2020 Taylor & Francis Group, LLC. Distinguishing between bull Y- and X-bearing sperm populations is advantageous for techniques using sexed bull semen. The aim of this study was to produce a single-chain fragment variable (scFv) antibody against plasma membrane epitopes on bull Y-bearing sperm. Variable heavy (VH)- and variable light (VL)-region genes generated from a hybridoma cell secreting a specific Y-bearing sperm monoclonal antibody (mAb-1F9) were cloned and expressed. The expected sizes of the DNA bands were ∼350 bp for the VH gene and ∼318 bp for the VL gene. The VH and VL genes were generated and used to construct an scFv gene (∼650 bp), which was expressed in E.coli TG1 cells and produced the corresponding soluble scFv antibody. Compared with the parent mAb-1F9, the scFv antibodies presented a high affinity for Y-bearing sperm and low cross-reactivity with X-bearing sperm. An immunofluorescence analysis confirmed that the scFv antibodies and mAb-1F9 recognize epitopes on the ...

White spot syndrome virus (WSSV) is one of the most important pathogens in shrimp farm throughout the world. Many researches on WSSV have been done, but no efficient approach has been gained to protect and cure the disease. In this study, we constructed a single-chain fragment variable (scFv) antibody library displayed on phage using spleen cells from mice immunized with denatured WSSV. After several rounds of panning respectively against purified intact WSSV virions and purified VP28 expressed in Escherichia coli, five novel scFv antibodies specifically against WSSV were selected, one of which, clone P75E8, recognized a linear epitope. The location in virions of the epitopes recognized by the five scFv clones was determined by immunoelectron microscopy. This study provides a new way to obtain more different antibodies specifically binding to WSSV, and especially provides a new strategy to obtain scFvs against linear epitopes.

Author Summary Antibody affinity maturation is a key aspect of an effective immune response to vaccines, likely to have an impact on clinical outcome following exposure to pathogens. Activation-Induced Cytidine Deaminase (AID) in B cells is a key enzyme involved in antibody class switching and somatic hypermutation, required for antibody affinity maturation. This human study demonstrated for the first time that induction of AID following H1N1pdm09 influenza vaccination directly correlated with in-vivo antibody affinity maturation against the hemagglutinin globular domain (HA1), containing most of the protective targets. Importantly, age differences were found. In younger adults, significant affinity maturation to the HA1 globular domain was observed, which associated with higher initial levels of AID and |2-fold-increase in AID after vaccination. With increased age, a drop in AID activity post-vaccination correlated with lower affinity maturation of the polyclonal antibody responses against the pandemic

To determine whether DNA polymerase plays a role in the hypermutation of immunoglobulin variable genes, we examined the frequency and pattern of substitutions in variable VH6 genes from the peripheral blood lymphocytes of three patients with xeroderma pigmentosum variant disease, whose polymerase had genetic defects. The frequency of mutation was normal but the types of base changes were different: there was a decrease in mutations at A and T and a concomitant rise in mutations at G and C. We propose that more than one polymerase contributes to hypermutation and that if one is absent, others compensate. The data indicate that polymerase is involved in generating errors that occur predominantly at A and T and that another polymerase(s) may preferentially generate errors opposite G and C.. ...

TY - JOUR. T1 - Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1. T2 - Conformational influence of an engineered disulfide bond. AU - Almog, Orna. AU - Benhar, Itai. AU - Vasmatzis, George. AU - Tordova, Maria. AU - Lee, Byungkook. AU - Pastan, Ira. AU - Gilliland, Gary L.. PY - 1998/5/1. Y1 - 1998/5/1. N2 - A recombinant Fv construct of the B1 monoclonal antibody that recognizes the Lewis(Y)-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the V(H) and V(L) domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues V(L)100 and V(H)44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023-1029, 1995). The B1 ...

