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Tauopathies, including Alzheimer's disease (AD), are a class of neurodegenerative diseases characterized by the presence of insoluble tau inclusions. Tau phosphorylation has traditionally been viewed as the dominant post-translational modification (PTM) controlling tau function and pathogenesis in tauopathies. However, we and others have identified tau acetylation as a primary PTM regulating both normal tau function as well as abnormal pathogenic features including aggregation. Prior work showed robust tau acetylation in aggregation hotspots located within the 2nd and 3rd repeat regions of tau (residues K280 and K311) in tauopathy brains, including AD, compared to non-tauopathy controls. By screening thousands of hybridoma clones, we generated site-specific and modification-specific monoclonal antibodies targeting acetylated tau at residues K280 or K311. To validate these antibodies in a bona fide neuronal system, we targeted the acetyltransferase CBP to the cytoplasm of neurons to promote tau acetylation. Several antibody clones specifically detected CBP-acetylated tau and co-localized with ac-tau in neurons. Additionally, our lead optimal anti-acetylated-tau monoclonal antibodies detected robust tau pathology in tangles and neuritic plaques of human AD brains. Given the now emerging interest in acetylated tau as critical regulator of tau functions, these sensitive and highly specific tools will allow us to further unravel the tau PTM code and, importantly, could be deployed as diagnostic or disease-modifying agents. [ABSTRACT FROM AUTHOR]

The article explores the application of nanomaterials in detecting harmful substances in food. It emphasizes the importance of identifying contaminants like drugs, toxins, metals, and allergens. The article presents various studies that utilize nanomaterials to detect specific contaminants, showcasing their effectiveness in providing fast and accurate analysis. It also discusses the development of biosensors for detecting food allergens. Overall, the article offers valuable insights into the use of nanomaterials in ensuring food safety. [Extracted from the article]

Using a quartz crystal microbalance with dissipation monitoring (QCM-D), the complex high-frequency viscosity, ñ = η′ − iη′′, of concentrated solutions of a monoclonal antibody (mAb) was studied with respect to its dependence on temperature, T, and concentration, c. Lysozyme and bovine serum albumin (BSA) served as reference materials. Viscoelasticity was found for the mAb solution, while the reference materials behaved like Newtonian liquids. The QCM-D probes the solution's dynamics on the time scale of a few tens of nanoseconds. The processes of relaxation accessed with the QCM-D are not amenable to standard viscometry. The inverse loss tangent at 15 MHz (equal to η′′/η′ at 15 MHz, quantifying the elastic contribution to the oscillatory stress) was between 0.1 and 0.5 for the concentrated mAb solutions. It decreased with increasing temperature and decreasing pH. Activation energies of viscous flow, Ea,η, were derived from the functions η′(T). Ea,η was found to be higher for the mAb solutions than for water. No such increase was found for the reference materials. This difference evidences protein–protein interactions (PPIs) between the mAb molecules, which do not exist in the same way for lysozyme and BSA. The excipients citrate and arginine did not noticeably affect the mAb's high-frequency viscosity as determined with the QCM-D. [ABSTRACT FROM AUTHOR]

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The presence of multiple pesticide residues in vegetables has become an increasingly critical concern. Immunochromatography has garnered significant interest for its convenience, sensitivity and speed. However, conventional immunochromatography was typically limited to single-target detection, exhibiting restricted efficiency and high costs in on-site large-scale detection scenarios. Dual-channel time-resolved fluorescent immunochromatography test strip (TRFITS) was developed for the simultaneous rapid detection of imidacloprid (IMI) and tolfenpyrad (TOL) in 15 min. Utilizing the excellent optical properties of time-resolved fluorescent microspheres, this TRFITS enabled better detection sensitivity (0.22 ng/mL for IMI, 11.85 ng/mL for TOL), which was approximately 20 times more sensitive than that of conventional immunochromatography, under optimal conditions. In addition, the TRFITS successfully detected IMI and TOL in vegetables samples with acceptable accuracy. The ability to simultaneously quantify multiple pesticide residues using a single test strip reduces material consumption and lowers operational costs, offering a practical solution for rapid on-site pesticide screening. [Display omitted] • Dual-channel TRFITS simultaneously detected imidacloprid and tolfenpyrad. • TRFMs coupled with monoclonal antibodies as detection probes. • TRFITS has the advantage of higher sensitivity and lower cost. [ABSTRACT FROM AUTHOR]

Cell-surface proteins, which are closely associated with various physiological and pathological processes, have drawn much attention in drug discovery and disease diagnosis. Thus, wash-free imaging of the target cell-surface protein under its native environment is critical and helpful for early detection and prognostic evaluation of diseases. To minimize the interference from autofluorescence and fit the penetration depth towards tissue samples, we developed a fluorogenic antibody-based probe, Ab-Cy5.5 , which will liberate > 5-fold turn-on near-infrared (NIR) emission in the presence of its target antigen within 10 min. By taking advantage of the fluorescence-quenched dimeric H-aggregation of Cy5.5, Ab-Cy5.5 with Cy5.5 attached at the N-terminus showed negligible background signal, allowing direct imaging of the target cell-surface protein in both living cells and tissue samples without washing. [Display omitted] • Developing a general strategy to change monoclonal antibodies into fluorogenic probes with NIR emission. • Wash-free imaging of endogenous cell-surface proteins with low background and high selectivity. • Compatible for both living cells and clinical tissue samples. [ABSTRACT FROM AUTHOR]

Thymidine kinase 1 (TK1) is a marker of cell proliferation that can be used for early screening, treatment monitoring, and evaluating the prognosis of patients with tumors. The main purpose of this study was to develop clinically applicable TK1 antibodies, establish an appropriate detection method, and provide material and technical support for the research and clinical application for different types of tumors. Experimental mice were immunized with the C-terminal 31 peptide of human TK1 to screen monoclonal cell lines capable of stably secreting specific antibodies. Monoclonal antibodies were then prepared, purified and screened for optimal pairing following the identification of purity and isotype. Finally, based on the principles adopted by the double-antibody sandwich detection method, we constructed a lateral flow immunochromatographic assay (LFIA) to quantify the concentration of TK1 in serum samples when using a gold nanoparticle-labeled anti-TK1 monoclonal antibody as a probe. The limit of detection for TK1 in serum was 0.31 pmol/L with a detection range of 0.31–50 pmol/L. The spiked recoveries ranged from 97.7% to 109.0% with an analytical precision of 5.7–8.2%; there was no cross-reactivity with common proteins in the serum. The established LFIA also exhibited good consistency with commercially available chemiluminescent immunoassay kits for the detection of clinical samples. The LFIA developed in this study has the advantages of high sensitivity, accuracy, reproducibility and strong specificity, and provides a new technical tool for the quantitative detection of TK1. • Material and technical support for the clinical detection of different tumors. • LFIA was set up to quantify the concentration of TK1 in serum samples. • The ultrasensitive LOD was 0.31 pmol/L with a detection range of 0.31–50 pmol/L. • LFIA exhibited good consistency with CLIA for the detection of clinical samples. [ABSTRACT FROM AUTHOR]

