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Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests. [Display omitted] [ABSTRACT FROM AUTHOR]

Long term stability of antibodies at room temperature is a major challenge in the commercialization of point-of-care devices for diagnostics. Since chitosan has been proven to be an excellent biofunctionalization material, the effects of four different biofunctionalization processes were studied to improve the room temperature stability of antibodies immobilized on chitosan modified paper-based microfluidic devices using blood typing antibodies as candidates. The devices used in this work have a flower-shaped design with 4 test zones at each corner. In three zones Anti-A, Anti-B, and Anti-D (Anti-Rh) antibodies are immobilized and the fouth zone represents the control (no antibodies) after biofunctionalization. The biofunctionalization of the paper devices was done with chitosan and chitosan cross-linked with sodium triphosphate pentabasic, glutaraldehyde, and sodium hydroxide. These devices were used for blood typing assays using real blood samples. A similar assay was also performed on unmodified (non-biofunctionalized) paper devices for comparison. Chitosan based biofunctionalized paper-devices showed better stability, up to 100 days as compared to 14 days on unmodified paper, at room temperature. Such biofunctionalized paper-based devices will be suitable for on-field and remote testing without any technical expertise and requirement for the cold chain. [ABSTRACT FROM AUTHOR]

This article presented the World Health Organization’s (WHO) recommendations on the use of Rabies vaccines excerpted from the Rabies vaccines: WHO position paper – April 2018 published in the Weekly Epidemiological Record [1] This position paper replaces the 2010 WHO position paper on rabies vaccines [2] . It presents new evidence in the field of rabies and the use of rabies vaccines, focussing on programmatic feasibility, simplification of vaccination schedules and improved cost-effectiveness. The recommendations concern the 2 main immunization strategies, namely vaccination for post-exposure prophylaxis and vaccination for pre-exposure prophylaxis. In the context of post-exposure prophylaxis, recommendations are also provided on the use of rabies immunoglobulins. Footnotes to this paper provide a number of core references including references to grading tables that assess the quality of the scientific evidence, and to the evidence-to-recommendation tables. In accordance with its mandate to provide guidance to Member States on health policy matters, WHO issues a series of regularly updated position papers on vaccines and combinations of vaccines against diseases that have an international public health impact. These papers are concerned primarily with the use of vaccines in large-scale immunization programmes; they summarize essential background information on diseases and vaccines, and conclude with WHO’s current position on the use of vaccines in the global context. Recommendations on the use of cholera vaccines were discussed by the Strategic Advisory Group of Experts (SAGE) in October 2017; evidence presented at these meetings can be accessed at: http://www.who.int/immunization/sage/meetings/2017/october/presentations_background_docs/en/ . [ABSTRACT FROM AUTHOR]

Total joint arthroplasty (TJA) has significantly improved the quality of life for millions suffering from end-stage arthritis. However, periprosthetic joint infections (PJI) remain a serious complication, necessitating extensive interventions and prolonged antimicrobial treatments. The aging population is expected to lead to a rise in TJA cases, subsequently increasing the incidence of PJI, particularly in the elderly who face higher mortality rates. Current diagnostic methods for suspected PJI, such as radiographs and biochemical markers like CRP and ESR, exhibit limited sensitivity. Therefore, there is a critical need for a specific synovial fluid biomarker assay to enhance PJI diagnosis using specific SF-based assay. This study introduces a novel microfluidic chip with a paper-based aptamer-sandwich assay for the quantitative detection of HNP 1, a crucial PJI biomarker, in synovial fluid. The assay leverages the advantages of aptamers over antibodies, demonstrating high selectivity and affinity for target molecules. The integration of a nitrocellulose (NC) membrane onto the microfluidic platform represents a significant advancement, reducing background signals and simplifying the assay procedure without intricate procedure and pre-treatment. The NC membrane-based microfluidic device offers rapid, cost-effective, and highly sensitive detection of HNP 1, with a limit of detection of 0.5 mg L−1. The microfluidic device demonstrates exceptional performance, detecting up to four clinical samples in approximately 42 min on a single chip with 100 % accuracy, as confirmed by analysis of 12 clinical samples and comparison with "gold-standard". Moreover, the assay exhibits a wide dynamic range of 0.5–100 mg L-1, underscoring its potential as a powerful tool for PJI diagnosis in clinical settings. This work introduces a paper-based microfluidic system tailored for rapid HNP 1 detection using synovial fluid near joint region (and not serum via blood) for better diagnosis. The innovative paper-based aptamer-sandwich assay yields results within 42-min. Significantly, it boasts a wide dynamic range, detecting levels from an impressive 0.5 mg L-1, crucial in the 2.6 mg L-1 threshold region. This heightened sensitivity and expansive detection capability establish our assay as a leader in PJI diagnostics, promising unmatched precision and efficiency in clinical applications. Schematic representation of the automated paper-based aptamer sandwich assay for periprosthetic joint infection (PJI) detection. The microfluidic chip features a reaction zone utilizing an NC membrane as the paper substrate, along with a dual aptamer assay for efficient aptamer-sandwich formation. Detection is achieved via the PMT module and subsequent analysis using fluorescent microscopy to differentiate PJI-positive and negative synovial fluid (SF) samples. [Display omitted] • We devised a novel paper-based aptamer-sandwich assay integrated into an automated microfluidic platform to detect HNP 1. • 42-minute swift operated assay, streamlined pretreatment steps, no extra quenching methods needed. • Clinical samples assessed on chip; results compared to gold standard ELISA (designed for serum, adjusted for SF). [ABSTRACT FROM AUTHOR]