Epitope prediction based on random peptide library screening has become a focus as a promising method in immunoinformatics research. Some novel software and web-based servers have been proposed in recent years and have succeeded in given test cases. However, since the number of available mimotopes with the relevant structure of template-target complex is limited, a systematic evaluation of these methods is still absent. In this study, a new benchmark dataset was defined. Using this benchmark dataset and a representative dataset, five examples of the most popular epitope prediction software products which are based on random peptide library screening have been evaluated. Using the benchmark dataset, in no method did performance exceed a 0.42 precision and 0.37 sensitivity, and the MCC scores suggest that the epitope prediction results of these software programs are greater than random prediction about 0.09-0.13; while using the representative dataset, most of the values of these performance measures are

TY - JOUR. T1 - Escherichia coli Skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments. AU - Hayhurst, Andrew. AU - Harris, William J.. PY - 1999/4. Y1 - 1999/4. N2 - Expression of single-chain antibody fragments (scAb) in the periplasm of Escherichia coli often results in low soluble product yield and cell lysis. We have increased scab solubility and prevented cell culture lysis by coexpressing the E. coli Skp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scab gene within an isopropyl β-D-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scab solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scab and should prove useful in expanding library sizes.. AB - ...

Kit 30500-050 Kit 30500-096 DNA marker 81-0020 DNA marker 81-0100 DNA marker 82-0100 DNA marker 82-0200 DNA marker 82-0500 DNA marker 82-1000 DNA marker 83-2500 DNA marker 83-5000 DNA Aptamers AD-155-B DNA Aptamers AD-155-F DNA Aptamers AD-155-U Peptide Aptamers AP-302-B Peptide Aptamers AP-302-F Peptide Aptamers AP-302-U Peptide Aptamers AP-304-B Peptide Aptamers AP-304-F Peptide Aptamers AP-304-U Peptide Aptamers AP-306-B Peptide Aptamers AP-306-F Peptide Aptamers AP-306-U Peptide Aptamers AP-308-B Peptide Aptamers AP-308-F Peptide Aptamers AP-308-U Peptide Aptamers AP-309-B Peptide Aptamers AP-309-F Peptide Aptamers AP-309-U Peptide Aptamers AP-310-B Peptide Aptamers AP-310-F Peptide Aptamers AP-310-U Peptide Aptamers AP-312-B Peptide Aptamers AP-312-F Peptide Aptamers AP-312-U Peptide Aptamers AP-315-B Peptide Aptamers AP-315-F Peptide Aptamers AP-315-U Peptide Aptamers AP-318-B Peptide Aptamers AP-318-F Peptide Aptamers AP-318-U Peptide Aptamers AP-319-B Peptide Aptamers AP-319-F Peptide Aptamers

Recently the minor B cell subpopulation that expresses the CD5 (Leu-1) antigen has been implicated as a source of IgM autoantibodies. Chronic lymphocytic leukemia (CLL), the most common leukemia in humans, represents a malignancy of small B lymphocytes that also express the CD5 antigen. However, little is known concerning the antibody variable region genes (V genes) that are used by these malignant CD5 B cells. We have found that a relatively high frequency of CLL patients have leukemic B cells with surface immunoglobulin (sIg) recognized by 17.109, a murine mAb specific for a kappa light chain associated crossreactive idiotype (CRI) associated with rheumatoid factor and other IgM autoantibodies. Flow cytometric analyses revealed that the relative expression of the 17.109-CRI by circulating leukemic B cells was directly proportional to the levels of sIg kappa light chain, indicating that there exists stable idiotype expression in the leukemic population. To examine this at the molecular level, ...

A monoclonal anti-idiotypic antibody specific to a human IgG 1 type monoclonal antibody possessing specificity to nicotinic acetylcholine receptor; a method for the production of the aforementioned monoclonal anti-idiotypic antibody by the steps of immunizing an animal with a human IgG 1 type monoclonal antibody specific to nicotinic acetylcholine receptor, collecting antibody-producing cells from the animal, fusing the collected cells with neoplastic cells, selecting from the product of fusion a hybridoma capable of producing a monoclonal anti-idiotypic antibody specific to the human IgG 1 type monoclonal antibody possessing specificity to nicotinic acetylcholine receptor, propagating the selected hybridoma thereby giving rise to said monoclonal anti-idiotypic antibody, and collecting the produced monoclonal anti-idiotypic antibody; and use of the monoclonal anti-idiotypic antibody as a reagent and as an adsorbent.

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen ...