Striated muscle contraction and relaxation is regulated by Ca2+ at the myofilament level via conformational modulations of the troponin complex. To understand the structure–function relationship of troponin in normal muscle and in myopathies, it is necessary to study the functional effects of troponin isoforms and mutations at the level of allosteric conformations of troponin subunits. Traditional methodologies assessing such conformational studies are laborious and require significant amounts of purified protein, while many current methodologies require non-physiological conditions or labeling of the protein, which may affect their physiological conformation and function. To address these issues, we developed a novel approach using site-specific monoclonal antibodies (mAb) as molecular probes to detect and monitor conformational changes of proteins. Here, we present examples for its application in studies of two subunits of troponin: the Ca2+-binding subunit, TnC, and the tropomyosin-binding/thin filament-anchoring subunit, TnT. Studies using a high-throughput microplate assay are compared with that using localized surface plasmon resonance (LSPR) to demonstrate the effectiveness of using mAb probes to assess ligand-induced conformations of troponin subunits in physiological conditions. The assays utilize relatively small amounts of protein and are free of protein modification, which may bias results. Detailed methodologies using various monoclonal antibodies (mAbs) are discussed with considerations for the optimization of assay conditions and the broader application in studies of other proteins as well as in screening of therapeutic reagents that bind a specific target site with conformational and functional effects. [ABSTRACT FROM AUTHOR]

The in vivo-generator radionuclides 140Nd (t1/2 = 3.4 d) and 134Ce (t1/2 = 3.2 d) were used to trace a urokinase-type plasminogen activator (uPA)-targeting mouse monoclonal antibody, ATN-291, in U87 MG xenograft tumor-bearing mice. ATN-291 is known to internalize on the uPA/uPA-receptor pair, making it an appropriate targeting vector for investigating the fate of in vivo generator daughters on internalizing probes. Ante-mortem and post-mortem PET imaging at 120 h post-injection gave no indication of redistribution of the positron emitting daughter nuclides 134La and 140Pr from tumor tissue (p > 0.5). The lack of redistribution indicates that the parent radionuclides 134Ce and 140Nd could be considered as long-lived PET-diagnostic matches to therapeutic radionuclides like 177Lu, 161Tb and 225Ac when internalizing bioconjugates are employed. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has magnified longstanding health care and social inequities, resulting in disproportionately high COVID-19-associated illness and death among members of racial and ethnic minority groups (1). Equitable use of effective medications (2) could reduce disparities in these severe outcomes (3). Monoclonal antibody (mAb) therapies against SARS-CoV-2, the virus that causes COVID-19, initially received Emergency Use Authorization (EUA) from the Food and Drug Administration (FDA) in November 2020. mAbs are typically administered in an outpatient setting via intravenous infusion or subcutaneous injection and can prevent progression of COVID-19 if given after a positive SARS-CoV-2 test result or for postexposure prophylaxis in patients at high risk for severe illness.† Dexamethasone, a commonly used steroid, and remdesivir, an antiviral drug that received EUA from FDA in May 2020, are used in inpatient settings and help prevent COVID-19 progression§ (2). No large-scale studies have yet examined the use of mAb by race and ethnicity. Using COVID-19 patient electronic health record data from 41 U.S. health care systems that participated in the PCORnet, the National Patient-Centered Clinical Research Network,¶ this study assessed receipt of medications for COVID-19 treatment by race (White, Black, Asian, and Other races [including American Indian or Alaska Native, Native Hawaiian or Other Pacific Islander, and multiple or Other races]) and ethnicity (Hispanic or non-Hispanic). Relative disparities in mAb** treatment among all patients†† (805,276) with a positive SARS-CoV-2 test result and in dexamethasone and remdesivir treatment among inpatients§§ (120,204) with a positive SARS-CoV-2 test result were calculated. Among all patients with positive SARS-CoV-2 test results, the overall use of mAb was infrequent, with mean monthly use at 4% or less for all racial and ethnic groups. Hispanic patients received mAb 58% less often than did non-Hispanic patients, and Black, Asian, or Other race patients received mAb 22%, 48%, and 47% less often, respectively, than did White patients during November 2020-August 2021. Among inpatients, disparities were different and of lesser magnitude: Hispanic inpatients received dexamethasone 6% less often than did non-Hispanic inpatients, and Black inpatients received remdesivir 9% more often than did White inpatients. Vaccines and preventive measures are the best defense against infection; use of COVID-19 medications postexposure or postinfection can reduce morbidity and mortality and relieve strain on hospitals but are not a substitute for COVID-19 vaccination. Public health policies and programs centered around the specific needs of communities can promote health equity (4). Equitable receipt of outpatient treatments, such as mAb and antiviral medications, and implementation of prevention practices are essential to reducing existing racial and ethnic inequities in severe COVID-19-associated illness and death. [ABSTRACT FROM AUTHOR]

