As an alternative, pAbs are produced as ascites in mice. Moreover, these antibodies have superior specificity compared with monoclonal antibodies because they are generated by a large number of B-cell clones each producing antibodies to a specific epitope Hudson et al. Monoclonal antibodies mAbs are clinically significant homogeneous and mono-specific scientific biomolecules produced from hybridoma cells by hybridoma technology Zhang, Since their discovery, these molecules have been used as research tools and have revolutionized the fields of biotechnology, immunology, diagnostics, and medicine.
Improvements in hybridoma technology are based on research demand, cost effectiveness, human labor, and reduced development time. Similarly, the production of mAbs requires multiple phases, long duration, and high cost. Currently, mAbs have been produced against a number of mycotoxins such as fumonisin B1 Yuan et al.
Similarly, mAbs against transmembrane enzymes have been produced Yuan et al. Monoclonal antibody products in the US, Europe, and global markets approved for diseases. Hybridoma technology has been a significant and essential platform for producing high-quality mAbs Zhang, It permits generation of therapeutic antibodies in a native form. However, technical difficulties in hybridoma production have updated the mainstream antibody production into new ways like display and transgenic mice techniques.
Nevertheless, hybridoma technology is a classical and established route of generating specific antibodies all around the globe Glukhova et al. The technology begins with immunization of test animals with an antigen of interest and serum antibody titer is determined by enzyme linked immunosorbent assay ELISA. Subsequently, the spleen is aseptically removed and splenocytes are fused with myeloma cells to produce hybridoma cells.
Hybridoma cells are then cultured in well plates in the presence of hypoxanthine-aminopterin-thymidine HAT selection medium for high throughput screening. Cell culture systems in vitro with specific mAb cell lines were then subjected to mass generation by media selection, shaker flasks, and bench-scale bioreactors Ling et al. Illustration showing the production route of hybridoma technology. Monoclonal antibodies are generated by immunizing laboratory animals with a target antigen.
B cells and myeloma cells are fused and then selected in HAT medium. Finally, hybridoma cells producing the desired antibodies are screened. The optimal conditions such as temperature, percent carbon dioxide, and humidity for cell cultures should be determined Sen and Roychoudhury, , and then transferred to a pilot scale for scalability and toxicology studies. In addition, clinical materials should be produced on a large scale under the existing good manufacturing practice cGMP regulations. After production on a small scale, products that are already cultured in a laboratory are then transferred to pilot scale commercialization.
The process then performs characterization, scaling, technology transfer, and validation. Commercial cell culture for the production of a biological product is completed by pilot scale laboratory methods Li et al.
Recently, commercialization is initiated by process characterization, scale-up, technology transfer, and validation of the manufacturing process Li et al. ELISA is enzyme-based colorimetric assay, requires large sample volumes, several incubation steps and has low detection sensitivity Tang and Hewlett, Conversely, nanotechnology and nanoparticles NPs use nanomaterials with length scale of 1— nanometers nm. Nanomaterials have unique biological properties such as small size, large surface-to-volume ratio, sharp melting temperature, magnetic properties, unusual target binding properties, and size based multi-coloring Qi and Wang, NPs such as gold particles have been used to improve assay sensitivity and specificity of antibodies, low limit of detection LOD , dose response over 10,fold and the detection sensitivity by 1,fold compared to ELISA Tang and Hewlett, Similarly, an essential factor of NPs selection is that nanometer-sized colloidal gold or silver particles can be conjugated with targeting ligands, including mAbs, peptides, or small molecules with functional and structural properties that are not available from either discrete molecules or bulk materials.
Nanoparticles are probed with mAbs that can be used to target any antigen of interest Ling et al. The use of colloidal gold is a rapid, less time consuming, and cost effective technique. This technology has been used in the development of immunochromatic strip assays based on mAb. The technique has widespread applications for the detection of a number of antigens exogenous antigens, endogenous antigens, autoantigens, neoantigens, viral antigens, and tumor antigens with high specificity and binding affinity Schumacher and Schreiber, Antigen preparation including quality and quantity is essential for the production of antibodies.
The purification of antigens is difficult and time consuming but antigen purity is vital for adequate immune responses Leenaars and Hendriksen, They show non-specific activity against the antigens of interest and specific activity against impurities. Higher concentrations of specific antibodies are obtained after purification and the exclusion of impurities by extensive absorption procedures such as antibody affinity chromatography Leenaars et al. Before scheduling immunization, antigen contamination should be considered by the researcher.
The antigen and diluents should be free of endotoxins such as lipopolysaccharide, or chemical residues that have been utilized to neutralize the microorganism. Additionally, the pH must be adjusted to prevent undesirable effects in the animal to be immunized Hendriksen and Hau, Moreover, sterile working conditions, antigen concentration, animal preparation, and injection quality must be confirmed.
These conditions are necessary to avoid suppression, sensitization, tolerance, or other superfluous immunomodulation and to induce effective immune responses. The inherent properties of antigen quantity include purification, animals to be immunized, type, and quality of the adjuvant to elicit high-titer serum responses , route and the immunization injection frequency Hanly et al. Antibody production involves the immunization or injection of laboratory animals with an immunogenic protein and test sampling of antiserum. The immunization is performed in specific animal species and the adjuvant is processed to form an immunogenic emulsion that is insoluble in water Leenaars and Hendriksen, Conjugation of smaller or less immunogenic antigens i.
After immunization, the animal is monitored daily or three times a week for side effects clinical and pathological examination. Severe pathological changes such as tissue reactivity, infection and anaphylactic reactions in case of booster injections have been reported in the absence of visual clinical or behavioral changes Leenaars et al. The immunization route is based on the choice of animal species, adjuvant, concentration and quality parameters of the antigen Apostolico Jde et al. Suitable immunization routes include subcutaneous s.
Blood sample handling should not interfere with the immunization site to avoid pain and distress to the animal while taking blood sample Leenaars et al. The i. The s. There is high risk of anaphylactic shock for booster injections by the i. The volume of the immunogenic mixture depends on the quality of the antigen and the degree of the lesions formed.
Therefore, smaller volumes of mixtures are injected to induce antibody responses except when increased concentrations of antigen are required Leenaars and Hendriksen, The immunization volume also depends on the animal species, route, and chemistry of the injection mixture. Booster injections have a significant effect on the outcomes of immunization and induction of B memory cells.