Engineered collaborative biochemical techniques and regulated nanomaterials (NMs) offer extraordinary opportunities for improving the analysis performance of lateral flow immunoassay (LFIA). Herein, inspired by the ability of macromolecules (e.g., proteins) to assemble into new functional units and the remarkable optical performance of engineered regulated NMs, goat anti-mouse immunoglobulin (GAMI) serves as the "crosslinker" integrate with gold‑manganese oxide (Au-MnO x) to assemble the "signal tracers (STs)-crosslinker-antibody (mAb)" for elevating the mAb utilization efficiency. Notably, the "STs-crosslinker-mAb" assembly shows ~13.33-folds mAb utilization efficiency enhance, which perfectly response the challenge between limited sensitivity and sufficient signal intensity in competitive-type LFIA. The black color and rough structure of Au-MnO x offer higher colorimetric brightness (~2-folds than AuNPs) and enhanced mAb coupling efficiency (up to 92.47%), which further improves sensitivity under the premise of functional assembly to intensify the competitive immunoreaction. Additionally, the convenient synthesis conditions (~13 min at room temperature) even comparable to direct purchase commercial products indicate that using Au-MnO x undoubtedly increases the cost-effectiveness. Encouragingly, the Au-MnO x -GAMI-mAb based LFIA exhibited high sensitivity (LOD: 0.063 ng mL−1 for clenbuterol (CLE) monitoring) by elevating mAb utilization efficiency with the attendant enhancing immune competition response in a cost-effective manner, which provides an invigorating reference pathway in point-of-care immunoassay. [Display omitted] [ABSTRACT FROM AUTHOR]

Signal amplification in colorimetric biosensors usually requires labile reagents and/or additional steps, which are not ideal for in-field measurements. Here we show that the limit of detection of a gold nanoparticle-labeled colorimetric immunosensor can be decreased 10-fold by performing the assay on a folded piece of paper. In this approach, the intrinsic porosity of the substrate enables generating multiple signals with a single drop of reagent. This yields combined signals with higher optical density thanks to the semitransparent nature of the wet material. It will be shown that this method can decrease the limit of detection 10 times in a model immunosensor for the detection of immunoglobulins. The procedure is so straightforward that it could be easily implemented to decrease the limit of detection of microfluidic paper-based analytical devices with colorimetric readouts. [ABSTRACT FROM AUTHOR]

Among the analytical tools, paper-based analytical devices (PADs) have become a leading alternative for point-of care testing (POCT). In this study, PADs were fabricated using an office laser printer. Then, the paper zone was modified with graphene oxide (GO) and pyrene derivatives, which provide a sufficient amount of carboxylic groups for conjugating antibodies. At an optimal pH, antibodies were covalently bound onto carboxylated cellulose surface in an oriented manner through a two-step strategy: electrostatic adsorption was followed by EDC/NHS coupling. α-fetoprotein (AFP) as a detection model, we compared with cellulose powder modified and unmodified paper zone. The results showed the color intensity and color uniformity on GO modified paper was improved. The activity of immobilized antibodies on GO/1-pyrenebutyric acid (GO/PBA) modified was three times higher than that of GO modified and about 1.8-fold higher than that of GO/1-pyrenecarboxylic acid (GO/PCA) modified. The GO/PBA modified paper-based immunoassay has enhanced sensitivity and low detection limit. A linear correlation between color intensity and concentration of AFP in the range of 0.01～16.5 ng mL−1 with a detection limit of 9.0 pg mL−1 were achieved, respectively. The obtained results point towards rapid, sensitive, and specific early diagnosis of liver cancer at the point of care and other low-resource settings. ● Paper-based micro-plates was developed using an office laser printer. ● GO/PBA nanocomposites improve the mechanical strength of cellulose to enhance color uniformity. ● Antibodies were immobilized using physical adsorption combined with EDC cross-linking. ● The activity of immobilized antibodies was significantly improved. ● The paper-based immunoassay showed excellent sensitivity and detection limit for AFP detection. [ABSTRACT FROM AUTHOR]