Radiobioconjugate therapy, recommended as one of the effective modalities for the treatment of cancer cells, is based on the concept of delivering the localized radiation at the cellular level to the disease site using a biological moiety. The high tumor/nontumor ratio is the essentially a sine qua non for the successful execution of targeted therapy which is highly desired. The central problem associated to radiobioconjugate therapy is the small fraction of a radiobioconjugate localized to the tumor, while the major fraction of it is delivered to the nontarget organs (reticuloendothelial system deposition). The current article focuses on the better perceptive of the factors of understanding, which includes the selection and expansion of sophisticated molecular carriers, assortment of a suitable radionuclide based on the class of emission, linear energy transmit, and the material radiophysical half-life. In addition, a concern to the biochemical interactions taking place at the molecular level, selection of a specific targeting strategy for designing effective treatment regimes, and importantly the challenges associated to it have also been discussed. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P) is a phospholipid that regulates pleiotropic biological activities and exerts extracellular functions by binding to five specific G-protein-coupled receptors, S1P receptors (S1PR) 1–5. When activated by S1P, S1PR promote the proliferation and invasion of tumor cells by inducing the formation of new blood vessels. We developed and assessed a new monoclonal antibody imaging probe 99mTc-HYNIC-S1PR1mAb, to explore the feasibility of targeting the S1PR1 in vitro and in vivo. S1PR1mAb was prepared and followed by technetium-99m labeling with succinimidyl 6-hydraziniumnicotinate hydrochloride. Cell uptake and blocking studies were performed to investigate the binding specificity of 99mTc-HYNIC-S1PR1mAb in vitro. 99mTc-HYNIC-S1P1mAb was also tested in vivo in mice xenografted with SK-HEP-1 (high-expression of S1PR1) and MCF-7 (low-expression of S1PR1) using single-photon emission-computed tomography (SPECT). Ex vivo gamma counting of tissues from tumor-bearing mice was used to evaluate 99mTc-HYNIC-S1PR1mAb biodistribution. The biodistribution study results showed significantly higher uptake in SK-HEP-1 tumors than in MCF-7 tumors (P < 0.001). Reduced uptake of 99mTc-HYNIC-S1PR1mAb in SK-HEP-1 was observed in tumor-bearing nude mice pretreated with fingolimod, which binds competitively to the receptors, especially S1PR1. 99mTc-HYNIC-S1PR1mAb can be synthesized and specifically targeted to S1PR1 in vitro and in vivo , allowing S1PR1 expression assessment with SPECT imaging. [ABSTRACT FROM AUTHOR]

Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram‐positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d‐alanine and N‐acetyl‐d‐glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA‐modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well‐defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA‐fragments, incorporating all possible glycosylation modifications (α‐GlcNAc at the RboP C4; β‐GlcNAc at the RboP C4; β‐GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA‐type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA‐hexamers. Automated solid phase syntheses were used to assemble a WTA‐dodecamer and glycosylated WTA‐hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA‐fragments were used to probe binding of monoclonal antibodies using WTA‐functionalized magnetic beads, revealing the binding specificity of these WTA‐specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA‐chains. [ABSTRACT FROM AUTHOR]

Analysis of therapeutic IgG aggregates in serum is a potential area of investigation as it can give deeper insights about the function, immunogenic issues and protein interaction associated with the aggregates. To overcome various complexities associated with the existing analytical techniques for analyzing aggregates in serum, a novel florescence microscopy-based image processing approach was developed. The monoclonal antibody (mAb) was tagged with a fluorescent dye, fluorescein isothiocyanate (FITC). Aggregates, generated by stirring, were spiked into serum and images were captured at various time points. After denoising, thresholding by weighted median, 1D Otsu, and 2D Otsu was attempted and a modified 2D Otsu, a new mode of thresholding, was developed. This thresholding method was found to be highly effective in removing noises and retaining analyte sizes. Out of 0–255, the optimized threshold value obtained for the images discussed in modified 2D Otsu was 9 while 2D Otsu's overestimated values were 38 and 48. Other morphological operations were applied after thresholding and the area, perimeter, circularity, and radii of the aggregates in these images were calculated. The proposed algorithm offers an approach for analysis of aggregates in serum that is simpler to implement and is complementary to existing approaches. [ABSTRACT FROM AUTHOR]

Aims: Vimentin citrullination, the calcium (Ca2+)‐dependent peptidylarginine deiminase (PAD)‐mediated conversion of an arginine residue of vimentin to a citrulline residue, has emerged as a pathophysiological outcome of autoimmune diseases and neurodegeneration. However, the roles, functions, and expression of citrullinated vimentin have not yet been elucidated because available antibodies are limited. Methods: We developed mouse monoclonal IgG1 and IgM specific for vimentin citrullinated at position R440 or R450 and applied Western blotting, immunohistochemistry, and immunofluorescent staining to investigate the pathogenesis of prion diseases in animal models, in patients with prion diseases, and in vitro. Results: Vimentin was found to be highly citrullinated at R440 and R450, and these citrullinated forms were mainly expressed in reactive astrocytes in the brain tissues of scrapie‐infected mice. Full‐length and cleaved forms of citrullinated vimentin were found in the cerebral cortices of sporadic Creutzfeldt‐Jakob disease (sCJD) patients. The distribution of citrullinated vimentin was mainly confirmed in vimentin‐, GFAP‐, and YKL‐40‐positive reactive astrocytes. Biochemically, citrullination promoted resistance to the caspase‐3‐ and caspase‐9‐mediated fragmentation of vimentin. Additionally, citrullination led to increased cytoplasmic and integral membrane/organelle vimentin enrichment, which indicated changes in the intrinsic solubility and distribution of vimentin. Conclusions: Our observations suggest that citrullinated vimentin acts as a specific indicator for the reactive state of astrocytes under abnormal neurological conditions. In addition, these novel antibodies will be helpful for studying the role of citrullinated vimentin in the pathogenesis of human disorders. [ABSTRACT FROM AUTHOR]

Small amounts of polysaccharides remaining tightly bound to cellulose microfibrils in the plant cell wall are significant to cell wall architecture and of importance to the rheological properties of plant-derived cellulose nanofibres (CNFs). A method for detecting these polysaccharides is presented. The method, Array-based Microfibril Surface Assessment (AMSA), uses monoclonal antibodies to probe polysaccharides that are exposed on fibrous material. Microfluidised alcohol insoluble residue from sugar beet pulp was used as control and baseline, representing a minimally processed material. To test the utility of AMSA, CNF prepared from sugar beet pulp were analysed. Surprisingly, nanofibrillation did not enhance binding of CBM3a (specific to crystalline cellulose) which indicates that bare cellulose is not easily made. However, nanofibrillation exposed a large amount of unbranched mannan, implying that mannan is closely associated with cellulose in these cell walls. Additional treatment of the CNF suspensions with pectinases removed the majority of homogalacturonan and rhamnogalacturonan I, which caused flocculation of the samples. This highlights the importance of preserving small quantities of these polysaccharides to promote CNF-water interactions and obtain well-dispersed fibres. In conclusion, it is demonstrated how AMSA has a utility in both biotechnological applications involving CNF as well as for exploring plant cell wall architecture. [ABSTRACT FROM AUTHOR]