Class switching of B cells depends on the time interval between two consecutive injections. A small volume of antigen can be used for a booster injection without adjuvant Leenaars et al.
A booster injection is used when the antibody titer has reached a plateau or begins to decline. Antibody titers typically peak at 2—3 weeks after immunization in the absence of the depot-forming adjuvant used in the first immunization. A booster injection is used after 4 weeks in the presence of depot-forming adjuvant. FCA should be used in the first s. Hybridoma technology is essential for the production of high-quality mAbs as well as research and diagnostic reagents, and it is currently the most rapidly growing class of therapeutics Hnasko and Stanker, MAbs can be effectively screened by a number of techniques such as ELISA, phage display, and other related technologies.
Screening identifies and picks only specific antibody producing hybridomas. Subsequently, expansions of clonal populations are produced from serially diluted sub-cloned individual hybridoma cells in a microtiter plate. After screening, hybridoma cells are expanded in bigger wells or in culture flasks to maintain the hybridoma and provide sufficient cells for cryopreservation and supernatants for later investigations Nelson et al. However, this technique restricts the number of clones that can be screened to no more than a few thousand.
Consequently, insufficient numbers of cells remain available for clonal expansion and cell immortalization. Nevertheless, these techniques only screen for binding activity and do not allow functional assays El Debs et al. MAb characterization is based on a determination of its physicochemical and immunochemical character, heterogeneity, purity, impurities, biological activity, potency, and concentration Berkowitz et al.
For physicochemical properties, the isotype class, subclass, light chain composition and primary structure of the mAb are determined. Immunological properties include binding assay, affinity, avidity, immunoreactivity cross-reactivity with other structurally homologous proteins , analysis of CDR, epitope characterization, and determination of effector functions CHMP, Heterogeneity including chromatographic and electrophoretic methods and quantity are determined for the characterization of mAb purity, impurity, contaminants, and concentration.
Specificity of mAbs is also checked. Peptide map, anti-idiotype immunoassay, potency, and other appropriate methods are used to determine its identity and biological activity CHMP, MAb-based products exhibit superior specificity for a particular antigen. Diagnostic applications include biochemical analysis and imaging. It involves a number of immunoassays for the detection of hormonal, tissue, and cell products. Imaging is carried out using radiolabeled mAbs for diagnostics of infectious diseases.
Therapeutic mAbs have a wide range of applications. They are used in the treatment of cancer, transplantation of bone marrow and organs, autoimmune diseases, cardiovascular diseases, and various infectious diseases. Therapy can be carried out by direct use of mAbs as therapeutic agents and as targeting agents respectively Modjtahedi et al. Computational and bioinformatics approaches play an essential role for antibody selection and epitope prediction. Recently, a simulation tool C-ImmSim was developed for the study of a number of different immunological processes.
The processes include simulations of immune response by representing pathogens, as well as lymphocytes receptors, amino acid sequences and T and B cell epitope prediction Rapin et al. The first fully human mAb was developed over 25 years ago by phage display and a selection of antigen-specific binders from blood lymphocyte libraries Gavilondo and Larrick, This technique employed transgenic animals such as mice and rabbits with integrated human immunoglobulin Ig loci.
Germline-configured chimeric constructs confirmed that human, mouse, and all mammalian Ig loci function in very similar ways Neuberger and Bruggemann, Antibody development has progressed from hybridoma technology to a recombinant deoxyribonucleic acid DNA approach. MAb immunoglobulins IgG are the starting material for the generation of smaller antibody fragments in lymphoid or non-lymphoid cells Klimka et al. Traditional hybridoma technology has several limitations such as being exclusively murine based, time consuming, and exhibiting low-affinity in conventional assays.
Therefore, antibody engineering, display system, and immunomodulation methods are now used to produce efficient therapeutic antibodies Klimka et al. The first study of recombinant antibodies in bacteria was difficult because of interference from disoriented proteins in the bacterial cytoplasm. A new antibody expression technique was developed to produce smaller antibody molecules Fab or Fv fragments; Okamoto et al. This technique involves the expression of antibody fragments for recombinant antibody construction.
In this way, a number of genetically engineered antibodies have been constructed, such as Fab fragments, Fv fragments, single-chain variable fragments, bivalent antibodies, and bispecific antibodies Little et al. Fv is used for the construction of V L and V H domains or their modifications such as scFv, which is the most popular fragment. A Gly4Ser 3 linker is used for the stabilization of V L —V H and proper antigen binding site formation without the loss of antibody affinity.
Chelating recombinant antibodies CRAbs are scFv segments with a high binding affinity. These constructs consist of two scFv fragments specific to the identical antigen and adjacent epitopes. These fragments are connected by a short linker up to 10 amino acids for the dimerization and formation of diabodies Wright and Deonarain, ; Nelson, Complete antibody and various types of antibody fragments. These fragments are constructed by antibody engineering techniques for enhanced therapeutic applications. ScFv has high affinity, highly solubility, multi-domains, and high binding specificity with their target antigen and they are used for antibody engineering, biotechnology, cancer research, and biomedical applications.
ScFv have been engineered to improve the effector functions of full-length antibodies, carrying toxins to kill cells, or cytokines to activate the immune system. Furthermore, bispecific antibodies have been constructed to target multiple receptors AlDeghaither et al. Recombinant antibody engineering and recombinant DNA technology has facilitated successful expression and cloning of widespread antibody fragments in bacteria E. These smaller fragments have several advantages over full-length antibodies such as tumor and tissue penetration, blood clearance, short retention times, and reduced immunogenicity.
Likewise, they have better fusion in bacteria, and display on a filamentous phage. These fragments permit the production of homogenous proteins for diagnostic and therapeutic purposes as well as structural studies Frenzel et al. ScFv fusion proteins are constructed by the association of heavy V H and light chains V L of immunoglobulins via a short peptide linker.
Antigen specific scFv can be easily generated by phage display. These fusion proteins have extensive applications in cancer therapeutics such as in lymphatic invasion vessels, colon cancer, hepatocarcinoma, and diagnostics of human disease Tonelli et al. Moreover, scFv have been widely used with phage display panning i. Similarly, co-expression vector systems have been used to prevent and cure diseases by scFv.