TY - JOUR. T1 - Intramuscular delivery of a single chain antibody gene prevents brain Aβ deposition and cognitive impairment in a mouse model of Alzheimer's disease. AU - Wang, Yan Jiang. AU - Gao, Chang. AU - Yang, Miao. AU - Liu, Xiao-Hong. AU - Sun, Annie. AU - Pollard, Anthony. AU - Dong, X. AU - Wu, Xiao-Bing. AU - Zhong, Jin Hua. AU - Zhou, Hua-Dong. AU - Zhou, Xin-Fu. PY - 2010/11. Y1 - 2010/11. N2 - Anti-beta-amyloid (Aβ) immunotherapy is effective in removing brain Aβ, but has shown to be associated with detrimental effects. We have demonstrated that Adeno-associated virus (AAV)-mediated delivery of an anti-Aβ single chain antibody (scFv) gene was effective in clearing brain Aβ without eliciting any inflammatory side effects in old APPSwe/PS1dE9 transgenic mice. In the present study, we tested the efficacy and safety of intramuscular delivery of the scFv gene in preventing brain Aβ deposition. The scFv gene was intramuscularly delivered to APPSwe/PS1dE9 transgenic mice at 3months ...

Mesothelioma is an intractable tumor with no curative treatment to date. In a first step toward developing targeted therapeutics against mesothelioma, we sought to identify internalizing antibodies that target mesothelioma-associated cell surface antigens. Taking a functional approach, we have used a nonimmune phage antibody library as an unbiased random-shaped affinity repertoire to select for tumor-targeting scFvs on live mesothelioma cells. The selection methodology was optimized to enrich for scFvs that efficiently target internalizing epitopes (17, 37, 38), providing a means of efficient intracellular payload delivery to mesothelioma cells. We identified 95 unique mesothelioma-targeting scFvs, 21 of which were further characterized by FACS profiling on tumor cell lines, immunohistochemistry on mesothelioma tissue samples, and in vitro internalization/payload delivery assays. All 21 scFvs bind to both epithelioid and sarcomatoid type mesothelioma cell lines. In addition, all 21 scFvs stain ...

Acute myeloid leukemia 2 protein antibody, Acute myeloid leukemia gene 2 antibody, AML 2 antibody, AML2 antibody, CBF alpha 3 antibody, CBF-alpha-3 antibody, CBFA 3 antibody, CBFA3 antibody, Core binding factor alpha 3 subunit antibody, core binding factor antibody, Core binding factor runt domain alpha subunit 3 antibody, Core binding factor subunit alpha 3 antibody, core-binding factor antibody, Core-binding factor subunit alpha-3 antibody, FLJ34510 antibody, MGC16070 antibody, Oncogene AML 2 antibody, Oncogene AML-2 antibody, PEA2 alpha C antibody, PEA2-alpha C antibody, PEBP2 alpha C antibody, PEBP2-alpha C antibody, Pebp2a3 antibody, PEBP2aC antibody, Polyomavirus enhancer binding protein 2 alpha C subunit antibody, Polyomavirus enhancer-binding protein 2 alpha C subunit antibody, runt domain alpha subunit 3 antibody, runt related transcription factor 3 antibody, Runt-related transcription factor 3 antibody, RUNX 3 antibody, Runx3 antibody, RUNX3_HUMAN antibody, SL3 3 enhancer factor 1 ...

Chronic lymphocytic leukemia is a disease with upregulated expression of the transmembrane tyrosine-protein kinase ROR1, a member of the Wnt/planar cell polarity pathway. In this study, we identified COBLL1 as a novel interaction partner of ROR1. COBLL1 shows clear bimodal expression with high levels in chronic lymphocytic leukemia patients with mutated IGHV and approximately 30% of chronic lymphocytic leukemia patients with unmutated IGHV. In the remaining 70% of chronic lymphocytic leukemia patients with unmutated IGHV, COBLL1 expression is low. Importantly, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 have an unfavorable disease course with short overall survival and time to second treatment. COBLL1 serves as an independent molecular marker for overall survival in chronic lymphocytic leukemia patients with unmutated IGHV. In addition, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 show impaired motility and chemotaxis towards CCL19 and ...

c met related tyrosine kinase antibody; CD136 antibody; CD136 antigen antibody; CDw136 antibody; Macrophage stimulating 1 receptor (c met related tyrosine kinase) antibody; Macrophage stimulating 1 receptor antibody; Macrophage stimulating protein receptor alpha chain antibody; MACROPHAGE STIMULATING PROTEIN RECEPTOR antibody; Macrophage stimulating protein receptor beta chain antibody; Macrophage-Stimulating 1 Receptor (MST1R) antibody; Macrophage-stimulating protein receptor beta chain antibody; MSP receptor antibody; Mst1r antibody; MST1R variant RON30 antibody; MST1R variant RON62 antibody; NPCA3 antibody; p185 RON antibody; p185-Ron antibody; Protein-tyrosine kinase 8 antibody; PTK 8 antibody; ptk8 antibody; PTK8 protein tyrosine kinase 8 antibody; Recepteur d'origine nantais (RON) antibody; RON antibody; RON protein tyrosine kinase antibody; RON variant E2E3 antibody; RON_HUMAN antibody; Soluble RON variant 1 antibody; Soluble RON variant 2 antibody; Soluble RON variant 3 antibody; Soluble ...