Granuloma annulare (GA) is a benign, usually selflimited inflammatory skin dermatosis characterized clinically by pink-red to brown dermal papules or annular plaques. The main histologic feature is the presence of palisading or interstitial granulomas composed of necrobiotic collagen, elastic fibers, and mucin surrounded by a lymphohistiocytic infiltrate. Granuloma annulare is commonly associated with trauma, infections, diabetes mellitus, dyslipidemia, malignancy, thyroid disease, and a variety of medications. Two cases of GA have been reported in association with the use of secukinumab, a monoclonal antibody directed against interleukin 17A (IL17A), for the treatment of moderate-to-severe plaque psoriasis. We report the third case of secukinumab-associated GA in a 52-year-old woman with a history of diabetes mellitus type II, dyslipidemia, and non-alcoholic steatohepatitis. After four months of therapy with secukinumab, she presented with pink papules coalescing to plaques involving the antecubital fossae. Histology demonstrated a lymphohistiocytic palisading granuloma with central necrobiotic collagen and mucin, consistent with GA. Physicians should be aware of the possibility of GA developing in patients receiving secukinumab, especially in those with predisposing factors for GA. A better understanding of secukinumab-associated GA may lead to discoveries in GA pathogenesis and reveal broader immunomodulatory effects of secukinumab. [ABSTRACT FROM AUTHOR]

Context: TMEM127 is a poorly known tumor suppressor gene associated with pheochromocytomas, paragangliomas, and renal carcinomas. Our incomplete understanding of TMEM127 function has limited our ability to predict variant pathogenicity.Purpose: To better understand the function of the transmembrane protein TMEM127 we undertook cellular and molecular evaluation of patient-derived germline variants.Design: Subcellular localization and steady-state levels of tumor-associated, transiently expressed TMEM127 variants were compared to the wild-type protein using immunofluorescence and immunoblot analysis, respectively, in cells genetically modified to lack endogenous TMEM127. Membrane topology and endocytic mechanisms were also assessed.Results: We identified 3 subgroups of mutations and determined that 71% of the variants studied are pathogenic or likely pathogenic through loss of membrane-binding ability, stability, and/or internalization capability. Investigation into an N-terminal cluster of missense variants uncovered a previously unrecognized transmembrane domain, indicating that TMEM127 is a 4- transmembrane, not a 3-transmembrane domain-containing protein. Additionally, a C-terminal variant with predominant plasma membrane localization revealed an atypical, extended acidic, dileucine-based motif required for TMEM127 internalization through clathrin-mediated endocytosis.Conclusion: We characterized the functional deficits of several germline TMEM127 variants and identified novel structure-function features of TMEM127. These findings will assist in determining pathogenicity of TMEM127 variants and will help guide future studies investigating the cellular role of TMEM127. [ABSTRACT FROM AUTHOR]

One of the possibilities of early detection of cancer disease is the analysis of the presence of protein biomarkers. Herein, we present the studies on the hybrid antibody—aptamer receptor layers for electrochemical detection of HER2 protein that is known as a biomarker of e.g. breast cancer. The application of MB-labelled aptamer did not allow to evidence the interaction with HER2 protein. Moreover, the use of unlabelled aptamer as a capture element and polyclonal antibody as a reporter probe did not show a dependence of current signal change vs HER2 protein concentration as well. On the contrary, the application of monoclonal antibody as a capture probe and aptamer labelled at 3′ end with MB as reporter probe enabled the determination of HER2 within 0.01–10 ng·ml−1 concentration range. The elaborated affinity assay also showed good selectivity towards HER2 biomarker and was used for HER2 determination in spiked serum sample [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) is a potent marine neurotoxin. Its frequent detection and widespread distribution pose a substantial health risk to consumers. The work presented herein describes the development of a lateral flow immunosensor based on a gold nanoparticle-labeled monoclonal antibody probe, which allows the affinity reaction of antigen–antibody and amplifies the reaction signal. The immunosensor assay was found to provide a visible limit of detection (vLOD) of 10 μg kg−1 in crucian and clam matrices without time-consuming pretreatment. Spiked samples were analyzed using the immunosensor and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The immunosensor gave average intra-assay recoveries of 84.0–103.9%, inter-assay recoveries of 76.1–105% for the crucian matrix and intra-assay recoveries of 78.5–93.0%, inter-assay recoveries of 74.8–85.7% for the clam matrix. The results obtained using the immunosensor and LC-MS/MS were in good agreement. The immunosensor test could be completed within 10 min, offering a convenient option or complementarity to the traditional methods of TTX detection. [ABSTRACT FROM AUTHOR]

• Multi-marker detection is performed based on SPR sensor in a simple and efficient way. • AFP and miRNA-125b are chosen for the early diagnosis of Hepatocellular Carcinoma. • DASM method is applied to improve the sensitivity and specificity of AFP detection. • Sensitive and specific detection of miRNA is achieved by S9.6 antibody enhanced method. Early diagnosis of Hepatocellular Carcinoma (HCC) is an important means to raise the survival rate of patients. Multi-marker combined detection is a powerful tool of early HCC diagnosis. Traditional detection methods are not effective and accurate because it is difficult to achieve combined detection of multiple markers. In this paper, we selected Alpha Fetoprotein (AFP) and miRNA-125b as the combined detection markers to improve the simultaneously diagnostic sensitivity and specificity. The anti-AFP monoclonal antibody and the DNA probes paired with the miRNA-125b were modified on the surface of surface plasmon resonance (SPR) sensor respectively to specifically recognize AFP and miRNA-125b in serum. In order to enhance the SPR response signal and detection sensitivity, Double Antibody Sandwich Method (DASM) and S9.6 antibody enhanced method were applied to achieve low detection limit of the two markers. Experimental results showed that AFP (25–400 ng/mL) was accurately detected by DASM and the detection limit of miRNA-125b by S9.6 antibody enhanced method reached 123.044 pM. These results verified the feasibility of the multi-marker detection method in early diagnosis of HCC. [ABSTRACT FROM AUTHOR]

The gall structure is built from redifferentiation of host plant tissues, changing the cell wall proprieties and associated functions. Because of the oxidative stress imposed by the galling insect on the plant tissues, some phenotypic traits associated with cell wall metabolism may be suppressed during gall development. Using an immunocytochemical approach we investigated the pectin and glycoprotein composition of the cell walls during non-galled leaf development and galls induced on Aspidosperma tomentosum (Apocynaceae). Embedding in historesin was used for anatomical characterization, as well as for immunocytochemical analyses. The samples were probed with the primary monoclonal antibodies JIM5, JIM7, LM1, LM2, LM5 and LM6. The gall develops especially from cell hypertrophy and hyperplasia of the spongy and palisade parenchyma, which determine its shape. Epitopes of β-D-galactan and α-L-arabinan in the galls were totally suppressed or less expressed. The increased labeling of homogalacturonan epitopes, especially in senescent galls, is a clear indicator that pectin methylesterase activity is maintained despite oxidative stresses imposed by the galling insect. Arabinogalactan proteins were not detected in any non-galled leaf or gall stages. The galling insect deeply changed the structure and deposition pattern of the cell wall, promoting new functionalities in the gall tissue compartments. • The galling insect is a phenotype manipulator of cell wall traits of the host organ. • Pectin dynamics is operational during A. tomentosum gall formation and suggest a linear process de-methyl esterification. • The gall cell walls have reduced abundance of galactan and arabinan epitopes. [ABSTRACT FROM AUTHOR]