In this paper, a new and facile method for the electrochemical determination of l -tyrosine was designed. First, 3-mercaptopropyl trimethoxysilane-functionalized silica nanoparticles were added to a paper disc. Then, the banana peel tissue and the mediator potassium hexacyanoferrate were dropped onto the paper, respectively. The modified paper disc was placed on the top of the graphite screen printed electrode and electrochemical characterization of this biosensor was studied by cyclic voltammetry and electrochemical impedance spectroscopy methods. The effective parameters like pH, banana peel tissue percentage, and the amount of mediator loading were optimized. l -tyrosine measurements were done by differential pulse voltammetry with a little sample (3 μL) for analysis. The biosensor showed a linear response for l -tyrosine in the wide concentration range of 0.05–600 μM and a low detection limit about 0.02 μM because of the co-catalytic effect of enzyme and nanoparticles. The stability of the biosensor and its selectivity were evaluated. This biosensor was applied for the voltammetric determination of l -tyrosine in the blood plasma sample. The results of the practical application study were comparable with the standard method (HPLC). In conclusion, a simple, inexpensive, rapid, sensitive and selective technique was successfully applied to the l -tyrosine analysis of the little samples. [ABSTRACT FROM AUTHOR]

Microfluidic paper-based analytical devices have been widely used for immunoassays, especially in resource-limited settings. Antibodies that have been immobilized on the cellulose surface by physical adsorption become unstable as a result of antibodies’ desorption caused by the washing process and lose their activity. In this paper, we present a new approach for the covalent immobilization of antibodies on the paper surface. More precisely, sodium periodate (NaIO 4 ) was used to activate the cellulose for introducing aldehyde groups. Antibodies were bound to the oxidized cellulose paper surface through imine formation (Schiff base) between the aldehyde groups and the primary amine groups of the antibody. Thus, using α-fetoprotein (AFP) as a model, a new paper-based sandwich ELISA was developed. The linear range of the assay for AFP was from 0.1 ng/mL to 11.2 ng/mL, the detection limit was 0.04 ng/mL, and the recovery rate of AFP spiked into human serum sample ranged from 89.2% to 101.5%. The intra- and inter-assay coefficients of variations obtained were 4.6–7.1% and 5.8–10.3% for standard AFP, respectively. Compared with the conventional paper-based ELISA, the developed assay exhibited superiority in terms of sensitivity, lower detection limit, stability and reliability. This simple and sensitive paper-based sandwich ELISA holds promise in clinical applications. [ABSTRACT FROM AUTHOR]

Highly sensitive and specific diagnostics for cannabis usage are essential for rapid on-site screening for illicit drug usage. To improve the sensitivity of THC immunoassays, a proper immobilization of the sensing elements on the sensor substrate is critical. In this work, we demonstrated the utilization of EDC/NHS coupling chemistry with nanocellulose to obtain efficient anchor layers for the immobilization of anti-immune complex antibodies on surfaces. In our approach, the high surface-to-volume ratio, OH-group-rich surface, and high hygroscopicity of TOCNF enable efficient surface functionalization and enhance water permeation inside the nanocellulose network structure, offering a hydrophilic spacer for the sensing antibodies. THC detection was shown in both SPR (surface plasmon resonance technique) and paper-based sensing systems. In SPR, antibody immobilization and the related interactions with the target molecule complex with 1–10 μg/mL THC were followed in-situ in aqueous environment, revealing robust attachment of the antibody to the nanocellulose layer and preserved bioactivity. Additionally, quantitative THC detection was enabled on paper substrate by colorimetric means by employing labeled anti-THC Fab antibody fragments as detection antibodies. THC detection efficiency of covalently linked biointerface was superior compared to the performance of physically linked biointerface. The chemical conjugation of anti-IC to nanocellulose allowed efficient binding, whereas supramolecular conjugation led to insufficient binding, highlighting the relevance of the developed nanocellulose-based anchor layer. Nanocellulose EDC/NHS coupling chemistry to obtain efficient immobilization of cannabis-specific antibodies on surfaces for solid sensors and paper-based immunoassays. [Display omitted] [ABSTRACT FROM AUTHOR]