Currently, various scFv fragments have been constructed against toxins and virulence factors of pathogens Tonelli et al. Additionally, a method for rapid and effective high-affinity GFP-based antibody production corresponding to scFv was developed. Skp co-expressing scFv has high solubility and binding activity to antigen thermolabile hemolysin TLH a pathogenic factor of Vibrio parahaemolyticus and was developed by using pACYC-Duet-skp co-expression vector. This scFv was constructed to detect TLH directly in real samples.
Phage-displayed antibody libraries have been widely used for the construction of high-affinity target-specific antibodies Chen et al. Moreover, antibody repertoires from phage-displayed libraries are constructed by harvesting messenger ribonucleic acids mRNAs from peripheral blood lymphocytes, hybridoma, spleen, bone marrow, tonsil, and similar other sources Chen et al.
Large libraries with a diverse range of antibodies and genes are created using reverse transcribed RT process into cDNA to function as a template for antibody gene amplification PCR Lim et al. Libraries are also created by PCR assembly, phagemid, and sequential cloning or combinatorial infection. The V H and V L chains are combined linker orientation dependent and cloned to construct a combinatorial scFv library for antigen selection Ahmad et al.
Another technique for recombinant antibody production is the utilization of phage recombinants displaying antibodies at their tips, and which undergo biopanning for the in vitro selection of scFv from large libraries of variable domains circumventing the traditional hybridoma method. Numerous scFv fragments have been constructed against haptens Wang et al. Phage display is a powerful biological technique for screening specific peptides or proteins.
Screening of antibody libraries by phage display permits the rapid selection of scFvs to isolate V H and V L chains for mAb transformation. Thus, therapeutic antibodies against noxious or highly conserved antigens, plasma membrane proteins, and receptors can be obtained in their native conformation while avoiding animal immunization Rader and Barbas, The peptide libraries are incubated on a plate coated with the antigen of interest.
Next, unbound phages are washed away. Numerous expression systems such as E. The expression and activation of scFv is performed by appropriate folding and in vitro refolding for aggregation. The expression systems for the production of active scFv antibody are selected, designed, and constructed based on hosts. The bacterial expression system is the most suitable and widely used method for the production of scFv antibody fragments compared to other available expression strategies Frenzel et al.
Additionally, it can be used for multi-plexed cloning, expression, and purification of proteins for structural genomics Rosano and Ceccarelli, Antibody characterization involves peptide mapping, glycan characterization analysis, purification, fragmentation of antibody pharmacokinetics, and quality assurance for many applications in basic research Roth et al. Biochemical characterization includes the expression of scFv antibody in a soluble form in infected E. In addition, further characterization is carried out by immunofluorescence antibody test IFAT , mass spectrometry, sequencing and indirect immunofluorescence IFI assays, cytotoxicity analysis, surface plasmon resonance, and NMR spectroscopy Wang et al.
Antibody engineering has become an aesthete discipline covering a wide range of production technologies. Moreover, techniques include the modification of clinically significant therapeutic drugs by antibody fragments especially for clinical imaging and to target multiple disease associated antigens Spiess et al. Fab antibody fragments are smaller with better tissue and tumor penetration than intact mAbs.
Fab lack a constant region and therefore, antibody effector functions Nelson, Moreover, Fab bind to specific antigens and are used in non-clinical studies e. This fragment has a 14 day serum half-life and is used to improve anti-tumor activity and to reduce immunogenicity Chames et al. The tumor necrosis factor TNF and interleukins 1 and 6 IL-1 and IL-6 proinflammatory cytokines cause multifactorial disease such as cancer and systemic inflammations.
Moreover, these factors are involved in redundancy of disease-mediation and crosstalk between signal cascades Arango Duque and Descoteaux, Similarly, upregulation of alternative receptors and pathway switching is often related to resistance to therapy Dong et al. The obstruction of several targets or multiple sites on one target is associated with improved therapeutic efficacy.
Over the past decade, dual targeting with bispecific antibodies has gradually switched to combinatorial or cocktail therapy. This targeting technique is based on the targeting of multiple disease-modifying reagents with one drug. The application of a single, bispecific molecule is advantageous because it is less complicated to administer to patients, requires reduced preclinical and clinical testing, and has cost effective manufacturing Kontermann and Brinkmann, Smaller antibody fragments permit in depth tissue penetration associated with the affinity of the antibody fragments.
Moreover, a high concentration of complete antibody restricts its ability to infiltrate tumors Weiner and Adams, Phage display is a selection technique for fusion proteins and phage coat proteins that are expressed on the phage surface. The bacteria are transformed with phagemid libraries, and then infected with a helper phage to assemble phage particles that express fusion proteins on their surface.
Various phage display systems. Gene pIII is represented as an orange box, the foreign DNA insert as a blue box, and the fusion products as a green circle. Filamentous bacteriophages used in phage display techniques are viruses that belong to the Inoviridae family. There are fewer of these filamentous phages in this genus compared with tailed phages. Inovirus virions are 7 mm in diameter, contain circular DNA enclosed in a protein capsid, and infect both Gram negative and positive bacteria.
They do not lyse host cells, instead, they are packed and extrude at the surface Marvin et al. The viruses enter host cells via pilli and are involved in genome replication, and virion structure, assembly and regulation Stassen et al. These viruses undergo extensive recombination, act as vectors phagemid for gene transfer and are closely related phenotypically and genotypically. Filamentous phages have three distinct classes, including M13, f1, fd, and M13 that infects E. This phage can be easily manipulated due to its small genome.
Therefore, these are the best-studied viruses and are extensively used in phage display technology. A number of in vitro techniques have been established for the development of antibodies in comparison with in vivo methods that involve animal immunization. PDT is a powerful tool for screening of specific recombinant protein binders against a large number of target antigens, including peptides, glycoproteins, glycolipids, saccharides, nucleic acids, and haptens. PDT is the most commonly used in vitro technology Bradbury et al.
Screening of the displayed protein by antigen in vitro is analogous to the selection of protein fragments in natural immunity Petrenko and Vodyanoy, Specific binder screening with biopanning allows the enrichment of the desired molecule Bazan et al. The first step is the incubation of the display library with an immobilized surface for example, microplate, magnetic beads, column matrix, PVDF membrane, or immunotubes of the entire cell. The non-binding phages are then removed by extensive washing and the binders are eluted by acid or salt buffer.