Having been researching in the field of phage display technology for years, Creative Biolabs is able to offer a series of comprehensive phage display library screening services by its own biopanning strategies and enhanced equipments.. Phage display technology is one of the major approaches applied in various protein interaction studies, especially in the discovery of specific antibodies, scaffolds and ligands. As binders with the desired specificity generally exist at low frequencies in the constructed libraries, phage display library screening has become an effective technique for the enrichment and identification of binders with high affinity and specificity from interested libraries.. Libraries from customized library construction services, in-house premade antibody libraries, peptide libraries or scaffold libraries, libraries provided by clients and other commercialized libraries can all be screened in Creative Biolabs.. "No matter it's purified or unpurified targets, we can screen for ...

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) γ-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA).While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas

Custom polyclonal antibody production, antibodies for cell signaling and signal transduction pathways, cellular organelle markers, 1-month antibody protocol,1-month antibody protocol sicgen,1-month short polyclonal antibody protocol,12-week antibody protocol, 12-week antibody protocol sicgen,12-week classic polyclonal antibody protocol,28-day antibody protocol, 28-day antibody protocol sicgen, 28-day short polyclonal antibody protocol, 3-month antibody protocol, 3-month antibody protocol sicgen, 3-month classic polyclonal antibody protocol, 4-week short polyclonal antibody protocol, 84-day antibody protocol, 84-day antibody protocol sicgen, 84-day classic polyclonal antibody protocol, actin, actin affinity antibodies,actin affinity antibodies sicgen, actin antibodies, actin antibodies sicgen, actin antibody, actin antibody sicgen, actin polyclonal antibodies,actin polyclonal antibodies sicgen,actin sicgen, affinity chromatography, affinity purified secondary antibodies, affinity purified secondary

Dogs spontaneously develop B-cell chronic lymphocytic leukemia (CLL), providing a naturally occurring model to study genetic risk factors and new therapeutics. CLL accounts for approximately 10% of canine lymphoma and leukemia samples submitted for flow cytometric immunophenotyping at the Colorado State University Clinical Immunology laboratory. There is a strong breed predilection in canine CLL, suggesting genetic predisposition (Bromberek et al, 2016). Our goals are to investigate the molecular mechanisms and clinical features of canine CLL, to evaluate this disease as a model for human CLL.. We previously investigated immunoglobulin heavy chain variable region (IGHV) mutation status in canine CLL patients and found breed-specific differences in mutation status. The majority of small-breed dogs, which have a strong predilection for CLL, had mutated IGHV genes (M-CLL), while Boxer dogs preferentially had unmutated IGHV genes (U-CLL)(79% of cases). We investigated the clinical presentation and ...

In this study, we developed a MAb that recognizes both FRα and FRβ (anti-FRαβ). The anti-FRαβ specifically stained trophoblasts and macrophages from human placenta, synovial macrophages from rheumatoid arthritis patient, liver macrophages from cynomolgus monkey and common marmoset, and cancer cells and tumor-associated macrophages from ovary and lung carcinomas. Su...

The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naïve and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a

A total of 52 serum samples from patients with symptoms suggestive of tick-borne encephalitis virus (TBEV) infection and positive IgM and/or IgG antibodies were tested for IgG avidity. Acute/recent TBEV infection was confirmed by low/borderline avidity index (AI) in 94.8% IgM positive/IgG positive samples, while in 5.2% high AI was found indicating persisting IgM antibodies. Majority of IgM negative/IgG positive samples (78.6%) showed high AI consistent with past TBEV infection. However, in 21.3% patients without measurable IgM antibodies current/recent infection was confirmed by AI. IgG avidity represents an additional serologic marker that improves diagnosis of TBEV, especially in cases of atypical antibody response.. Key words: IgG avidity, serology, tick-borne encephalitis virus. Tick-borne encephalitis virus (TBEV) is a small, enveloped virus that belongs to the family Flaviviridae, genus Flavivirus, tick-borne encephalitis serocomplex. There are three subtypes of TBEV: the European, the ...