Background Context: Adolescent idiopathic scoliosis (AIS) is the leading cause of spinal deformity in adolescents globally. Recent evidence from genome-wide association studies has implicated variants in or near the ladybird homeobox 1 (LBX1) gene, encoding the ladybird homeobox 1 transcription factor, in AIS development. This gene plays a critical role in guiding embryonic neurogenesis and myogenesis and is vital in muscle mass determination. Despite the confirmation of the role for LBX1 gene variants in the development of AIS, the biological basis of LBX1 contribution to AIS remains mostly unknown.Purpose: To investigate the potential role of LBX1 in driving spinal curving, curve laterality, and progression through muscle-based mechanisms in AIS patients by analyzing its gene and protein expression.Study Design: This is a cross-sectional study using clinical data and biological samples from the Immunometabolic CONnections to Scoliosis study (ICONS study).Patient Sample: Twenty-five patients with AIS provided informed consent. Paraspinal muscle biopsies from the maximal points of concavity and convexity for gene expression and protein analysis were obtained at the start of corrective spinal surgery.Outcome Measures: The outcome measures included the detection of paraspinal muscle LBX1 mRNA abundance and LBX1 protein expression and the correlation of the latter with age, sex, and curve severity.Methods: The measurement of mRNA abundance was done using quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, protein lysates from the biopsied muscle samples were probed with a monoclonal LBX1 antibody to compare the muscle protein levels on either side of the scoliotic curve by western blot. This study received funding from the Division of Orthopedics, Department of Surgery, McMaster University, Hamilton, Ontario, Canada ($39,900 CAN for 2 years). The authors have no conflicts of interest to disclose.Results: LBX1 mRNA abundance (concave 2.98±0.87, convex 3.40±1.10, p value 0.73) and protein expression (concave 1.20±0.13, convex 1.21±0.10, p value 0.43) were detected on both sides of the scoliotic curve at equivalent levels. The expression of LBX1 protein did not correlate with age (concave: correlation coefficient 0.32, p value 0.12; convex: correlation coefficient 0.08, p value 0.69), sex (concave: correlation coefficient -0.03, p value 0.08; convex: correlation coefficient 0.07, p value 0.72), or the severity of spinal curving measured using the Cobb angle (concave: correlation coefficient -0.16, p value 0.45; convex: correlation coefficient -0.08, p value 0.69).Conclusions: LBX1 is expressed in erector spinae muscles, and its levels are equal in muscles on both sides of the scoliotic curve in AIS. The expression of LBX1 on the convex and concave sides of the scoliotic curve did not correlate with age, sex, or the severity of spinal curving. The molecular mechanisms by which LBX1contributes to the development and propagation of AIS need to be explored further in muscle and other tissues. [ABSTRACT FROM AUTHOR]

The article discusses how DNA damage corrects the aberrant cytoplasmic localisation of nucleophosmin in NPM1 mutated acute myeloid leukaemia. Topics include how nucleophosmin (NPM1) is an abundant phosphoprotein involved in multiple cellular functions and its gene (NPM1) is the most commonly mutated gene in acute myeloid leukaemia.

IPO-38 is a potential biomarker for early diagnosis of gastric cancer that we recently identified. Although we characterized its chemical nature as a nucleosome histone, we suspected the existence of histone modification for the IPO-38 antibody-labeled protein. Here, we used a commercially available modified histone peptide array to identify the type and site of histone modification labeled by the IPO-38 monoclonal antibody. In protein array analysis, the citrulline modification of histone 3 on arginine 26 (H3R26Cit) yielded the strongest signal. Although peptidyl arginine deiminase-2 and -4 (PADI2 and PADI4, respectively) can catalyze the conversion of arginine to citrulline, we observed that only PADI4 expression correlated with the citrulline histone modification of H3R26Cit. Overexpression of PADI4, via transfection of a eukaryotic expression vector, and knockdown of PADI4 gene expression, by a PADI4 CRISPR/Cas9 vector, confirmed the crucial function of PADI4 on the increased level of H3R26Cit in gastric cancer cell lines. By immunoprecipitation and immunoblotting, we found an interaction between H3R26Cit and H3K27me3. Our study established the first link between the IPO-38 antigen and citrullinated histone 3, and clarified the upstream regulatory enzyme PADI4. The new findings suggest an important role for the citrullination modification of histone in gastric cancer biology, and should help us optimize the development of a sensitive and specific diagnostic reagent. [ABSTRACT FROM AUTHOR]

In order to achieve a highly sensitive detection of procymidone in vegetables, three paper-based biosensors based on a core biological immune scaffold (CBIS) were developed, which were time-resolved fluorescence immunochromatography strips with Europium (III) oxide (Eu-TRFICS). Goat anti-mouse IgG and europium oxide time-resolved fluorescent microspheres formed secondary fluorescent probes. CBIS was formed by secondary fluorescent probes and procymidone monoclonal antibody (PCM-Ab). The first type of Eu-TRFICS (Eu-TRFICS-(1)) fixed secondary fluorescent probes on a conjugate pad, and PCM-Ab was mixed with a sample solution. The second type of Eu-TRFICS (Eu-TRFICS-(2)) fixed CBIS on the conjugate pad. The third type of Eu-TRFICS (Eu-TRFICS-(3)) was directly mixed CBIS with the sample solution. They solved the problems of steric hindrance of antibody labeling, insufficient exposure of antigen recognition region and easy loss of activity in traditional methods. They realized multi-dimensional labeling and directional coupling. They replaced the loss of antibody activity. And the three types of Eu-TRFICS were compared, among which Eu-TRFICS-(1) was the best detection choice. Antibody usage was reduced by 25% and sensitivity was increased by 3 times. Its detection range was 1–800 ng/mL, the limit of detection (LOD) was 0.12 ng/mL with the visible LOD (vLOD) of 5 ng/mL. [Display omitted] • Time-resolved fluorescent microspheres were applied to CBIS. • The monoclonal antibody usage was reduced by 25% prepared by the CBIS. • The CBIS connect multiple microspheres to one antibody molecule. • The linear range of Eu-TRFICS-(1) used to detect procymidone is 1–800 ng/mL. • Three kinds of Eu-TRFICS used to detect procymidone are constructed and compared. [ABSTRACT FROM AUTHOR]