Then, binders are amplified using an appropriate bacterial host cell such as E. Finally, DNA sequencing of the primary structure is carried out to produce individual clones of the target protein Bazan et al. Schematic illustration of the biopanning technique. The target is attached to a phage library that is immobilized on a solid surface. Unbound phages are washed out, and specific phages are eluted and amplified. After several rounds of biopanning, the phages are analyzed to obtain diagnostic and therapeutic agents.
Many factors including proper biopanning design, type of immobilized surface, binding time, washing, and antigen concentration affect the level of selection and the screening of antibodies to unique epitopes Bazan et al. Numerous screening cycles are essential in biopanning to attain the preferred binding activity of the acquired monoclonal phage antibodies. The development of an antibody library from immunized animals has become obsolete because of the construction of diverse universal single-pot libraries for the isolation of numerous antigens.
PDT has many applications in biotechnology, production of recombinant multifunctional antibodies, cancer, immunotherapeutics, and the enhanced validity of protein fragments Yau et al. The study of epitopes and mimotopes in the interactions of antigen-antibody binding was the earliest application of PDT Wu et al. Mimotopes are miniscule peptides that mimic linear, intermittent, or non-peptide epitopes.
This laid the foundation of new molecular recognition techniques to determine protein folding, stability, structure-to-function relationships, and other related protein-protein interactions. The fusion of many ligands with phage particles has enrich phage displayed cDNA libraries significantly Vithayathil et al. Several novel molecular techniques have been established for screening functional molecules. These techniques include the identification of peptide agonists, receptor antagonists, the determination of binding specificity of domains, mapping of simple carbohydrates and functional epitopes, the identification of tumor inhibitor targets, and molecular imaging by fluorescently labeled phages Fukuda, Recently, PDT has been widely used in medical sciences for the production of a large number of humanized antibodies and the production of new therapeutics.
These antibodies have preclinical and clinical applications Rothe et al. A large number of antibody reagents are being developed for hematological applications such as cell subpopulation identification, directed therapeutics, and in vivo imaging. Anti-ABO, anti-Rh, and anti-Kell hemagglutination antibodies have been developed against red blood antigens Marks et al. These diabodies are being used for the treatment of neonatal alloimmune thrombocytopenia Watkins et al. Moreover, various antibody reagents have been raised against fetal red blood cells Huie et al. Additionally, this technique has helped the production of antibodies against dendritic cells, white blood cells WBC Fitting et al.
Human immune libraries developed by PDT facilitate the study of autoimmune and neurological disorder physiology, clinical diagnostics, and the treatment of AITP platelet disorder caused by anti-platelet autoantibodies , MS, myasthenia gravis MG [antibodies against nicotinic acetylcholine receptor AcChoR ], thrombotic thrombocytopenic purpura TTP , Cogan's syndrome CS caused by systemic vasculitis, acute anterior uveitis AAU , ocular inflammation, insulin dependent diabetes mellitus IDDM caused by the destruction of pancreatic beta cells, Wegener's granulomatosis Finnern et al.
Additionally, the technique has therapeutic uses in blistering skin diseases, pemphigus vulgaris PV Payne et al. Neurological disorders are treated by intracellular antibody fragments intrabodies , which are potentially therapeutic. Intrabodies select abnormal intracellular proteins. However, there are several limitations in the extracellular binding and internalization of DNA transfected by viral based vectors, lipofection or electroporation Jazi et al.
These are not efficient in vivo techniques and can alter cell viability. This problem can be overcome by fusing protein transduction domains PTD to antibodies Langedijk et al. They are also used for kuru disease, familial fatal insomnia by the accumulation of abnormal prion protein PrPSc Thanongsaksrikul and Chaicumpa, , Huntington's disease, and Parkinson's disease. Biopanning in vivo with phage display libraries has facilitated the isolation of peptides homing to all types of organs in the human body. Phage display is applied to stem cells for cell based regenerative medicine Gothard et al.
The binding of peptides with the extracellular domain of the LOX-1 receptor is associated with hypertension and atherogenesis Nixon et al. Other studies have reported that the homing of an RGD-motif-containing peptide to angiogenic vasculature was linked to a proapoptotic peptide and was successfully used for the treatment of collagen-induced arthritis in mice.
Phage libraries have also been used for anti-obesity, microparticle MP , avb3 integrin angiogenesis therapy, and in targeting vascular endothelial growth factor VEGF Cooke et al. Similarly, phage displays are used for tumor targeting agents e. This technique has replaced radiolabeled antibodies that have multiple disadvantages including reduced natural immunity Adachi et al. Furthermore, PDT has been used to isolate a number of peptides for molecular imaging. Its advantages are small size, rapid blood clearance, lack of immunogenicity, tissue penetration, and increased diffusion.
Numerous peptides for tumor targeting were isolated using human B cell lymphoma McGuire et al. Hybridoma technology is a well-established method for the generation of murine mAb cell lines by the fusion of splenocytes harvested from immunized mice with myeloma cells. The technology remains a feasible method for laboratories that implement basic cell biological research. Hybridoma technology is a comparatively simple procedure with minimal cost for the steady production of native whole immunoglobulins Tomita and Tsumoto, Nevertheless, this technology has various limitations such as antibodies produced by the hybridoma technique are strictly murine proteins that limits their therapeutic use in humans.
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Moreover, indefinite production costs, low fusion efficiency, limited number of mAbs, difficulty in developing mAbs against strictly conserved and toxin antigens and time consumption are other disadvantages Hnasko and Stanker, The production and amplification of antibodies in vitro using bacteria by PDT has a low turnaround time compared with other methods. Additionally, the library comprises of diverse variants up to 10 13 , which can be selected against a varied range of biological and inorganic targets Sblattero and Bradbury, The experimental conditions can be controlled, and the required equipment and libraries are available commercially.
Disadvantages include a difficult procedure and lack of antibodies displayed on the surface of each bacteriophage yielding a small number of mAbs Willats, Antibody engineering is a remarkable modification technique for production of highly specific and efficient antibody products. However, antibodies are bulky macromolecules that encompass challenges in construction, optimal pharmacokinetics, manufacturing, stability, and process development.