TY - JOUR. T1 - Comparison of Six-Month Outcomes for Primary Percutaneous Revascularization for Acute Myocardial Infarction With Drug-Eluting Versus Bare Metal Stents (from the APEX-AMI Study). AU - Patel, Manesh R.. AU - Pfisterer, Matthias E.. AU - Betriu, Amadeo. AU - Widmisky, Petr. AU - Holmes, David R.. AU - O'Neill, William W.. AU - Stebbins, Amanda. AU - Van de Werf, Frans. AU - Armstrong, Paul W.. AU - Granger, Christopher B.. PY - 2009/1/15. Y1 - 2009/1/15. N2 - We evaluated the use and outcomes of drug-eluting stents (DESs) and bare metal stents (BMSs) in a large primary percutaneous coronary intervention (PCI) acute ST-elevation myocardial infarction (MI) trial. Recently concerns have been raised with "off-label" use of DESs for short- and long-term clinical outcomes. Limited randomized data exist evaluating DESs versus BMSs in ST-elevation MI. Patients (n = 5,745) in the Assessment of Pexelizumab in Acute Myocardial Infarction (APEX-AMI) trial were categorized by stent type used. ...

Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and selection. Selection involves B cells competing for T cell help based on the amount of antigen they capture and present on their MHC class II (MHCII) proteins. How GC B cells are able to rapidly and repeatedly transition between mutating their B cell receptor genes and then being selected shortly after is not known. We report that MHCII surface levels and degradation are dynamically regulated in GC B cells. Through ectopic expression of a photoconvertible MHCII-mKikGR chimeric gene, we found that individual GC B cells differed in the rates of MHCII protein turnover. Fluctuations in surface MHCII levels were dependent on ubiquitination and the E3 ligase March1. Increases in March1 expression in centroblasts correlated with decreases in surface MHCII levels, whereas CD83 expression in centrocytes helped to stabilize MHCII at that stage. Defects in MHCII ubiquitination caused GC B cells to

The exterior of bacteriophage T4 capsid is coated with two outer capsid proteins, Hoc (highly antigenic outer capsid protein; molecular mass, 40 kDa) and Soc (small outer capsid protein; molecular mass, 9 kDa), at symmetrical positions on the icosahedron (160 copies of Hoc and 960 copies of Soc per capsid particle). Both these proteins are nonessential for phage infectivity and viability and assemble onto the capsid surface after completion of capsid assembly. We developed a phage display system which allowed in-frame fusions of foreign DNA at a unique cloning site in the 5' end of hoc or soc. A DNA fragment corresponding to the 36-amino-acid PorA peptide from Neisseria meningitidis was cloned into the display vectors to generate fusions at the N terminus of Hoc or Soc. The PorA-Hoc and PorA-Soc fusion proteins retained the ability to bind to the capsid surface, and the bound peptide was displayed in an accessible form as shown by its reactivity with specific monoclonal antibodies in an ...

Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and ...

Intracellular expression of Ab fragments has been efficiently used to inactivate therapeutic targets, oncogene products, and to induce viral resistance in plants. Ab fragments expressed in the appropriate cell compartment may also help to elucidate the functions of a protein of interest. We report in this study the successful targeting of the protein tyrosine kinase Syk in the RBL-2H3 rat basophilic leukemia cell line. We isolated from a phage display library human single-chain variable fragments (scFv) directed against the portion of Syk containing the Src homology 2 domains and the linker region that separates them. Among them, two scFv named G4G11 and G4E4 exhibited the best binding to Syk in vivo in a yeast two-hybrid selection system. Stable transfectants of RBL-2H3 cells expressing cytosolic G4G11 and G4E4 were established. Immunoprecipitation experiments showed that intracellular G4G11 and G4E4 bind to Syk, but do not inhibit the activation of Syk following FcepsilonRI aggregation, ...