Adenosine deaminase acting on RNA-1 (ADAR1) is an RNA-editing enzyme which catalyzes the deamination of adenosine to inosine in double-stranded RNA substrates during post-transcription processing [[1]]. In this study, we found serum autoantibodies to ADAR1 in patients with dermatomyositis (DM) (Case 1) and systemic lupus erythematosus (SLE) (Case 2), in which a pathogenetic role for type I interferon is implicated [[5]]. Twenty-two sera from SSc patients, 10 from SLE patients, and 7 from DM patients were tested by ELISA (Supplementary file) with the standard curve estimation using sera from Case 1. [Extracted from the article]

An ultrasensitive monoclonal antibody-based gold nanoparticle immunochromatographic strip assay and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) were developed to detect florfenicol (FF) and thiamphenicol (TAP) in egg samples. The ic-ELISA, with optimized pH, methanol content and sodium chloride content, exhibited an IC50 value of 0.2 ng/mL for FF and 0.27 ng/mL for TAP, with the working range of 0.05-0.77 and 0.05-1.42 ng/mL, respectively. The optimized ic-ELISA showed negligible cross-reactivity with other phenols and broad-spectrum antibiotics. The recoveries in egg samples using the ic-ELISA ranged from 84% to 115% with a coefficient of variation of less than 5%. Based on this monoclonal antibody, a rapid and ultrasensitive immunochromatographic strip assay was developed with a cutoff value of 1 ng/mL for FF and TAP. Our results indicated that both developed methods were highly useful for screening FF and TAP in eggs. [ABSTRACT FROM AUTHOR]

A monoclonal antibody (mAb) directed against phenylethanolamine A (PEA) was successfully prepared with the immunization of mice and cell fusion, and used to develop a detection method for a colloidal gold immunoassay. The 50% inhibitory concentration of mAb PEA-2G10 was 0.213 ng/mL, the limit of detection was 0.033 ng/mL, and its detection range 0.033-0.679 ng/mL. The recovery rate for PEA in swine urine was 84.5-90.8%, with good stability and accuracy. “Dry” immunoassay test strips were successfully prepared based on antibody 2G10. When the strip was tested in swine urine, the cutoff value for PEA was 5 ng/mL, with highly sensitivity. The method developed here can be used to test swine urine on-site for the rapid detection of PEA. [ABSTRACT FROM AUTHOR]

In this study, a sensitive monoclonal antibody (mAb) 3D5 against sparfloxacin (SPFX) was generated. Based on the mAb 3D5, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed for the detection of SPFX in honey. The half maximal inhibitory concentration and limit of detection for the ic-ELISA method were 0.12 and 0.02 ng/mL, respectively. The experiments showed a negligible cross-reaction with other drugs. The average recovery rates for spiked SPFX honey extracts ranged from 90% to 101%, indicating an accepted accuracy. Furthermore, a lateral-flow immunochromatographic assay strip method with a cut-off value of 2 ng/mL was developed. Therefore, both of the developed methods are suitable for future use as rapid screening methods to detect and control the content of SPFX residues in honey samples. [ABSTRACT FROM AUTHOR]

Vitamin B2 (riboflavin) is a water-soluble vitamin that has important roles in human health. In this study, we developed a sensitive and specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) strip for the rapid detection of vitamin B2. Following routine fusion and selection, the optimum monoclonal antibody against vitamin B2 was obtained. The 50% inhibitory concentration and limit of detection of ic-ELISA were 8.18 and 1.80 ng/mL, respectively. The cut-off value of the lateral-flow ICA strip was 50 ng/mL. The results revealed that our developed methods are suitable for the on-site detection and mass screening of vitamin B2 in food and pharmaceutical products. [ABSTRACT FROM AUTHOR]

The presence of pesticide residues in crops is a significant public concern. Due to the large number of samples and pesticides, a reliable, simple, cost-effective, and high-throughput analytic method is required. In this study, we developed a gold nanoparticle (GNP) immunochromatographic assay (ICA) for the rapid detection of 6-benzylaminopurine (6-BA) residues in bean sprouts. A monoclonal antibody against 6-BA, having a half-maximum inhibition concentration of 2.25 ng/mL, was labeled with GNPs and fixed onto a glass fiber membrane to create a conjugate pad. Coating antigen and goat anti-mouse antibody were sprayed onto a nitrocellulose membrane to form the test line and control line, respectively. The visual limit of detection and cutoff limit of ICA were 10 and 80 ng/g in bean sprouts, respectively. The results of ICA were obtained within 10 min with the naked eye. Therefore, ICA is a rapid tool for the on-site screening and detection of 6-BA in bean sprouts. [ABSTRACT FROM AUTHOR]

A novel and highly sensitive immunochromatographic strip based on a monoclonal antibody (3A9) was developed for the detection of cadmium in tap water. The 50% inhibition concentration of the antibody, which showed no cross-reactivity with other heavy metal ions, was 0.45 ng/mL and it recognized Cd(II)-1-(4-isothiocyanobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA) (Cd(II)-ITCBE) and not metal-free ITCBE and EDTA. The cutoff value for semi-quantitative detection of the strip was 5 ng/mL and the lower limit of detection for quantitative detection was 0.2 ng/mL using a scanning reader. The percent recovery ranged from 107.6% to 132% in tap water samples. [ABSTRACT FROM AUTHOR]

Abstract An affinity sensor based on capacitive transduction was developed to detect benzo(a)pyrene (BaP) in river water. Two types of recognition elements, the synthetic receptor analogues molecularly imprinted polymers (MIPs) and natural monoclonal antibody (mAb) were tested for this type of biosensor. Different polymerization strategies to obtain MIPs were compared. One approach comprised a preliminary batch synthesis of beads that were subsequently coupled covalently to an electrode surface. Another approach consisted of the in-situ synthesis of MIPs directly onto the electrode surface using electropolymerization. The latter approach provided the best results. To choose the optimal recognition element mAb and MIP-modified electrodes different sets were evaluated with regards to their sensitivity, selectivity, linear range and re-usability. The mAb-modified electrodes were considerably more sensitive toward BaP (ng L−1 range vs µg L−1 range for the MIP-modified one), and showed a broader linear working range than the MIP-modified electrodes. The latter revealed more suitable for group-selective determination of PAHs. The developed capacitive sensor was tested for the detection of BaP in naturally-contaminated water samples collected in different places of Ghent, Belgium. The results obtained with the sensor were coherent and reproducible, and were in a good agreement with the confirmation technique, HPLC-FlD. Graphical abstract fx1 Highlights • An affinity capacitive sensor was developed to determine benzo(a)pyrene in water. • As a receptor the antibody was compared to molecularly imprinted polymers. • Two strategies for antibody immobilization on the electrode surface were compared. • To obtain MIPs, different polymerization strategies were tested. • The receptors were evaluated regard their sensitivity and re-usability. [ABSTRACT FROM AUTHOR]

