feasibility of producing these antibodies in rice plants. Two scFv constructs, with or without the endoplasmic reticulum (ER) targeting KDEL sequence, were introduced into rice for either ER-retention of the recombinant antibody or its secretion. In agreement with several other published reports, extremely low-levels of scFv were produced in rice plants transformed with the construct lacking the ER-targeting sequence. Constructs containing the KDEL sequence resulted in significantly higher levels of the antibody in rice leaves. Although scFv transcripts were found in all three rice tissues analyzed, scFv protein was detected only in the leaf and embryo tissues and not in the endosperm portion of the seed. Functionality of the rice-produced scFv was tested in two in vitro assays, i.e., inhibition of K99-induced horse red blood cell agglutination and inhibition of the attachment of enterotoxigenic Escherichia coli (ETEC) to calf enterocytes. Rice-scFv was found to be functionally equivalent to anti-K99 monoclonal antibody (mAb) in both the assays. The results obtained in this investigation provide valuable information and in combination with other studies of this kind, will be helpful in devising strategies to improve production of useful recombinant proteins in the seeds.

Assembly of a functional immunoglobulin Fv fragment in Escherichia coli.

An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli. The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the Fv fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The Fv fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant Fv fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the Fv fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.

Isolation, expression and characterization of two single-chain variable fragment antibodies against an endo-polygalacturonase secreted by Sclerotinia sclerotiorum.

Canola is a very important economic crop in the world and canola stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic, highly destructive and non-host-specific fungus, can reduce yield significantly. This fungus secretes numerous cell wall degrading enzymes including an endo-polygalacturonase, SSPG1d, which has been detected at early stages of infection. In this report we describe the isolation of two recombinant antibodies of the single-chain variable fragment (ScFv) format from RNA of mice immunized with recombinant SSPG1d (rSSPG1d) or a peptide derived from SSPG1d (peptide 3796) that was predicted to be antigenic. The ScFvs were isolated using the established phage display technology. These recombinant antibodies were expressed, purified and refolded to functional antibodies with a yield of 120-500mug per liter of cell culture. Recombinant antibodies were characterized using various techniques including enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Of the two ScFvs, it appears that only ScFv-rSSPG1d is able to detect whole SSPG1d produced by the fungus. Thus our results indicate that this ScFv may have utility in the detection of the SSPG1d enzyme in an antibody-based diagnostic test for S. sclerotiorum infection.

High-level periplasmic expression and purification of scFvs.

The isolation of recombinant antibodies by phage display naturally leads to experiments to evaluate their biological and immunological properties. Although crude preparations may have their value in initial studies, the need often exists for highly purified protein that can be tested in vivo. This chapter describes methods to generate high yields of scFv from bacterial cultures and to purify protein to the degree of homogeneity required for the most exacting analysis.