TY - JOUR. T1 - Identification of peptide mimotopes of gp96 using single-chain antibody library. AU - Shanmugam, Arulkumaran. AU - Suriano, Robert. AU - Goswami, Neha. AU - Chaudhuri, Devyani. AU - Ashok, Badithe T.. AU - Rajoria, Shilpi. AU - George, Andrea L.. AU - Mittelman, Abraham. AU - Tiwari, Raj K.. N1 - Funding Information: Acknowledgments We gratefully acknowledge the financial assistance of AVT and the National Cancer Institute RO1 grant 1RO1CA131946 (RKT).. PY - 2011/3. Y1 - 2011/3. N2 - Heat shock proteins such as gp96 are immunogenic and are widely used as vaccines in immunotherapy of cancers. The present study focuses on the use of peptide mimotopes as immunotherapeutic vaccines for prostate cancer. To this end, we developed a 15-mer gp96 peptide mimotope specifically reactive to MAT-LyLu gp96-peptide complex using combinatorial single-chain antibody and peptide phage display library. The immunogenicity of the synthesized gp96 mimotope was analyzed initially in normal BALB/c mice ...

Polyclonal antibodies are a pool of antibodies originating from different B cells in a host animal that recognize more than one epitope on the same protein or antigen. In contrast, a monoclonal antibody is an antibody originating from a single B cell in a host animal that recognizes only one epitope or binding site on a protein or other antigen. Polyclonal antibodies are usually purified from the serum of an immunized host animal such as a rabbit, chicken or goat. Whereas monoclonal antibodies are usually purified from cell culture media supernatant from the growth of a hybridoma cell line. Monoclonal antibodies are divalent but monospecific. Polyclonal antibodies are also divalent but consist of a pool of antibodies with multiple epitope specificities.. ...

Background: Prolonged cross-clamp time during cardiac surgery increases the risk of postoperative mortality and myocardial injury. This subanalysis from the pexelizumab for reduction of infarction and mortality in coronary artery bypass grafting surgery (PRIMO-CABG) trial, a phase III double-blind, placebo-controlled study of 3,099 patients undergoing on-pump coronary artery bypass graft surgery with or without valve surgery, assessed the impact of pexelizumab, an investigational C5 complement inhibitor, on postoperative outcomes after prolonged aortic cross-clamp time. Methods: The composite endpoint of death or myocardial infarction through postoperative day 30 and death alone through days 30, 90, and 180 were examined in subpopulations of patients across different cross-clamp times. Results: After prolonged cross-clamping (≥90 minutes), death, or myocardial infarction through day 30 and death through days 30, 90, and 180 were significantly increased in the intent-to-treat population and ...

To obtain thermostable immunoreagents specific for the spore form of Bacillus anthracis two llamas were immunized with a combination of six different recombinant proteins. These proteins BclA, gerQ, SODA1, SOD15, BxpB and the protein p5303 have all been shown as components of the B. anthracis spore and could potentially serve as targets for the detection of spores in multiplexed biosensors. Peripheral blood lymphocytes were used to construct a phage display library from which single domain antibodies (sdAbs) targeting each of the proteins were isolated. Unique sdAbs exhibiting nanomolar or better affinities for the recombinant proteins were obtained and most of the isolated sdAbs retained their ability to bind antigen after cycles of heating as determined by enzyme linked immunosorbent assay (ELISA). SdAbs targeting the BclA and gerQ proteins were able to successfully detect bacterial spores, whether broken or intact, using a direct ELISA; the sdAbs were specific, showing binding only to B. anthracis

Read or download book entitled Therapeutic Antibody Engineering written by William R Strohl which was release on 16 October 2012, this book published by Elsevier. Available in PDF, EPUB and Kindle Format. Book excerpt: The field of antibody engineering has become a vital and integral part of making new, improved next generation therapeutic monoclonal antibodies, of which there are currently more than 300 in clinical trials across several therapeutic areas. Therapeutic antibody engineering examines all aspects of engineering monoclonal antibodies and analyses the effect that various genetic engineering approaches will have on future candidates. Chapters in the first part of the book provide an introduction to monoclonal antibodies, their discovery and development and the fundamental technologies used in their production. Following chapters cover a number of specific issues relating to different aspects of antibody engineering, including variable chain engineering, targets and mechanisms of ...

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle

Based on years of experience in phage display technology, Creative Biolabs now is able to provide services for the construction and screening of immune antibody library.. With years of research and development in the past decades, phage display has been a powerful technology to display millions or even billions of different peptides or proteins. It is now a common choice for the studies of protein-protein, protein-peptide and protein-DNA interactions. Among all the applications of phage display technology, one of the most successful ones is the isolation of monoclonal antibodies utilizing large capacity phage antibody libraries.. Thanks to the rapid development of this technology, it has become a reliable tool for the production of monoclonal antibody with high specificity and affinity. "Compared with conventional hybridoma technology, the generation of immune antibody libraries is not limited by the requirement of fusion partners. This expands the possibility to develop monoclonal antibodies ...

