Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile

Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility

Автоматическая оптимизация технологического комплекса обогащения железных руд по сигналам магнитной индукции сепаратора

Recombinant antibodies can be used to diagnose, treat and prevent disease by exploiting their specific antigen-binding activities. A large number of drugs currently in development are recombinant antibodies and most of these are produced in cultured rodent cells. Although such cells produce authentic functional products, they are expensive, difficult to scale-up and may contain human pathogens. Plants represent a cost-effective, convenient and safe alternative production system and are slowly gaining acceptance. Five plant-derived therapeutic recombinant antibodies (plantibodies) are undergoing clinical evaluation, three of which can be used as prophylactics

Expression of Recombinant Antibodies

Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications

Antibody fragments as probe in biosensor development

Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed

Modern affinity reagents: Recombinant antibodies and aptamers

AbstractAffinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research

Fv antibodies to aflatoxin B1 derived from a pre-immunized antibody phage display library system

The production and characterization of recombinant antibodies to aflatoxin B[SUB1] (AFB[SUB1]), a potent mycotoxin and carcinogen is described. The antibody fragments produced were then applied for use in a surface plasmon resonance-based biosensor (BIAcore), which measures biomolecular interactions in 'real-time'. Single chain Fv (scFv) antibodies were generated to aflatoxin B1 from an established phage display system, which incorporated a range of different plasmids for efficient scFv expression. The scFv's were used in the development of a competitive ELISA, and also for the development of surface plasmon resonance (SPR)-based inhibition immunoassays. They were found to be suitable for the detection of AFB[SUB1], in this format, with the assays being sensitive and reproducible

DEVELOPING RECOMBINANT ANTIBODIES FOR SORTING RECEPTOR SORLA

Breast cancer is the most common malignancy and the leading cause of cancer-related death among women. About 15 to 20% of breast cancers has amplification of the HER2 oncogene. HER2 is a member of the epidermal growth factor receptor (EGFR) family that promotes cell survival, proliferation, motility, and resistance to apoptosis. Despite many advances in treatment options targeting HER2, anti-HER2 therapy resistance remains a relevant issue. SorLA is a sorting receptor of the vacuolar protein sorting 10 proteins (vps10p) domain receptor family, a family of sorting receptors transporting cargo between cell surface and intracellular compartments such as the trans-Golgi network, endosomes, and lysosomes. A link between HER2 cancer signaling and the expression of SorLA has recently been discovered. SorLA was shown to promote HER2 recycling by preventing its lysosomal degradation. Depletion of SorLA decreased HER2 signaling, promoted HER2’s accumulation in dysfunctional lysosomes and sensitized the cancer cells to a lysosometargeting cationic amphiphilic drug ebastine. High SorLA expression correlated with poor patient outcomes in HER2-amplified breast cancer patients. These findings make SorLA an attractive target for drug therapy. The aim of this study was to develop anti-SorLA antibody fragments (binders), the most promising of which will later be developed into a full-length humanized anti-SorLA antibody ultimately testable in patients. The binders were developed using phage display targeting either full-length SorLA or its binding domain. In the first round 576 clones were screened. In the second round 68 clones were screened and subsequently sequenced, producing 41 different sequences. These most promising 34 clones were then used to immunoprecipitated SorLA to further test for binding activity. Out of these 34 binders 23 successfully immunoprecipitated SorLA. The most promising clones will be tested in vitro in HER2 positive cancer cell lines to assess their cancer growth -halting capabilities. The most successful clone will be developed into a full-length human antibody that can be tested in patients

Selection of recombinant antibodies against Lawsonia intracellularis

Proliferative enteropathy, caused by the obligate intracellular bacterium Lawsonia intracellularis (McOrist et al., 1995), is a widely distributed disease throughout the world causing substantial economic loss. The diagnostics of the disease is complicated by limited value of serological surveys as well as the fact that L. intracellularis can only be cultured in cell cultures

Recombinant antibodies against Clostridium difficile toxin A

The carboxy-terminal region of Clostridium difficile Toxin A was expressed as a recombinant protein (ToxinA-CT), purified, and shown to behave as would be expected of the receptor-binding domain. ToxinA-CT was then used to isolate scFv antibodies from a phage display library using conventional bio-panning methods. Progressive enrichment of ToxinA-CT-specific scFvs through 3 rounds of selection was observed. Those scFvs that showed strongest reaction with ToxinA-CT in ELISA were sequenced, expressed as soluble antibodies and purified. A panel of diverse scFvs was established that appeared to bind to non-overlapping epitopes on ToxinA-CT. To assay for protective activity, scFvs were mixed with native Toxin A, added to cells in culture, and the response monitored over a 2 hour period. The scFvs were added to consistently delay the cytopathic activity of Toxin A but were unable to match the neutralizing activity of a polyclonal serum or its Fab derivatives. The epitopes recognised by the scFvs were localized using a sub-cloning strategy. Defined parts of the ToxinA-CT reading frame were recovered by PCR and fusions created with maltose binding protein. scFvs were tested for recognition of the maltose binding fusion proteins. In one instance, a scFv reacted with multiple fusion proteins suggesting recognition of a repeated peptide motif. A maltose binding protein fusion carrying a candidate sequence was successfully recognised by this scFv. Overall, phage display enabled assembly of a panel of scFv antibodies against epitopes in the carboxy-terminal domain of Toxin A. The inability of these antibodies to block the activity of Toxin A may be due to the multivalent interaction between the toxin and its receptor. Alternatively, the ToxinA-CT used in scFv isolated may lack functions that are crucial for receptor interaction and hence potential targets for antibody-mediated inhibition

Seed-produced anti-globulin VHH-Fc antibodies retrieve globulin precursors in the insoluble fraction and modulate the Arabidopsis thaliana seed subcellular morphology

Key message Nanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins. Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination