Antibody Molecules Research Articles

Construction of a highly sensitive immunosensor based on antibody immunoglobulin G/3-(trimethoxysilyl) propylamine/graphene oxide for antigen-specific immunoglobulin G detection

The aim of this study is to develop an immunosensor that has the ability to detect antigen G with properties such as very low detection of limit, remarkable with no bifunctional crosslinking species and excellent activity at physiological pH values. Immunoglobulin G (IgG) is found in all body fluids. When a specific bond occurs between the anti-immunoglobulin G and the immunoglobulin G (anti-IgG), the result is activation of the reaction against viral and bacterial diseases. For detection and determination of antibody–antigen (Rabbit IgG–AntiRabbit IgG) interactions, a novel electrochemical immunosensor based on antibody immunoglobulin G/3-(trimethoxysilyl)propylamine/graphene oxide/glassy carbon (Ab/TMSPA/GO/GCE) was fabricated by using of carboxylic groups (–COOH) on graphene oxide (GO) and by exploiting 3-(trimethoxysilyl)propylamine (TMSPA) to covalently conjugate antibody molecules. For characterization of the modification process of the surface of glassy carbon electrode(GCE), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used. The change of impedance response was used for the monitoring of the interactions of antibody with different concentrations of antigen. Based on the results obtained, with increasing concentrations of antigen, the electron transfer resistance increased. The limit of detection (DL) and linear range 400 ng mL−1 and 2–1500 µg mL−1 were obtained.

Creation of Multimeric Single-Domain Antibodies Using Bacterial Superglues.

Multimerization of single-domain antibodies (sdAbs) is instrumental for construction of antibody molecules with high avidity, extended in vivo half-life, and tailor-made biological activity. Two-component superglues, based on bacterium-derived peptides (Tags) and small protein domains (Catchers) that form isopeptide bonds when in close proximity, enable the creation of multimers by simply mixing of the individual components. Here, we provide detailed methods for the construction of sdAbs and scaffolds bearing genetically fused superglue components and their assembly into multimeric complexes.

Development of a potent high-affinity human therapeutic antibody via novel application of recombination signal sequence–based affinity maturation

Therapeutic antibody development requires discovery of an antibody molecule with desired specificities and drug-like properties. For toxicological studies, a therapeutic antibody must bind the ortholog antigen with a similar affinity to the human target to enable relevant dosing regimens, and antibodies falling short of this affinity design goal may not progress as therapeutic leads. Herein, we report the novel use of mammalian recombination signal sequence (RSS)–directed recombination for complementarity-determining region–targeted protein engineering combined with mammalian display to close the species affinity gap of human interleukin (IL)-13 antibody 731. This fully human antibody has not progressed as a therapeutic in part because of a 400-fold species affinity gap. Using this nonhypothesis-driven affinity maturation method, we generated multiple antibody variants with improved IL-13 affinity, including the highest affinity antibody reported to date (34 fM). Resolution of a cocrystal structure of the optimized antibody with the cynomolgus monkey (or nonhuman primate) IL-13 protein revealed that the RSS-derived mutations introduced multiple successive amino-acid substitutions resulting in a de novo formation of a π–π stacking–based protein–protein interaction between the affinity-matured antibody heavy chain and helix C on IL-13, as well as an introduction of an interface-distant residue, which enhanced the light chain–binding affinity to target. These mutations synergized binding of heavy and light chains to the target protein, resulting in a remarkably tight interaction, and providing a proof of concept for a new method of protein engineering, based on synergizing a mammalian display platform with novel RSS-mediated library generation.

Functional Magnetic Microdroplets for Antibody Extraction

AbstractAntibodies play an essential role in modern medicine for diagnostic and therapeutic applications. Even though the production of antibodies is the fastest growing pharmaceutical industry area, the cost of antibodies remains high, which limits access to antibody‐based medicine both in developing and developed countries. The bottleneck and major cost factor in the production is purification of the antibody. Here, a proof‐of‐concept is presented for antibody extraction using ferrofluid microdroplets. An external magnetic field splits oil‐based ferrofluid droplets into an array of daughter microdroplets, which serve as a magnetically tunable, hydrophobic, liquid substrate with a relatively large surface area. A fusion protein (HFBI‐Protein A), added to the solution surrounding the magnetic droplets, adsorbs strongly at the liquid‐liquid interface by the hydrophobin HFBI moiety, creating a bifunctional monolayer that can catch antibody molecules. After adsorption at the liquid‐liquid interface, these antibody molecules can be released by decreasing the pH of the solution. The antibody extraction process is investigated using confocal microscopy and gel electrophoresis. In addition, the effect of HFBI on the field‐induced ferrofluid droplet splitting is examined. This study provides a proof of concept for utilizing liquid‐liquid instead of a solid‐liquid system in antibody handling.

Further preclinical development of a clinically effective bio‐therapeutic against Porphyromonas gingivalis

AbstractBackgroundThe ever increasing list of broad adverse medical conditions associated with Porphyromonas gingivalis (Pg) infection associated with the long term, oral, biofilm‐associated colonization in humans leading to a state of chronic systemic inflammation with multiple organ system diseases (atherosclerosis, cardiovascular, stroke, diabetes type 2/metabolic syndrome, cancer, Alzheimer, etc.), stands in stark contrast to a grossly lacking set of effective treatments and/or prophylactic interventions.MethodUsing multiple technologies in microbiology, protein chemistry, proteomics, molecular biology, nucleic acid and protein sequencing, immunochemistry, scanning electron microscopy, biologics development, mid and large scale up hybridoma monoclonal antibody production and human chimeric antibody development and enzymology, an updated status of a newly developed precision biological will be presented.ResultKB‐001 a murine IgG1 binds directly to a unique hetero‐multimer antigen involved in the bacterial cargo IX transporter secretion protein complex through a high affinity bi‐valent binding (kD 10‐8‐9). About 40‐60 antibody molecules bind to emerging OMVs per bacterial are observed. In addition isolated OMVs demonstrate binding to the outer and inner membranes. Mechanism of action studies demonstrate that the antibody interferes with the proteolytic processing of the larger parent protein required for subsequent endo‐peptidase activity and assembly. More specifically, the binding of antibody to this complex prevents the maturation of the gingipains/LPS endo‐protease/peptidase system‐needed for its absolute survival and the production of its secreted outer membranes vesicles (OMVs) responsible for the majority of its systemic multi‐systems pathology. In an earlier human clinical trial, following its topical oral administration in advanced Pg infected periodontal patients, long term (12 months) prevention of re‐colonization of P. gingivalis following standard dental therapy was observed. In addition, the paratope binding domain from this murine Mab was successfully grafted onto a human IgG1 framework thus creating a second generation human‐chimeric, bio‐therapeutic antibody.ConclusionThese data now demonstrate the two precision bio‐therapeutics could be valuable for treating and/or preventing P. gingivalis infection in humans.

Novel electrogravimetric biosensors for the ultrasensitive detection of plasma matrix metalloproteinase-2 considered a potential tumor biomarker

In this study, we developed novel, simple gravimetric and voltammetric sensors for the ultrasensitive detection of active matrix metalloproteinase (MMP)-2 in plasma. The developed sensors are cost-effective, require a very less amount of reagents, and are time-saving. They detect MMP-2 based on antigen–antibody recognition and its ability to cleave glycine-leucine peptide bond. The three-dimensional bioplatform of the sensors consisted of a cationic polyethyleneimine (PEI) polymer that facilitated robust immobilization of the dipeptide labeled with anthraquinone (AQ), or antibody molecules in appropriate density, which was crucial for biosensing. Detection was performed using quartz crystal microbalance with dissipation and voltammetry. The results showed that the developed sensors were characterized by high stability, wide analytical range (2.0 pg mL−1 to 5.0 μg mL−1), and low detection limit (ca. 10 fg mL−1). They also exhibited excellent efficiency in the determination of active MMP-2 in real samples, such as blood plasma. The developed sensors may hold great promise for the early diagnosis of cancers.

Multifunctional Gold Nano-Cytosensor With Quick Capture, Electrochemical Detection, and Non-Invasive Release of Circulating Tumor Cells for Early Cancer Treatment.

Circulating tumor cells (CTCs) are metastatic tumor cells that shed into the blood from solid primary tumors, and their existence significantly increases the risk of metastasis and recurrence. The timely discovery and detection of CTCs are of considerable importance for the early diagnosis and treatment of metastasis. However, the low number of CTCs hinders their detection. In the present study, an ultrasensitive electrochemical cytosensor for specific capture, quantitative detection, and noninvasive release of EpCAM-positive tumor cells was developed. The biosensor was manufactured using gold nanoparticles (AuNPs) to modify the electrode. Three types of AuNPs with controllable sizes and conjugated with a targeting molecule of monoclonal anti-EpCAM antibody were used in this study. Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) of the cytosensors were performed to evaluate the cell capture efficiency and performance. The captured 4T1 cells by the AuNPs hindered electron transport efficiency, resulting in increased EIS responses. The cell capture response recorded using EIS or DPV indicated that the optimal AuNPs size should be 17 nm. The cell capture response changed linearly with the concentration range from 8.0 × 10 to 1 × 107 cells/mL, and the limit of detection was 50 cells/mL. After these measurements, glycine-HCl (Gly-HCl) was used as an antibody eluent to destroy the binding between antigen and antibody to release the captured tumor cells without compromising their viability for further clinical research. This protocol realizes rapid detection of CTCs with good stability, acceptable assay precision, significant fabrication reproducibility with a relative standard deviation of 2.09%, and good recovery of cells. Our results indicate that the proposed biosensor is promising for the early monitoring of CTCs and may help customize personalized treatment options.

Application of Single-Domain Antibodies ("Nanobodies") to Laboratory Diagnosis.

Antibodies have proven to be central in the development of diagnostic methods over decades, moving from polyclonal antibodies to the milestone development of monoclonal antibodies. Although monoclonal antibodies play a valuable role in diagnosis, their production is technically demanding and can be expensive. The large size of monoclonal antibodies (150 kDa) makes their re-engineering using recombinant methods a challenge. Single-domain antibodies, such as “nanobodies,” are a relatively new class of diagnostic probes that originated serendipitously during the assay of camel serum. The immune system of the camelid family (camels, llamas, and alpacas) has evolved uniquely to produce heavy-chain antibodies that contain a single monomeric variable antibody domain in a smaller functional unit of 12–15 kDa. Interestingly, the same biological phenomenon is observed in sharks. Since a single-domain antibody molecule is smaller than a conventional mammalian antibody, recombinant engineering and protein expression in vitro using bacterial production systems are much simpler. The entire gene encoding such an antibody can be cloned and expressed in vitro. Single-domain antibodies are very stable and heat-resistant, and hence do not require cold storage, especially when incorporated into a diagnostic kit. Their simple genetic structure allows easy re-engineering of the protein to introduce new antigen-binding characteristics or attach labels. Here, we review the applications of single-domain antibodies in laboratory diagnosis and discuss the future potential in this area.

New Model to Predict the Concentration-Dependent Viscosity of Monoclonal Antibody Solutions.

Concentrated monoclonal antibody solutions exhibit high solution viscosity, which is experimentally measured to be ∼1-2 orders of magnitude higher than the viscosity of water. However, physical processes responsible for the high antibody viscosity are not fully understood. We show that fluid occlusion due to the trapped solvent molecules within the boundaries formed by the aggregated antibodies is responsible for the elevated solution viscosity. We develop a theory to predict the viscosity of monoclonal antibodies based on the geometry of the antibody molecule and the aggregate morphology. We validate our theory with experiments and highlight useful insights obtained from the viscosity equation which could help in controlling the drug viscosity at the molecular design stage itself.

Activation of Cascade-Like Antitumor Immune Responses through In Situ Doxorubicin Stimulation and Blockade of Checkpoint Coinhibitory Receptor TIGIT.

Although T cell centered immune checkpoint blockade (ICB) therapies have shown unprecedented success in various cancer types, only a minority of patients benefit from these treatments due to the lack of neoantigen burden and exhaustion of tumor-infiltrating immune stimulating cells. Inspired by the crucial role of NK cell based immunity and potential immunostimulating effect of chemotherapeutic drugs, the therapeutic efficiency on tumor inhibition through combination of doxorubicin (DOX) and blockade of TIGIT (T-cell Ig and ITIM domain), a coinhibitory receptor expressed by both NK and T cells, in a matrix metalloproteinase 2 (MMP-2)-degradable hydrogel is thoroughly evaluated in this study. Due to the distinct release kinetics from destructed framework of hydrogels, the differentially released DOX and anti-TIGIT monoclonal antibody (aTIGIT) molecules can elicit immunogenic tumor microenvironment and reverse the exhaustion of NK and effector T cells to realize not only durable localized tumor inhibition but also systemic and long-lasting immune memory responses. Thus, this work pioneers the union of chemotherapeutic drugs and NK cell centered ICB therapies for activating cascade-like antitumor immune responses through eliciting immunogenic tumor microenvironment and boosting both innate and adaptive immunity.

An Ultrasensitive Virus ELISA Based on a magnetic Mesoporous Silica Nanoprobe

AbstractDetection of infectious viruses relies on quantitative polymerase chain reaction (qPCR). However, qPCR requires costly equipment, a clean operating environment and experienced technicians, limiting its wide applicability. On the other hand, enzyme‐linked immunosorbent assay (ELISA) is widely used in biological laboratories due to its relatively high sensitivity and ease of operation. However, ELISA‐based detection of the virus is hampered because it is lower sensitive than qPCR. Herein, a nanoprobe ELISA (NP‐ELISA) based on a mesoporous silica nanoprobe, which is constructed by first being loaded with peroxidase and further coated with positively charged polymer polyethyleneimine, and finally functionalized with antivirus antibodies, is designed. Results show that each NP probe is encapsulating 170 peroxidase molecules and presents 200 antibody molecules on the surface. The limit of detection (LOD) of NP‐ELISA (LOD = 1450 PFU mL−1) for the detection of real virus samples is tenfold sensitive than that of standard ELISA (LOD = 14, 414 PFU mL−1) and the assay time for NP‐ELISA is reduced by 1 h as compared with standard one. Therefore, the NP‐ELISA provides a rapid and sensitive immunoassay platform that can readily be implemented for biological laboratory research as well as for on‐site clinical diagnostics.

1540 Genome-Wide Association Analysis In 401,583 Individuals Identifies Novel Therapeutic Targets for Haemorrhoids

Abstract Aim To elucidate the genetic architecture of haemorrhoids and identify genes and biological pathways central to their pathobiology. Method We report the first ever genome-wide association study of haemorrhoids in 31,652 cases and 369,931 controls from UK Biobank. Genes and biological pathways were prioritised using several bioinformatic approaches, and potential therapeutic targets were identified in the Open Targets Platform. A weighted genetic risk score (wGRS) for haemorrhoids was constructed to compare genetic susceptibility in surgical vs non-surgical haemorrhoids patients. Results Twelve novel genome-wide significant susceptibility loci were discovered to be associated with haemorrhoids. Seventeen genes were mapped to these loci, and gene sets in biological pathways relating to extracellular matrix regulation and TGF-β signalling were strongly implicated. Seven gene-products (41.2%) were predicted tractable to antibody and/or small molecule targeting, and three products (17.6%) have known pharmaceutical interactions (ACHE, ADRA2B, ELN). The wGRS analysis demonstrated that haemorrhoid patients requiring surgery have a higher inherent genetic susceptibility than those managed non-surgically (P = 4.58 × 10-27). Conclusions This study has advanced our understanding of haemorrhoids pathobiology with the identification of several biologically plausible genes and pathways, many of which demonstrate strong therapeutic potential. The wGRS correlated with disease severity, representing a first step in personalised medicine approaches to haemorrhoids.

Identification and Characterization of a Monoclonal Antibody Variant Species with a Clipping in the Complementarity Determining Region Isolated by Size Exclusion Chromatography Under Native Conditions.

The content of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Peptide bonds in the hinge region of mAbs are susceptible to hydrolysis, generating Fc-Fab fragments, which are associated with lower efficacy than the intact antibody. Fc-Fab fragments can be separated from intact antibody molecules under native conditions by size exclusion chromatography (SEC). Although several fragments generated by a clip in the complementarity determining region (CDR) have been reported, their efficacies have not been analyzed. This is because these fragments could not be separated from intact antibodies under native conditions owing to their similar molecular sizes. Here, we report that bevacizumab variant with clipping in the CDR, with the resulting fragments remaining intact in the variant, can be isolated under native conditions by selecting an adequate SEC column.

Ultrasensitive capacitance sensor to detect amyloid-beta 1-40 in human serum using supramolecular recognition of β-CD/RGO/ITO micro-disk electrode

In this paper, we developed a new ultrasensitive capacitance sensor for detection of amyloid beta 1-40 (aβ40) protein (one of Alzheimer's disease core biomarkers) in human serum based on the high supramolecular recognition of the β-cyclodextrin/reduced graphene oxide (β-CD/RGO) nanohybrid toward the anti-aβ40 antibody molecule. The sensor was established by immobilizing specific anti-aβ40 antibody onto the β-CD/RGO nanohybrid functionalized on indium tin oxide micro-disk electrode (anti-aβ40/β-CD/RGO/ITO). Detection of aβ40 in the human serum (HS) using the sensor anti-aβ40/β-CD/RGO/ITO is carried out by capacitance measurement without a redox probe to prevent protein denaturation, serving as a convenient strategy for point-of-care diagnosis. In comparison with other studies, the sensor shows a very low limit of detection of 0.69 fg mL−1 in HS, demonstrating its ability for the ultrasensitive detection of aβ40. Using this sensor, the dissociation constant KD of the binding interaction between anti-aβ40 and aβ40 in HS is found to be 2.9 × 10−7 nM, indicating the high binding affinity of antibody–antigen and the suitability of the anti-aβ40/β-CD/RGO/ITO sensor for aβ40 protein detection. The good selectivity of the anti-aβ40/β-CD/RGO/ITO sensor in the presence of differential analytes was also performed in this paper.

Helicase antigen (HAGE)-derived vaccines induce immunity to HAGE and ImmunoBody®-HAGE DNA vaccine delays the growth and metastasis of HAGE-expressing tumors invivo.

The management of patients with triple-negative breast cancer (TNBC) continues to pose a significant clinical challenge. Less than 30% of women with metastatic TNBC survive 5years, despite adjuvant chemotherapy and the initial higher rates of clinical response that can be achieved with neoadjuvant chemotherapy. ImmunoBody is a plasmid DNA designed to encode a human antibody molecule with complementarity-determining regions engineered to express cytotoxic and helper T-cell epitopes derived from the cancer antigen of interest. The helicase antigen (HAGE) is a cancer testis antigen, which is expressed in TNBC. Herein, we have identified a 30-amino-acid-long HAGE-derived sequence containing human leukocyte antigen (HLA)-A2- and HLA-DR1-restricted epitopes and demonstrated that the use of this sequence as a peptide (with CpG/incomplete Freund's adjuvant) or incorporated into an ImmunoBody vaccine can generate specific interferon-γ-secreting splenocytes in HHDII+ DR1+ mice. T-cell responses elicited by the ImmunoBody-HAGE vaccine were superior to peptide immunization. Moreover, splenocytes from ImmunoBody-HAGE-vaccinated mice stimulated invitro could recognize HAGE+ tumor cells and the human TNBC cell line MDA-MB-231. More importantly, the growth of implanted HHDII+ DR1+ HAGE+ Luc+ B16 cells.

Venomics of the poorly studied hognosed pitvipers Porthidium arcosae and Porthidium volcanicum

We report the first proteomics analyses of the venoms of two poorly studied snakes, the Manabi hognosed pitviper Porthidium arcosae endemic to the western coastal province of Manabí (Ecuador), and the Costa Rican hognosed pitviper P. volcanicum with distribution restricted to South Pacific Costa Rica and western Panamá. These venom proteomes share a conserved compositional pattern reported in four other congeneric species within the clade of South American Porthidium species, P. nasutum, P. lansbergii, P. ophryomegas, and P. porrasi. The paraspecific immunorecognition profile of antivenoms produced in Costa Rica (ICP polyvalent), Perú (Instituto Nacional de Salud) and Brazil (soro antibotrópico pentavalente, SAB, from Instituto Butantan) against the venom of P. arcosae was investigated through a third-generation antivenomics approach. The maximal venom-binding capacities of the investigated antivenoms were 97.1 mg, 21.8 mg, and 25.7 mg of P. arcosae venom proteins per gram of SAB, ICP, and INS-PERU antibody molecules, respectively, which translate into 28.4 mg, 13.1 mg, and 15.2 mg of total venom proteins bound per vial of SAB, ICP, and INS-PERU AV. The antivenomics results suggest that 21.8%, 7.8% and 6.1% of the SAB, ICP, and INS-PERU antibody molecules recognized P. arcosae venom toxins. The SAB antivenom neutralized P. arcosae venom's lethality in mice with an ED50 of 31.3 mgV/g SAB AV. This preclinical neutralization paraspecificity points to Brazilian SAB as a promising candidate for the treatment of envenomings by Ecuadorian P. arcosae. Biological significanceAssessing the preclinical efficacy profile of antivenoms against homologous and heterologous medically relevant snake venoms represents an important goal towards defining the biogeographic range of their clinical utility. This is particularly relevant in regions, such as Mesoamerica, where a small number of pharmaceutical companies produce antivenoms against the venoms of a small number of species of maximum medical relevance among the local rich herpetofauna, leaving a wide range of snakes of secondary medical relevance, but also causing life-threatening human envenomings without nominal clinical coverage. This work is part of a larger project aiming at mapping the immunological characteristics of antivenoms generated in Latin American countries towards venoms of such poorly studied snakes of the local and neighboring countries' herpetofauna. Here we report the proteomics characterization of the Manabi hognosed pitviper Porthidium arcosae endemic to the western coastal province of Manabí (Ecuador), and the Costa Rican hognosed pitviper P. volcanicum with distribution restricted to southwestern Costa Rica, the antivenomics assessment of three bothropoid commercial antivenoms produced in Costa Rica, Perú, and Brazil against the venom components of P. arcosae, and the in vivo capacity of the Brazilian soro antibotrópico pentavalente (SAB) from Instituto Butantan to neutralize the murine lethality of P. arcosae venom. The preclinical paraspecific ED50 of 31.3 mg of P. arcosae venom per gram of antivenom points to Brazilian SAB as a promising candidate for the treatment of envenomings by the Manabi hognosed pitviper P. arcosae.

A Fully Integrated Online Platform For Real Time Monitoring Of Multiple Product Quality Attributes In Biopharmaceutical Processes For Monoclonal Antibody Therapeutics

In response to FDA's call for Quality by Design (QbD) in biopharmaceutical product development, the biopharmaceutical industry has been developing highly sensitive and specific technologies in the monitoring and controlling of product quality attributes for bioprocesses. We previously published the successful application of an off-line multi-attribute method (MAM) to monitor more than 20 critical quality attributes (CQA) with superior sensitivity for the upstream process. To further remove the hurdles of laborious process sampling and sample preparation associated with the offline method, we present here a fully integrated MAM based online platform for automated real time online process monitoring. This integrated system includes Modular Automated Sampling Technology (MAST) based aseptic sampling, multi-function Sequential Injection Analysis (SIA) sample preparation, UHPLC separation and high-resolution mass spectrometry (HRMS) analysis. Continuous automated daily monitoring of a 17-day cell culture process was successfully demonstrated for a model monoclonal antibody (mAb) molecule with similar specificity and sensitivity as we reported earlier. To the best of our knowledge, this is the first report of an end-to-end automated online MAM system, which would allow the MAM to be applied to routine bioprocess monitoring, potentially replacing multiple conventional low resolution and low sensitivity off-line methods. The online HPLC or HPLC/MS platform could be easily adapted to support other processing steps such as downstream purification with minimal software re-configuration.

Use of Intravitreal Bevacizumab Injection among Patients Undergoing Surgical Retinal Interventions at Tertiary Eye Hospital: A Descriptive Cross-sectional Study.