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Nonetheless, progress in antibody engineering technologies such as phage display, yeast display, bacterial display, and ribosomal or cell-free display continue to advance our capacity to rapidly screen and refine stable binding immunoglobins. These engineering techniques further improve biological properties significantly in the effector domains of the mAbs Filpula, Immunomodulatory techniques are persistently progressing to expand the clinical efficacy of therapeutic antibodies. Cell surface antigens exhibit a wide array of targets that are overexpressed, mutated or selectively expressed, and selected for modulated antibody-based therapeutics.
The technology functions through engineering alterations in antigen or receptor function, the immune system i. Immunomodulatory antibodies have gained significant clinical success Scott et al. The half-life can be extended by improving affinity of Fc for Fc neonatal receptor FcRn. Moreover, it can be prolonged by engineering pH-dependent antigen binding to enhance recycling of IgG via FcRn, and effective binding to the target molecule. Engineering the Fc region permits the development of molecules that are better suited to the pharmacology activity required of them Vincent and Zurini, ; Rath et al.
It involves modulation of constant Fc part of monoclonal human IgG1 hIgG1 antibodies to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics Grevys et al. Similarly, new opportunities have been created by the development of antibody-drug conjugates ADCs to treat the infectious diseases or target cancer cells. ADCs are being developed by progressing in antibody generation, selection of exceedingly cytotoxic molecules, and construction of stable linkers that can be investigated in clinical trials Vincent and Zurini, Cytotoxic therapeutic mAbs often help target cell-killing by eliciting immune effector functions.
Natural killer NK cells exhibit essential role in immunity in the context of mAb treatment by exerting direct cytotoxicity toward infected or tumor cells and contributing in modeling the adaptive response Cheng et al. Several T- or NK-cell modulators such as ipilimumab and nivolumab were approved for the treatment of metastatic melanoma Berman et al. Consequently, they enhance the therapeutic potential of mAbs. Several studies have established a link between activating Fc receptors and the efficacy of mAb therapy using mouse tumor models Romain et al.
Recently, glyco-engineering technique has been used to produce recombinant therapeutic proteins with optimized efficacy, half-life, specificity, and antigenicity. Glyco-engineering of expression platforms is progressively documented as an essential approach to advance biopharmaceuticals Ferrer-Miralles et al. The technique has been applied to in vivo expression systems that include mammalian cells, insect cells, yeast, and plants for the production of recombinant proteins. The underlined approaches aim at developing glycoproteins with homogeneous N- and O-linked glycans of defined composition Dicker and Strasser, Moreover, multi-level glyco-engineering techniques have been investigated to generate IgG with defined Fc-glycans in eukaryotic cells Dekkers et al.
Additionally, E. Antibodies are engineered with superior properties such as binding affinity, stability, and catalytic activity by several other display tools for example, yeast and bacterial display for broad spectrum of biotechnology, medicine, and biomedical applications. Yeast surface display exhibit development of recombinant antibodies by displaying on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein Cherf and Cochran, Yeast display technique has been used for engineering protein affinity, stability, and enzymatic activity.
Moreover, it is extensively applied in protein epitope mapping, identification of protein-protein interactions, and uses of displayed proteins in industry and medicine Cherf and Cochran, Several recombinant antibodies have been generated by yeast display for lethal infections such as highly pathogenic H5N1 avian influenza virus Lei et al. Similarly, several bacterial display systems have been established for Gram-negative bacteria and Gram-positive bacteria Lee et al. The display systems comprised of a carrier protein as an anchoring motif, a target protein, and a host strain.
Proteins developed for use as anchoring motifs include outer membrane proteins, lipoproteins, autotransporters, subunits of surface appendages, and S-layer proteins Han and Lee, Bacterial display has widespread applications including live vaccine development, screening-displayed peptide libraries, biosorbents, whole-cell biocatalysts, and biosensor development. Moreover, the promising technology is helping in the remediation of pollutants, biofuel production, and production of enantiomerically pure compounds Han and Lee, ; Ramanan et al.
Ribosome display is a cell-free display system, and a technique to perform entirely in vitro selection of proteins or peptides to bind desired ligand. Ribosome display consists of both prokaryotic and eukaryotic display systems Zahnd et al. Ribosome display allows synchronized isolation of a functional nascent protein, through affinity for a ligand together with the encoding mRNA.
The encoding mRNA is then transformed and amplified as DNA for further manipulation, including repeated cycles or protein expression. The advantages of ribosome display over other cell based methods include displaying very large libraries, generating toxic, proteolytically sensitive and unstable proteins, and incorporation of modified amino acids or mutations at distinct positions He and Taussig, ; Zahnd et al. Ribosome display systems have been investigated to identify potential antigens of Clonorchis sinensis Kasi et al. The large size of mAbs limits tumor penetration, and their long serum half-life is not suitable for therapy and imaging applications.
Therefore, antibody fragments have been constructed in various formats as they are small, monovalent, penetrate tumor tissues efficiently, and are rapidly eliminated by renal clearance Chames et al. Similarly, recombinant antibodies have several advantages: i bacteria, yeast, plants, or animals can be used to produce antibodies, ii no need for immunization, and iii intrinsic properties immunogenicity, binding affinity, pharmacokinetics, specificity, and stability of antibodies can be modified easily using mutagenesis techniques. Genetically engineered antibodies have integral characteristics that suit various downstream applications or can be converted into functional whole immunoglobulins Bradbury et al.
Antibodies exhibit strong immunity to defend against foreign antigens and non-self-agents. However, a variety of recombinant antibodies is needed to interact these hostile antigens. Over the last decade, the use of antibody engineering or recombinant antibody technology has shaped the genetic manipulation of a diverse range of antibody fragments for research, diagnosis, and therapy Kontermann and Muller, This technology has resulted in better affinity and specificity of manipulated antibody fragments and has facilitated the replacement of hybridoma technology with various display systems for unlimited antibody production against any known antigen Gram et al.
Conversely, engineered antibodies have various disadvantages such as they exhibit greater expense and complexity in manufacturing compared to antibodies developed by hybridoma technology Spiess et al. Due to their foreign nature, engineered therapeutic antibodies lead to allotypic immune responses that results in rapid clearance from body by kidney, elicit on T-cell help, and have reduced antibody avidity.
Moreover, engineered antibodies exhibit reduced half-life due to lack of an Fc domain and prevention of FcRn-mediated recycling. Likewise, antibody based therapies have more limitations based on the fact that many targets sometimes in low level have not yet been determined for various diseases Chames et al. In the s, von Behring and Kitasato worked on tetanus antitoxin that lead to the development of a new discipline, immunology.