Bispecific antibodies are generally prepared by co‐expression of mixtures of recombinant monoclonal antibodies. Because of increased molecular complexity, characterization of a desired bispecific antibody or a mixture of monoclonal antibodies is more challenging than characterization of a conventional single monoclonal antibody. The purpose of this study is to develop a fast and simple method for qualitative/semi‐quantitative analysis of antibody mixtures using liquid chromatography–electrospray triple time‐of‐flight mass spectrometry (LC‐ESI‐TOF/MS) to complement the enzyme‐linked immunosorbent assay. To demonstrate the proof of concept for the analysis of antibody mixtures, three different tool monoclonal antibodies (trastuzumab, rituximab, and cetuximab) with various mixture ratios were treated by either PNGase or Fabricator to simplify the antibody structures without glycans. After deglycosylation, the mixtures of antibodies were analyzed by LC‐ESI‐TOF/MS in the positive ion mode. The m/z scan range of 2000–4000 was used for the deconvolution of each peak from antibodies. Because each antibody could show different ionization efficiency in TOF MS, the peak intensities obtained from various mixture antibodies (1:6:3 or 3:1:6 or 6:3:1) were normalized by the peak intensities of 1:1:1 mixture of three antibodies. Overall, two different methods treated by either PNGase or Fabricator were comparable in estimating the mixture ratios; however, the accuracy and precision data from the Fabricator group were slightly better than PNGase group possibly due to the generation of smaller fragments by Fabricator. [ABSTRACT FROM AUTHOR]

Novel fluorescent antibody probes, which show antigen-dependent fluorescence responses, were developed by selective N-terminal fluorescent labeling of IgG monoclonal antibodies using a reductive alkylation. Several kinds of IgG against tag peptides and small molecules were successfully utilized to detect antigens in a rapid and quantitative manner. [ABSTRACT FROM AUTHOR]

Canakinumab, a high‐affinity human anti‐interleukin‐1β monoclonal antibody, is being used for the treatment of a broad spectrum of inflammatory diseases. This phase 1 study compared the relative bioavailability of a single dose of subcutaneous canakinumab either self‐administered with an autoinjector (AI) or administered by a health care professional (HCP) with a prefilled safety syringe (SS) in healthy subjects. The study enrolled 80 subjects randomized 1:1 to receive 150 mg/mL of a liquid formulation of canakinumab via an AI or SS into either the thigh or abdomen. The geometric mean ratio (90% confidence interval [CI]) of Cmax was 1.09 (0.97‐1.21), AUClast was 1.06 (0.96‐1.17), and AUCinf was 1.05 (0.94‐1.18) for the AI versus SS. The 2‐sided 90%CIs that compared self‐administration with the AI versus administration by an HCP using the SS were entirely within the bioequivalence limits of 80%‐125%. Two subjects in the AI group and 1 in the SS group experienced mild injection‐site reactions. No immunogenicity response was detected in the 307 samples analyzed for immunogenicity. No discontinuations because of adverse events were reported in this trial. Canakinumab administration with an AI or SS has a comparable bioavailability, meeting bioequivalence criteria. [ABSTRACT FROM AUTHOR]

There is a paucity of the investigation of risk factors associated with foot-and-mouth disease (FMD) in Egypt. Subsequently, in the present study the impact of risk factors associated with FMD infection was analyzed in cattle and buffalo reared in Qalyubia governorate, Egypt. Serum samples were collected from 288 animals (n = 90 water buffalo (Bubalus bubalis) and n = 198 cattle) and adapted for detection of FMD virus infection by ELISA assay with monoclonal antibody (MELISA) to type serotype O. Direct FMD virus serotype was detected in 115 animals (n = 82 cattle and n = 33 buffalo). Univariate analysis revealed a significant association between FMD infection and the fattening animals (P = 0.024; odds ratio (OR) 2.118; confidence interval (CI) 1.105-4.058). Multivariate statistics demonstrated that locality was the potential risk factors for infection by FMD virus (P = 0.001; OR 31.613; CI 13.791-72.466). The present investigation draws attention and gives the impression to the potential risk factors associated with FMD-infected cases in cattle and buffalo in Qalyubia governorate in Egypt. Recognition of the potential risk factors associated with FMD-infected cases offer a novel approach to construct the best preventive measures. [ABSTRACT FROM AUTHOR]

The present study represents fabrication of nonstructural antibody (NS1) based immunosensor coupled with bovine serum albumin (BSA) modified screen printed carbon electrodes (SPCE) as transducing substrate for the early diagnosis of dengue virus. The anti-NS1 monoclonal antibody was immobilized on electro grafted BSA surface of working electrode. The electrons transfer resistance before and after NS1 attachment was monitored as a function of its concentration to perform the qualitative and quantitative analysis. The as prepared impedimetric immunosensor successfully detected the dengue virus protein with enhanced limit of detection (0.3 ng/mL) and linear range (1–200 ng/mL). The selectivity of the designed device was further elaborated with several interfering analytes and was finally demonstrated with human serum samples. The extravagant selectivity, sensitivity and easier fabrication protocol corroborate the potential applications of such immunosensor for practical diagnosis of dengue virus. [ABSTRACT FROM AUTHOR]

IL-2 induces regulatory T cells (Tregs) and reduces disease severity, such as in graft-versus-host disease and systemic lupus erythematosus. To investigate the regulatory network of IL-2 in rheumatoid arthritis, we examined the effects of IL-2-anti-IL-2 mAb immune complexes (IL-2ICs) in a rheumatoid arthritis model of collagen-induced arthritis (CIA). CIA was induced in male DBA/1 mice by two immunizations with type II collagen at 3-wk intervals. IL-2ICs were prepared by mixing 5 μg of an anti-IL-2 mAb (clone JES6-1D) with 1 μg of mouse IL-2 and were injected i.p. every day for 3 d. Mouse paws were scored for arthritis using a macroscopic scoring system. Th1, Th2, Th17, and Tregs were analyzed by flow cytometry. Joint histopathology was examined by H&E and immunohistochemical staining. Treg functions were examined by studying in vitro suppression using flow cytometry. IL-2IC administration effectively elicited a 1.6-fold expansion of CD4+Foxp3+ Tregs in peripheral blood cells relative to that found in control mice. IL-2IC treatment significantly inhibited arthritis in CIA mice. Histopathological examination of joints revealed inhibited synovial cell proliferation and IL-17, IL-6, and TNF-α levels but increased Foxp3+ Tregs after IL-2IC treatment. Flow cytometric examination of spleen cells revealed reduced IFN-γ- and IL-17-producing cells and increased IL-10-producing Tregs after IL-2IC treatment. The suppressive activities of CD4+CD25+ Tregs induced by IL-2ICs were stronger than those in untreated mice. IL-2ICs inhibited arthritis by augmenting not only Treg numbers but also Treg functions, which play regulatory roles in autoimmune arthritis. [ABSTRACT FROM AUTHOR]