Intravitreal Bevacizumab injection has now become a routine procedure for retina specialists throughout the world. Easy availability of this monoclonal antibody molecule even in Nepal has brought a revolution in the management of various retinal diseases. This study aims to find out the prevalence of the use of intravitreal Bevacizumab for retinal diseases at the tertiary eye hospital. This descriptive cross-sectional study was carried out in the retina department at a tertiary care hospital from January 2017 to December 2019 after obtaining ethical clearance from Nepal Health Research Council (Ref: 125/2020P). The sample size was calculated and the study enrolled all patients who received intravitreal Bevacizumab for retinal diseases using convenience sampling technique. Data were analyzed using Statistical Package for Social Science Version 21. Point estimate at 95% Confidence Interval was calculated, along with frequency and percentage for binary data. Out of 959 total surgical retinal interventions done 296 (30.86%) at 95% Confidence Interval (27.93-33.78) patients received intravitreal Bevacizumab. Out of total intravitreal Bevacizumab injections, 143 (36.7%) injections were given to retinal vein occlusions patients, 127 (32.6%) injections were given to diabetic retinopathy patients and 66 (17%) injections was given to age-related macular degeneration patients. Males 176 (59.5%) outnumbered the females 120 (40.5%) in receiving intravitreal Bevacizumab. Mean baseline Logarithm of the Minimal Angle of Resolution visual acuity, 1.1, improved to, 0.75, after 3 months of intravitreal Bevacizumab. Intravitreal Bevacizumab was one of the commonest retinal interventions used. Retinal vein occlusion, diabetic retinopathy, and age-related macular degeneration were the commonest retinal diseases needing intravitreal Bevacizumab.

Nitrogen‐doped TiO2 Nanocrystals for Highly Sensitive Electrochemical Immunoassay of Carcinoembryonic Antigen

AbstractNitrogen‐doped TiO2 nanocrystals (N−TiO2 NCs) were simply synthesized and then functionalized with streptavidin for highly sensitive electrochemical immunoassay of tumor marker. Scanning electron microscopy, transmission electron microscopy, static water contact angle, and cyclic voltametric measurement were adapted to examine the properties of N−TiO2 NCs and resultant immunosensor. The functionalized N−TiO2 NCs sensing platform shows high electrochemical conductivity, large surface area and excellent hydrophilicity. The features make them to produce high current response, capture more antibody molecules, and maintain the bioactivity of immobilized antibodies. By means of carcinoembryonic antigen (CEA) as model tumor marker, a wide linear range of 0.005–3 ng/mL and a low detection limit of 0.005 ng/mL (signal‐to‐noise ratio of 3) were achieved by the proposed CEA immunosensor. Furthermore, the resultant CEA immunosensor displays high specificity and was employed to determine CEA in clinical serum samples.

Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion

Monoclonal antibodies, antibody fragments and fusion proteins derived thereof have revolutionized the practice of medicine. Major challenges faced by the biopharmaceutical industry are however high production costs, long processing times and low productivities associated with their production in mammalian cell lines. The yeast Saccharomyces cerevisiae, a well-characterized eukaryotic cell factory possessing the capacity of post­translational modifications, has been industrially exploited as a secretion host for production of a range of products, including pharmaceuticals. However, due to the incompatible surface glycosylation, few antibody molecules have been functionally expressed in S. cerevisiae. Here, three non-glycosylated antibody fragments from human and the Camelidae family were chosen for expression in a S. cerevisiae strain (HA) previously evolved for high α-amylase secretion. These included the Fab fragment Ranibizumab (Ran), the scFv peptide Pexelizumab (Pex), and a nanobody consisting of a single V-type domain (Nan). Both secretion and biological activities of the antibody fragments were confirmed. In addition, the secretion level of each protein was compared in the wild type (LA) and two evolved strains (HA and MA) with different secretory capacities. We found that the secretion of Ran and Nan was positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. To investigate the mechanisms for different secretion abilities in these selected yeast strains for the different antibody fragments, RNA-seq analysis was performed. The results showed that several bioprocesses were significantly enriched for differentially expressed genes when comparing the enriched terms between HA.Nan vs. LA.Nan and HA.Pex vs. LA.Pex, including amino acid metabolism, protein synthesis, cell cycle and others, which indicates that there are unique physiological needs for each antibody fragment secretion.