They described antibodies for the first time and discovered that inactive toxins can elicit a protective immune response against active toxins in animals Kantha, The transfusion of serum from these protected animals elicited an immune response in other animals. Similarly, antibodies have remarkable applications in the field of diagnostics, therapeutics and targeted DDS.
They have been used to study various diseases such as cancer, metabolic and hormonal disorders, and infections caused by bacteria, viruses, fungi, algae, protozoa and other agents Sundar and Prajapati, Moreover, these biomolecules have numerous application in the diagnosis of lymphoid and myeloid malignancies. Likewise, immunoglobins are used in tissue typing, ELISA, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, analyzing a patient's antibody profile, and antiidiotype inhibition Siddiqui, The detection of antibodies against infectious agents and the construction of rapid and sensitive antibody-based immunodiagnostic test kits is an important part of basic and clinical research.
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The production of antibodies depends on specific protein titers, extent of immunity, and identification of B cell responses Burbelo et al. Accessibility of full length DNA sequences is useful for the rapid and robust systematic identification of antigens by recombinant proteins using protein arrays for complete proteome analysis Kierny et al.
Furthermore, numerous novel high-throughput dominant immunoassays are used to detect antibody responses against antigens Burbelo et al. ELISA is the most common method for the quantitation of pathological antigens. It is rapid, consistent, relatively easy to analyze, and adaptable to high-throughput screening Alonso et al.
Then, plates are blocked with irrelevant protein e. Additionally, addition of 0. In addition, the incubation conditions and reagent formulations should be preferably optimized based on the type of ELISA Grange et al. Antigen is attached to the solid phase followed by an enzyme-labeled antibody. This assay is used to detect various pathogenic antigens Brasino et al. Instead, a peroxidase enzyme-conjugated secondary antibody is added onto the first antibody. Specific antibodies to the analyte of interest are immobilized on a micro-titer plate.
Then, enzyme-conjugated antigen is incubated with capture antibody and the same antigen in its unconjugated form. This step yields a color following the addition of substrate. The signals produced are directly proportional to the quantity of conjugated enzyme bound and inversely proportional to the quantity of unconjugated antigen present Dupont-Deshorgue et al. The capture antibody is immobilized on a microtiter plate. Then, unknown or known samples are added into the matrix to minimize attachment to the solid phase. Peroxidase-enzyme labeled antibody is then added for coloration, which is directly proportional to the amount of antigen present Qu et al.
ELISA has been extensively used in the detection of various pathological antigens from viral, bacterial, fungal, protozoa, algal, and numerous other sources Thavaselvam and Vijayaraghavan, An improved ELISA has been used for detecting anti-melanoma differentiation-associated gene 5 MDA5 antibodies that are expressed in patients with dermatomyositis.
Western blot assay WBA is also called immunoblotting. This technique is used for the determination of molecular weight and amount of relevant proteins present in a sample. Proteins in a sample are first separated by electrophoresis and then transferred to a nitrocellulose or polyvinylidene difluoride PVDF membrane for the detection of bound primary proteins with antibodies specific to the protein of interest Ness et al. Nonspecific sites in the membrane are then blocked with BSA, non-fat milk powder, or casein. Finally, a labeled secondary antibody is added for detection by chemiluminescence or fluorescence Lewis et al.
WBA has been used for the confirmation of presence of purified proteins produced against various pathological antigens such as the lethal toxin of Clostridium sordellii Arya et al. Dot Blot DB assays are used to measure protein concentrations semi-quantitatively. It is slightly different from the WBA. Proteins in the sample are not separated by electrophoresis but are spotted directly on a membrane and hybridized with an antibody probe Emmerich and Cohen, This technique is cost effective and uses avidin-biotin technology with diaminobenzidine as a chromogen.
It is used for the analysis and quantitation of protein in cerebrospinal fluid CSF samples from cases of Creutzfeldt-Jakob disease CJD , and for disease control of other neurodegenerative diseases such as Alzheimer's disease AD and Parkinson's disease PD Subramanian et al. Immunohistochemistry IHC is used for the detection and identification of proteins and their localization in tissues.
It is essential to retain the morphology of tissues, cells and the availability of antigen sites. Fresh, rapidly frozen tissue sections are preferably used for IHC by chemically fixing tissues in formalin and embedding in wax Zhu et al. Additionally, fixing crosslinks amino acids in the tissue that block access to the epitope sites and prevent the action of any protein specific antibodies.
The exposure of hidden epitope regions is performed by digestion with an enzyme or by heat treatment, which removes endogenous peroxidase activity and non-specific sites are blocked. A labeled antibody or an unlabeled primary antibody specific to the protein of interest is used, followed by the addition of a secondary labeled antibody specific to the primary antibody Diorio et al. IHC has recently been used for determination and identification of expressed proteins such as lysosome-associated protein transmembrane-4 beta LAPTM4B associated with the prognosis of several human malignancies Xiao et al.
Immunoprecipitation IP is used for the study of protein-protein interactions, specific enzyme activity, posttranslational modifications, protein quantification, and determination of molecular weight of protein antigens. Rare proteins can be accumulated up to 10,fold by IP Dwane and Kiely, Flow cytometry FC is used to study antibodies on the cell surface and their related physiochemical properties. This technique was developed over 40 years ago Robinson et al.
Proteins produce fluorescence or scattered light when passed through the machine sensing point to generate quantitative data on a large number of cells. FC has been used for the visualization of pulmonary clearance Zhou et al. Fluorescence Activated Cell Sorting FACS is used for the detection of specific cells from a mixed population of cells based on their distinctive fluorescence or light scattering characteristics Yilmaz et al. The technique allows the isolation of cells by flow cytometry. In addition, the technology has been used for quantification of the cellular uptake of cell-penetrating peptides and mRNA Date et al.
Enzyme linked immunospot Elispot assay is used for monitoring cellular immune responses in humans and other animals Whiteside et al. It involves a polyvinylidene difluoride PVDF assisted microtiter plate pre-coated with antibodies specific to the antigen of interest. A capture antibody binds to the analyte of interest under precise conditions. Then, a biotinylated antibody specific to the analyte of interest is added to detect the original antibody after a wash to remove cell debris.