Paper has been used to engineer many types of bio-diagnostics. A major issue to most paper-based bio-diagnostics is the biomolecule instability causing the short shelf-life of the diagnostics. Commercial papers contain various polymeric additives. Polyamidoamine-epichlorohydrin (PAE), a polyelectrolyte typically used as wet-strength agent, is commonly used in filter papers and paper towels, which are often used as substrate in bioactive paper. However, the effect of cellulose or polymeric additives on antibody bioactivity is unknown. This limits paper optimization for diagnostic applications. In this study, model papers were made with and without PAE addition. IgM Anti-A blood typing antibody was physisorbed and dried on paper, aged for up to 9 weeks at different relative humidity (RH) conditions and the antibody activity was measured. The antibody bioactivity was represented as blood typing efficiency measured by image analysis. The surface chemical composition was measured using X-ray photoelectron spectroscopy (XPS). Antibody bioactivity loss was promoted by elevated RH, corresponding to increased paper water content. PAE significantly reduces the paper water content under ambient environment. Antibody bioactivity is higher on paper made with PAE under the high humidity conditions (57.6%–84.3% RH). However, under conditions of humidity saturation (100%RH), PAE shows little effect on reducing paper water content nor on protecting antibody bioactivity. These results demonstrate the water content of paper to be associated with antibody bioactivity loss. [ABSTRACT FROM AUTHOR]

Abstract: Background: Measurements of human growth hormone (hGH) are a prerequisite for identifying a deficiency or excess. Our study is the first to investigate the reliability of a very sensitive assay for the quantification of GH in dried blood spots on filter paper. Objective: Validation of a commercially-available enzyme-linked immunoassay (ELISA) for measuring hGH from filter paper samples of dried blood. Methods: We used an assay system (ELISA, E022, Mediagnost) based on polyclonal rabbit antibodies. Its suitability is ascribable to its very high sensitivity (1.6ng/L) and virtual absence of interfering factors, excepting for a cross-reactivity with high pegvisomant concentrations. Results: hGH was found to be stable in dried blood spots on filter paper (No. 903, Whatman) over eight days at 37°C. Extraction of hGH from filter paper, in comparison to EDTA plasma, was 107% (SD 8.1%; n =6) over a range from 2.4 to 34.5μg/L. Linear regression analysis (n =119) showed a correlation of R 2 =0.97 for the hGH concentration in serum and on filter paper samples. Conclusion: Our findings demonstrate the reliability of measurements of hGH in dried blood spots on filter paper. The advantages of this method are the low sample volume and the easy transport, storage, and handling of samples. This method contributes to the standardisation of diagnostics pertaining to abnormal hGH secretion as it facilitates the comparison of decisive measurements. [Copyright &y& Elsevier]

A novel protein immobilization method based on plasma treatment of paper on the low-cost paper-based immunodevice was established in this work. By using a benchtop plasma cleaner, the paper microzone was treated by oxygen plasma treatment for 4 min and then the antibody can be directly immobilized on the paper surface. Aldehyde group was produced after the plasma treatment, which can be verified from the fourier transform infrared spectroscopy (FT-IR) spectra and x-ray photoelectron spectroscopy (XPS) spectra. By linked to aldehyde group, the antibody can be immobilized on the paper surface without any other pretreatment. A paper-based immunodevice was introduced here through this antibody immobilization method. With sandwich chemiluminescence (CL) immunoassay method, the paper-based immunodevice was successfully performed for carcinoembryonic antigen (CEA) detection in human serum with a linear range of 0.1–80.0 ng/mL. The detection limit was 0.03 ng/mL, which was 30 times lower than the clinical CEA level. Comparing to the other protein immobilization methods on paper-based device, this strategy was faster and simpler and had potential applications in point-of-care testing, public health and environmental monitoring. [ABSTRACT FROM AUTHOR]