Abstract Drug resistance remains a formidable challenge to cancer therapy. P-glycoprotein (Pgp) contributes to multidrug resistance in numerous cancers by preventing accumulation of anticancer drugs in cancer cells. Strategies to overcome this resistance have been vigorously sought for over 3 decades, yet clinical solutions do not exist. The main reason for the failure is lack of cancer specificity of small-molecule Pgp inhibitors, thus causing severe toxicity in normal tissues. In this study, Pgp-targeted photodynamic therapy (PDT) was developed to achieve superior cancer specificity through antibody targeting plus locoregional light activation. Thus, a Pgp monoclonal antibody was chemically modified with IR700, a porphyrin photosensitizer. In vitro studies showed that the antibody-photosensitizer conjugates specifically bind to Pgp-expressing drug resistant cancer cells, and caused dramatic cytotoxicity upon irradiation with a near infrared light. We then tested our Pgp-targeted approach in mouse xenograft models of chemoresistant ovarian cancer and head and neck cancer. In both models, targeted PDT produced rapid tumor shrinkage, and significantly prolonged survival of tumor-bearing mice. We conclude that our targeted PDT approach produces molecularly targeted and spatially selective ablation of chemoresistant tumors, and thereby provides an effective approach to overcome Pgp-mediated multidrug resistance in cancer, where conventional approaches have failed. Graphical abstract Unlabelled Image Highlights • Pgp-targeted PDT was developed to achieve high cancer specificity via antibody targeting plus locoregional light ablation; • Antibody-photosensitizer conjugates provided Pgp-specific delivery in chemoresistant tumors; • Locoregional light activation of photosensitizer further enhanced cancer specificity; • Pgp-targeted PDT produced rapid tumor shrinkage, and significantly prolonged survival of tumor-bearing mice. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is the main site of protein synthesis, folding, and secretion to other organelles. The capacity of the ER to process proteins is limited, and excessive accumulation of unfolded and misfolded proteins can induce ER stress, which is associated with plant diseases. Here, a transgenic Arabidopsis system was established to express anti-cancer monoclonal antibodies (mAbs) that recognize the tumor-associated antigen GA733-2. Monoclonal antibody (mAb) CO17-1A recognize a tumor-associated epitope expressed on the colorectal cancer cell surface. The ER retention Lys-Asp-Glu-Leu (KDEL) motif sequence was added to the C-terminus of the heavy chain to retain anti-colorectal cancer mAbs in the ER, consequently boosting mAb production. Agrobacterium-mediated floral dip transformation was used to generate T1 transformants, and homozygous T4 seeds obtained from transgenic Arabidopsis plants expressing anti-colorectal cancer mAbs were used to confirm the physiological effects of KDEL tagging. Germination rates were not significantly different between both plants expressing mAb CO without KDEL mAb CO (CO plant) and mAb CO with KDEL mAb COK (COK plant). However, COK plants primary root lengths were shorter than those of CO plants and non-transgenic Arabidopsis plants in in vitro media. Most ER stress-related genes, with the exception of bZIP28 and IRE1a, were upregulated in COK plants compared to CO plants. Western blot and SDS-PAGE analyses showed that COK plants exhibited up to five times higher expression and mAb amounts than plants. Enhanced expression in mAb COK plants was confirmed by immunohistochemical analyses. mAb COK was distributed across most of the area of leaf tissues, whereas mAb CO was mainly distributed in extracellular areas. Surface plasmon resonance analyses revealed that mAb CO and mAb COK possessed equivalent or slightly better binding activities to antigen EpCAM compared to a commercially available parental antibody. N-glycosylation analysis showed that mAb CO had plant specific residues whereas mAb COK mainly showed an oligo-mannose N-glycan structure without the plant specific glycan residues. In this study, the reduction of plant growth and biomass induced by ER retention signal peptide might be only in in vitro conditions, and thus should be carefully considered for the initial screening for transgenic lines on culture media. Taken together, nevertheless the fusion of ER retention signal peptide is an effective approach for enhancing the yields of recombinant proteins in vivo. [ABSTRACT FROM AUTHOR]

Freeze-drying is the method of choice to dry formulations with biopharmaceutical drugs, to enhance protein stability. This is usually done below the glass transition temperature of maximally freeze-concentrated solutions (T g ′), to avoid protein aggregation, preserve protein activity, and obtain pharmaceutically ‘elegant’ cakes. Unfortunately, this is a lengthy and energy-consuming process. However, it was recently shown that drying above T g ′ or even above the collapse temperature (T c ) is not necessarily detrimental for stability of biopharmaceuticals, and hence provides an attractive option for freeze-drying cycle optimisation. The goal of the present study was to optimise the freeze-drying cycle for a model IgG monoclonal antibody (20 mg/mL) in sucrose and sucrose/glycine formulations, by reducing primary drying time. To study the impact of shelf temperature (T s ) and chamber pressure on product temperature (T p ), one conventional and five aggressive cycles were tested. Aggressive conditions during primary drying were achieved by increasing T s from −20 °C (conventional cycle) to 30 °C, with chamber pressure set to 0.1 mbar, 0.2 mbar or 0.3 mbar. These combinations of T s and chamber pressure resulted in T p well above T g ′, and in some cases, even above T c , without causing macrocollapse. Other critical quality attributes of the products were also within the expected ranges, such as reconstitution time and residual water content. Physical stability was tested using size exclusion chromatography, dynamic light scattering, and micro-flow imaging. All of the lyophilised samples were exposed to stress and the intended storage conditions, with no impacts on the product seen. These data show that implementation of aggressive conditions for the investigated formulations is possible and can significantly contribute to the reduction of primary drying times by up to 54% (from 48 to 22 h) in comparison to conventional freeze-drying. [ABSTRACT FROM AUTHOR]