Finally, an enzyme labeled conjugate is added after a second wash to remove unbound antibody and to visualize a colored product. The product is typically a black spot representing a single cell that produces the antigen of interest Janetzki et al. This technique was used in the development of a coxsackievirus A16 neutralization test Hou et al.
Diagnostic applications include diagnosing sensitization to house dust mites Chang et al. The lateral flow immunochromatographic test LFT is a simple and cost effective device used to detect the presence or absence of a target antigen. It is widely used for medical diagnostics at home pregnancy test or in a laboratory testing. The technology involves the transportation of fluids e. The technique comprises various components and steps. A sample pad acts as a sponge and absorbs the fluids.
Then, fluids migrate to a conjugate pad containing protein conjugates immobilized on the surface of bio-active particles in a salt-sugar matrix that reacts with the antigen. Next, sample fluids dissolve the conjugate salt-sugar matrix and antibody-particles. The fluid mix flows through the porous structure causing the analyte to bind with particles while migrating further through the third capillary bed. Finally, there is a third capture molecule in striped areas that binds to the fluid mix containing analytes and the particle consequently changes color Yu et al.
LFTs can be used as a competitive or sandwich assay. Latex blue color , nanometer-sized particles of gold red color , or fluorescent or magnetic labeled particles are also used Seydack, The technique is qualitative, nevertheless, the quantity of analyte in a sample can be measured by the intensity of the test line color.
Progress in hybridoma technology and the production of highly specific mAbs has revolutionized the therapeutic use of antibodies for the diagnosis and cure of infections, the development of vaccines, antigenic characterization, and genetic manipulation. Antibodies have widespread applications in diagnostics, therapeutics and targeted DDS against potent pathogens, cancer, and physiological disorders Tiwari et al. They are used for the diagnosis of lymphoid and myeloid malignancies, tissue characterization, ELISA, radiolabeled immunoassay, and serotyping of microbes.
In addition, they are used in the diagnosis of immunological interpolation with passive antibodies, anti-idiotype suppression, or magic bullet treatments with cytotoxic agents coupled to anti-mouse specific antibodies Keller and Stiehm, Similarly, recombinant DNA technology rDNA has revolutionized the reconstruction of mAbs by genetic engineering using chimeric antibodies, humanized antibodies, CDR grafted antibodies for therapeutic use Dimitrov and Marks, ; Shen et al.
Molecular imaging provides a sensitive, non-invasive method for the molecular characterization of a cell surface phenotype for disease diagnosis and treatment. It is a rapidly growing multidiscipline that involves molecular biology, immunology, and medicine Massoud and Gambhir, The therapeutic applications of antibodies include clinical diagnosis and treatment in hematology, cardiology, immunology, autoimmunity, infectious diseases, and oncology Neves and Kwok, MAbs can be used as molecular imaging probes for investigating cell surfaces in vivo.
Coupling of cell surface targets with advances in antibody technology have facilitated the production of antibodies optimized for non-invasive imaging Manning et al. Antibodies select peptides according to the affinity of paratopes their combining sites from the libraries Forsstrom et al. Antibodies protect a host against invading microbes. These natural vaccines neutralize toxins and induce microbicidal effector functions. Antibody-mediated identification of pathogens during infection is essential for revealing immunoprotective responses in the host Sharon et al.
B cell epitope variable region of protective antibodies in contact with infectious antigens mapping is important for the development of effective vaccines in support of sero-diagnostics. This technique identifies protective epitopes for vaccine development and the estimation of conventional vaccines killed or attenuated pathogens; Malito et al. B and T cell epitope conformation is determined by pepscan technique, which has led to the development of antibody therapeutics, vaccine design, and recognition of protective antibody responses Ahmad et al.
The technique also targets pathogens with novel antimicrobials. Peptides are screened by biopanning with antibodies from the sera of various human diseases, including severe acute respiratory syndrome SARS , human papillomavirus HPV , and avian influenza viruses AIV. Moreover, peptide-based antigens are also used for serological diagnosis and development of vaccines Wu et al. The surface plasmon resonance SPR technique was first used in the year , for the epitope mapping of polysaccharides in L.
This technique screens molecular interactions in real-time with complex physical principles. SPR was used for the epitope mapping of vitamin B12 holo-transcobalamin , hemophilia disease, ricin toxin and the manganese transport protein C MntC of Staphylococcus sp. Phage display panning methods have failed to yield specific results because of the presence of parasitic phage clones that are often not removed by washing. To overcome these limitations, next generation sequencing NGS technology is used to sequence sub-libraries in biopanning experiments Naqid et al.
Next generation phage display NGPD can sequence 3, up to a million reads per panning round. NGPD has been used to identify target specific ligands by a single panning round using a library or ligand motif Ravn et al. This technique has widespread applications for the identification of thousands of ligands and mapping antibodies in response to infectious particles. Clinical targets and antigen-related ligands have been discovered with the help of genetically encoded peptide libraries. Correspondingly, ligands are selected using NGPD by the detection of low abundant copy clones without numerous rounds of selection.
Subsequently, NGS has facilitated the quantification of gene expression, genome assembly, and metagenome analysis. Antibodies are critical for immunity against infectious diseases, and extensively used for prevention and treatment of infection caused by bacteria, virus, and other infectious agents to improve public health.
Emil von Behring was awarded the first Nobel Prize in Physiology or Medicine in for his discovery of serum therapy for diphtheria. The emergence of recombinant technologies has revolutionized the selection, humanization, and development of therapeutic antibodies and permitted the strategic design of antibody-based elements for specificity and diversity.
A number of genetically engineered mAbs have been used for the treatment of numerous infectious diseases Hudson and Souriau, Ebola virus disease EVD is a severe, often fatal, zoonotic infection documented with most recent widespread outbreak in west Africa. It is caused by a virus of the Filoviridae family genus Ebolavirus. A recent study investigated the treatment of EVD patient with ZMapp, a buffy coat transfusion from an Ebola survivor, and the broad-spectrum antiviral GS Therapeutic antibodies have been generated to fight off several infectious cancers.
Cancer is a group of diseases that involve abnormal cell growth and proliferation caused by alterations, or mutations, in the genetic material of the cells. The cell outgrowths or lumps may be benign or metastatic Marusyk and Polyak, Cancer outpaces the immune system, avoids detection, or blocks immune system activity. Thus, numerous therapeutic antibodies such as necitumumab, ramucirumab, GRN, ganitumab, cixutumumab MM, BIIB , mapatumumab, trebananib, BHQ, and carlumab have been investigated for the successful treatment of human cancers.