Plasmodium vivax Duffy binding protein (DBP) is an essential ligand for reticulocyte invasion making it a premier asexual blood stage vaccine candidate. However, strain-specific immunity due to DBPII allelic variation may complicate vaccine efficacy, suggesting that an effective DBPII vaccine needs to target immune responses to conserved epitopes that are potential targets of strain-transcending neutralizing immunity. Anti DBPII monoclonal antibodies, which were previously characterized by COS7 cell binding assay as inhibitory and non-inhibitory to DBPII-erythrocyte binding, were mapped to DBPII gene fragment libraries using phage display. Inhibitory mAb 3C9 binds to a conserved conformation-dependent epitope in subdomain 3 while non-inhibitory mAb 3D10 binds to a linear epitope in subdomain 1 of DBPII. More definitive epitope mapping of mAb 3D10 was achieved using a random peptide library displayed on phage. Since DBP region II is mostly made up of alpha-helices, we used a randomized helical scaffold

1A14: COMPLEX BETWEEN NC10 ANTI-INFLUENZA VIRUS NEURAMINIDASE SINGLE CHAIN ANTIBODY WITH A 5 RESIDUE LINKER AND INFLUENZA VIRUS NEURAMINIDASE

Using cosmids covering about 117 Kb upstream of the human immunoglobulin chain C mu gene, we have identified a potentially functional VH gene, belonging to the VHVI subgroup. This VHVI gene is only about 95 Kb from the C mu gene and is probably the first functional VH segment of the Igh locus. These results illustrate the proximity of the human VH, DH and JH segments involved in creation of the complete heavy chain genes.

Streptococcus pneumoniae is a serious worldwide pathogen and the focus of numerous vaccine development projects. Currently the most widely accepted surrogate marker for evaluating the efficacy of a given vaccine is to utilize ELISA. Measurement of antibody concentration by ELISA without reduction in cross-reactive antibodies causes an overestimation of antibody concentration and therefore protection, this is most notable in the aged, an at risk group for this infection. We compared the immune response to the pneumococcal polysaccharides (PPS) 4 and 14 of 20 young to 20 elderly adults. Pre-and post-vaccination IgG antibody concentrations and antibody avidity against PPS4 and PPS14 were measured using two different enzyme-linked immunosorbant assay (ELISA) absorption protocols. All sera were pre-absorbed with either cell-wall polysaccharide (CPS), or CPS and serotype 22F polysaccharide. Pre- and post-vaccination IgG antibody concentrations for serotype 4, but not 14, were significantly lowered with the

We determined that, as compared with human naïve B cells, human GC B cells have a higher intrinsic affinity threshold for antigen. We observed that independently of other extrinsic factors, such as competition among B cells for antigen, the intrinsic affinity of GC B cells for an antigen dictated each subsequent step in B cell activation from the magnitude of BCR signaling to the receptivity of BCR-stimulated GC B cells to Tfh cell signals that drive IRF-4 expression and PC differentiation. We provided evidence that BCRs on LZ GC B cells are concentrated in distinct, highly dynamic, ezrin- and actin-rich pod-like structures through which the BCRs engage antigen, signal, exert pulling forces, and extract antigen from membranes. In contrast, the BCRs on naïve B cells function in flat, stable membrane contacts, with antigen-containing surfaces displaying the well-described features of immune synapses and cSMACs. The role of these pod-like structures in establishing high-affinity thresholds for GC ...

Transgenic mosquitoes resistant to malaria parasites are being developed to test the hypothesis that they may be used to control disease transmission. We have developed an effector portion of an antiparasite gene that can be used to test malaria resistance in transgenic mosquitoes. Mouse monoclonal antibodies that recognize the circumsporozoite protein of Plasmodium gallinaceum can block sporozoite invasion of Aedes aegypti salivary glands. An anti-circumsporozoite monoclonal antibody, N2H6D5, whose corresponding heavy- and light-chain gene variable regions were engineered as a single-chain antibody construct, binds to P. gallinaceum sporozoites and prevents infection of Ae. aegypti salivary glands when expressed from a Sindbis virus. Mean intensities of sporozoite infections of salivary glands in mosquitoes expressing N2scFv were reduced as much as 99.9% when compared to controls.