A novel device for targeting proteins with a Molecularly-Imprinted Polymer (MIP) recognition material is presented herein, by employing wax paper with conductive ink as background support. Cellulose paper was modified with paraffin wax to make it waterproof, and afterwards covered by a carbon ink to make it conductive, achieving the goal to make easy and low cost supports, and, in this case, with good detection features. Bovine Serum Albumin (BSA) was used herein as target protein. Albumin has an important role in the human body and BSA is normally used as its model protein because of its good stability and low cost. The development of BSA detection systems is important in various fields, such as biochemistry, medicine and pharmacy. Raman measurements were conducted during all preparation stages of the recognition material and electrochemical assays were used to evaluate the analytical performance of the sensor. The assembly of the device was optimized by employing different polymerization timings and monomers of different chemistries and charges. In general, longer polymerization periods and the use of a positively charged monomer benefited the analytical response. The results obtained within different calibrations indicated a linear response over a wide concentration range, from 0.005 to 100 mg/mL of BSA in buffer solution. In addition, the analytical evaluation of the device under several background solutions, including synthetic and real urine, supported the good performance of the sensor and pointed out that the principle of sensor assembly may be extended to other proteins in urine. In addition, the detection capabilities of the device proposed herein are better than other BSA sensing devices relying on electrochemical transduction. [ABSTRACT FROM AUTHOR]

Paper with immobilized protein can provide a low-cost platform for diagnostics, but evidence is emerging that the instability of protein on paper during shipping and storage severely limits its commercial development. Of these, paper-immobilized antibody, though widely used, is in urgent need of improvement in the shelf life. Herein we provided a detailed investigation of a simple and versatile approach for achieving stable antibody on paper to improve bioassay performance. Anti-Blood Group A IgM was chosen as the model antibody and its activity was tested via agglutination reactions. Representative polysaccharides (i.e., chitosan, sodium alginate and dextran) were used as protectants and their ability to stabilize antibody under heat and desiccation stresses was investigated. It was found that the presence of dextran with high concentration markedly stabilized paper-immobilized IgM for at least 120 days. Our results indicate that dextran with good film-forming ability could be a protectant for paper-immobilized antibody. [ABSTRACT FROM AUTHOR]

This article describes a 3D microfluidic paper-based analytical device that can be used to conduct an enzyme-linked immunosorbent assay (ELISA). The device comprises two parts: a sliding strip (which contains the active sensing area) and a structure surrounding the sliding strip (which holds stored reagents—buffers, antibodies, and enzymatic substrate—and distributes fluid). Running an ELISA involves adding sample (e.g. blood) and water, moving the sliding strip at scheduled times, and analyzing the resulting color in the sensing area visually or using a flatbed scanner. We demonstrate that this device can be used to detect C-reactive protein (CRP)—a biomarker for neonatal sepsis, pelvic inflammatory disease, and inflammatory bowel diseases—at a concentration range of 1–100 ng/mL in 1000-fold diluted blood (1–100 µg/mL in undiluted blood). The accuracy of the device (as characterized by the area under the receiver operator characteristics curve) is 89% and 83% for cut-offs of 10 ng/mL (for neonatal sepsis and pelvic inflammatory disease) and 30 ng/mL (for inflammatory bowel diseases) CRP in 1000-fold diluted blood respectively. In resource-limited settings, the device can be used as a part of a kit (containing the device, a fixed-volume capillary, a pre-filled tube, a syringe, and a dropper); this kit would cost ~ $0.50 when produced in large scale (>100,000 devices/week). This kit has the technical characteristics to be employed as a pre-screening tool, when combined with other data such as patient history and clinical signs. [ABSTRACT FROM AUTHOR]

Abstract: Dried blood spot samples (DBSS) on filter paper offer several advantages compared to conventional serum/plasma samples: they do not require any phlebotomy or separation of blood by centrifugation; they are less invasive; they allow sample stability and shipment at room temperature; and they pose a negligible risk of infection with blood-borne viruses, such as HIV, HBV and HCV, to those who handle them. Therefore dried blood spot samples (DBSS) on filter paper can be a quick, convenient and inexpensive means of obtaining blood samples for biomarker discovery, disease screening, diagnosis and treatment monitoring in non-hospitalized, public health settings. In this study, we investigated for the first time the potential application of dried blood spot samples (DBSS) in protein expression profiling using antibody array technology. First, optimal conditions for array assay performance using dried blood spot samples (DBSS) was established, including sample elution buffer, elution time, elution temperature and assay blocking buffer. Second, we analyzed dried blood spot samples (DBSS) using three distinct antibody array platforms, including sandwich-based antibody arrays, quantitative antibody arrays and biotin-label-based antibody arrays. In comparison with paired serum samples, detection of circulating proteins in dried blood spot samples (DBSS) correlated well for both low- and high-abundance proteins on all three antibody array platforms. In conclusion, our study strongly indicates the novel application of multiplex antibody array platforms to analyze dried blood spot samples (DBSS) on filter paper represents a viable, cost-effective method for protein profiling, biomarker discovery and disease screening in a large, population-based survey. [Copyright &y& Elsevier]