The malignancies include NSCLC, metastatic gastric adenocarcinoma, brain metastasis, breast, ovarian, peritoneal, fallopian tube, prostate, pancreatic, and colorectal cancers, melanoma of the eye and liver, non-Hodgkin lymphoma, and multiple myeloma Dantas-Barbosa et al. Similarly, engineered antibodies have been used for the treatment of various types of arthritis. Therapeutic antibodies such as adalimumab, mavrilimumab, and GSK have been developed for the treatment of rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, and ankylosing spondylitis Dantas-Barbosa et al.
Therapeutic antibodies developed for the treatment of other infectious diseases include prophylaxis, anthrax, autoantibody-positive, lupus, angioedema, immune thrombocytopenic purpura, macular degeneration, hemophilia A, psoriasis, alzheimer, muscle loss and weakness, optic neuritis, ulcerative colitis, pulmonary fibrosis, and asthma Dantas-Barbosa et al. Prognosis of a disease is a complex process that involve deduction from several observable symptoms and the choice of treatment based on therapeutic effectiveness.
Antibodies, because of their exquisite specificity, and mAbs in general exhibit greater specificity. Therefore, they are used widely in a variety of assay formats, in the diagnosis and treatment of infectious diseases Zola and Thomson, Moreover, they show rapid identification of new or rare infectious agents that is an important public health measure. Shortly, development of novel mAbs are helping to monitor and lessen the likelihoods of epidemics and other disease threats imposed on human health by prevalent infectious agents Zola et al.
In the past few decades, antibodies have been developed by using conventional techniques such as hybridomas with significant applications in therapeutics. However, more recently, there have been advancements in recombinant technologies that have enhanced the generation of efficient immunoglobulins and their fragments. This powerful technology is used in a variety of systems to approach different questions from a background of cell biology and biotechnology.
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Moreover, it is used for the production of different types of engineered antibodies against any target molecule or highly unique conserved antigens. Additionally, it is facilitating in diagnosis and treatment of infections to improve human health. Antibody engineering possesses broad spectrum of biological, biotechnological, medical, and antibody applications for the development of novel therapeutics in various disease fields. Numerous novel therapeutic drugs have been developed by in vitro screening and selection techniques based on several high throughput immune effector functions, engineered antibodies, and high-affinity antibody fragments.
This review has comprehensively described hybridoma technology, advances in antibody engineering techniques, engineered antibodies, antibody fragments, display technologies, and applications of antibodies. In conclusion, the study will help in understanding the significance of antibody fabrication approaches with extensive uses in molecular, immunological, diagnostic, imaging, biomedical, and biotechnological fields, pursuing a healthier future for humans.
AS and SW wrote the paper and all the authors approved its submission. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. National Center for Biotechnology Information , U. Journal List Front Microbiol v. Front Microbiol. Published online Mar Abdullah F. Author information Article notes Copyright and License information Disclaimer.
This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology. Received Jan 26; Accepted Mar 9. The use, distribution or reproduction in other forums is permitted, provided the original author s or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. This article has been cited by other articles in PMC. Abstract Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods.
Keywords: antibody engineering, hybridoma technology, antibody fragments, scFv, phage display technology, immunomodulation, immunology. Introduction In recent years, the development of polyclonal and monoclonal antibody by means of laboratory animals has become a vital approach to protect against a number of pathogenic contagions Marasco and Sui, Polyclonal antibody Antigen interactions are essential for the normal functions of antibodies that are widely used in research or therapeutics. Open in a separate window.
Figure 1. Monoclonal antibody Monoclonal antibodies mAbs are clinically significant homogeneous and mono-specific scientific biomolecules produced from hybridoma cells by hybridoma technology Zhang, Table 1 Monoclonal antibody products in the US, Europe, and global markets approved for diseases. Eli Lilly and Co. Takeda Pharmaceutical Co. Method of antibody production Hybridoma technology has been a significant and essential platform for producing high-quality mAbs Zhang, Figure 2.
Structure Fold. Cortez-Retamozo, V. Lauwereys, G. Hassanzadeh Gh, M. Gobert, K. Conrath, S. Muyldermans, P. De Baetselier, and H. Revets Efficient tumor targeting by singledomain antibody fragments of camels. Reiter, Y. Schuck, L. Boyd, and D. Plaksin An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface. Huston, J. Levinson, M. Mudgett-Hunter, M. Tai, J. Movotny, M. Margolies, R. Ridge, R. Bruccoleri, E. Haber, and R. Crea Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.
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Methods 43— Sauer Optimizing the stability of single-chain proteins by linker length and composition mutagenesis. Carter, P. Kelley, M. Rodrigues, B. Snedecor, M. Covarrubias, M. Velligan, W. Wong, A. Rowland, C. Kotts, M. Carver, and et al. Biotechnology — McCafferty, J. Griffiths, G. Winter, and D. Chiswell Phage antibodies: filamentous phage displaying antibody variable domains.
Hoogenboom, H. Griffiths, K. Johnson, D. Chiswell, P. Hudson, and G. Winter Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody Fab heavy and light chains. Nucleic Acids Res. Garrard, L. Yang, M. O'Connell, R. Kelley, and D. Henner Fab assembly and enrichment in a monovalent phage display system. Biotechnology 9: — Chang, C.
Landolfi, and C. Queen Expression of antibody Fab domains on bacteriophage surfaces. Potential use for antibody selection. Google Scholar. McGuinness, B. Walter, K. FitzGerald, P. Schuler, W. Mahoney, A. Duncan, and H. Hoogenboom Phage diabody repertoires for selection of large numbers of bispecific antibody fragments. Nature Biotechnol. Holt, L. Enever, R. Tomlinson The use of recombinant antibodies in proteomics.
Knappik, A. Ge, A. Honegger, P. Pack, M. Fischer, G. Wellnhofer, A. Hoess, J. Wolle, A. Pluckthun, and B. Virnekas Fully synthetic human combinatorial antibody libraries HuCAL based on modular consensus frameworks and CDRs randomized with trinucleotides. Hanes, J. Schaffitzel, A. Knappik, and A.
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