英) We have established a method for selecting binding phages from a phage immunoglobulin heavy chain variable region (VH) library by panning with nitrocellulose membranes (membrane panning). To evaluate the concentrating ability of membrane panning for binding phages, a phage VH library containing clones that bind to hen egg white lysozyme (HEL) was used for panning against HEL. The efficiency of our method was as high as that of panning with magnetic beads. In addition, we performed membrane panning against target proteins and isolated the binding phages. The human VH genes of these phages were cloned and expressed as VH-bacterial alkaline phosphatase (PhoA) conjugates (VH-PhoA) in Escherichia coli. The dose-dependent binding of VH-PhoA to target proteins was confirmed by dot blotting. When applied to disease-associated antibodies, these methods will likely benefit clinical research. In addition, these techniques may be applicable to systematic analysis in proteome studies. (日) ...

Selective suppression of IgE antibody response was demonstrated. Preadministration of DNP-coupled mycobacterium (DNP-Tbc) inhibited the formation of anti-DNP IgE antibody induced by DNP-OA without any suppressive effect on anti-DNP IgG antibody response. Secondary anti-DNP IgE antibody response by DNP-OA was also significantly depressed by the preadministration of DNP-Tbc. Anti-OA IgE antibody response induced by DNP-OA was also depressed by DNP-Tbc, whereas anti-OA IgE antibody response induced by PAB-OA was not affected by the preadministration of DNP-Tbc. Preimmunization with DNP-MGG induced much higher anti-DNP IgG antibody response than DNP-Tbc, but DNP-MGG did not suppress the induction of anti-DNP IgE antibody. The transfer of DNP-Tbc-primed spleen cells into normal mice depressed anti-DNP IgE antibody response. B cell-depleted cell populations also showed a comparable inhibitory effect to that of unfractionated DNP-Tbc primed cells. In the adoptive cell transfer experiment, ...

TY - JOUR. T1 - Automated panning and screening procedure on microplates for antibody generation from phage display libraries. AU - Turunen, Laura. AU - Takkinen, Kristiina. AU - Söderlund, Hans. AU - Pulli, Timo. PY - 2009. Y1 - 2009. N2 - Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay ...

CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized

The invention provides recombinant antibody molecules comprising antigen binding regions derived from the heavy and/or light chain variable regions of a donor anti-CD3 antibody, e.g. OKT3, and which have anti-CD3 binding specificity, preferably of affinity similar to that of OKT3. The recombinant antibody is preferably a humanized antibody and may be a chimeric or CDR-grafted antibody. A method is disclosed for preparing CDR-grafted humanized antibodies in which, in addition to the CDR's, non-human antibody residues are preferably used at positions 23, 24, 49, 71, 73 and 78 of the heavy chain variable region and at positions 46, 48, 58, and 71 of the light chain variable region. The recombinant, especially the humanized, anti-CD3 antibodies may used for in vivo therapy or diagnosis.

Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering

Activation-induced deaminase (AID) is expressed only in germinal center B cells. There, it is required for somatic hypermutation, gene conversion and class switch recombination of antibody variable region segments, three processes that diversify antibodies during immune responses. Although AID has homology to RNA-editing enzymes, three recent reports suggest it could initiate the diversification processes by deaminating cytidine residues within the antibody genes themselves.

Phage-displayed single domain antibodies (sdAb) were compared to monomeric solubly expressed sdAb and llama polyclonal antibodies for the detection of ricin. SdAb are comprised of the variable domain derived from camelid heavy chain only antibodies (HcAb). Although HcAb lack variable light chains, they as well as their derivative sdAb are able to bind antigens with high affinity. The small size of sdAb (~16 kDa), while advantageous in many respects, limits the number of labels that can be incorporated. The ability to incorporate multiple labels is a beneficial attribute for reporter elements. Opportunely, sdAb are often selected using phage display methodology. Using sdAb displayed on bacteriophage M13 as the reporter element gives the potential for incorporating a very high number of labels. We have demonstrated the use of both sdAb and phage- displayed sdAb for the detection of ricin using both enzyme linked immunosorbent assays (ELISAs) and Luminex fluid array assays. The phage-displayed sdAb led to

Fine specificity of anti-V3 antibodies induced in chimpanzees by HIV candidate vaccines.: The fine specificity of the anti-V3 antibody responses induced in chim