Aflatoxingenetic fungi producing highly toxic and carcinogenic mycotoxins and threatening human health widely exist in our living environment and food. Taking aflatoxingenetic fungi as an analyte example, here we report a way to improve the sensitivity of paper-based sandwich immunosensor for macromolecule. With a biomarker-specific nanobody and polyclonal antibody (IgG), two modes of paper-based immunosensors were constructed, immobilization nanobody mode and immobilization IgG mode. It was found that the sensitivity of the immobilization nanobody mode was at least 500 times higher than that of the immobilization IgG mode. The reasons for the large difference in sensitivity were also theoretically. Under the optimized conditions, the immobilization nanobody mode sensitivity reached 0.035 μg/mL aflatoxingenetic mycelia, each assay was finished within 15 min, and the spiked recoveries of aflatoxingenetic mycelium reference in peanuts were 77.0∼91.6%. And then, two important application cases were successfully demonstrated to identify the abundance of aflatoxingenetic fungi in market peanuts and peanut rhizosphere soil for environmental safety evaluation. This is the first report of time-resolved paper-based sandwich immunosensor for aflatoxingenetic fungi with nanobody immobilization, and the first sensitivity comparison between nanobody immobilization and IgG immobilization, which provides a new approach to develop highly sensitive immunosensor for macromolecular hazard materials. • The sensitivity of the immobilization nanobody mode was over 500 times higher than that of the immobilization IgG mode. • It provides a new approach to develop ultrahigh sensitive immunosensor for macromolecular hazard materials. • The developed paper-based sandwich immunosensor with LOD for aflatoxingenetic fungi reference was 0.035 μg/mL. • Evaluating risk of aflatoxingenetic fungi in food and environment. [ABSTRACT FROM AUTHOR]

Highlights: [•] The current assessment of rabies immunity in wildlife faces technical difficulties. [•] Field sampling of fox and raccoon dog blood on filter papers (FP) is feasible. [•] The FP method allows the storage, transport and ELISA titration of blood samples. [•] The FP sampling method was found highly specific and repeatable in both wild species. [•] We report a reliable alternative to assess effectiveness of oral rabies vaccination. [ABSTRACT FROM AUTHOR]

Abstract: A novel lab-on-paper device combining the simplicity and low-cost of microfluidic paper-based analytical devices (μPADs) and the sensitivity and selectivity of chemiluminescence ELISA (CL-ELISA) for the high-throughput, rapid, stable and reusable point-of-care testing is presented here. Chitosan was used to modify μPADs to covalently immobilize antibodies on μPADs. Thus, sandwich CL-ELISA on μPADs can be easily realized for further development of this technique in sensitive, specific and low-cost application. The paper device was fabricated by a low-cost, simple, and rapid wax-screen-printing method. Using tumor markers and paper microzone plate as model, the application test of this paper-based CL-ELISA was successfully performed with a linear range of 0.1–35.0ngmL−1 for α-fetoprotein, 0.5–80.0UmL−1 for cancer antigen 125 and 0.1–70.0ngmL−1 for carcinoembryonic antigen. Since the cutoff values of the three tumor markers in clinical diagnosis are 25ngmL−1, 35UmL−1 and 5ngmL−1, the sensitivity and linear ranges of the proposed method were enough for clinical application. In addition, this lab-on-paper immunodevice can provide reproducible results upon storage at 4°C (sealed) for at least 5 weeks. Ultimately, this novel chitosan modification and wax-screen-printing methodology for μPADs can be readily translated to other signal reporting mechanism including electrochemiluminescence and photoelectrochemistry, and other receptors such as enzyme receptors and DNA receptors for determination of DNA, proteins and small molecules in point-of-care testing. [Copyright &y& Elsevier]

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a global pandemic outbreak. To date, approximately one million deaths and over 32 million cases have been reported. This ongoing pandemic urgently requires an accurate testing device that can be used in the field in a fast manner. Serological assays to detect antibodies have been proven to be a great complement to the standard method of reverse transcription-polymerase chain reaction (RT-PCR), particularly after the second week of infection. We have developed a specific and sensitive immunosensor for immunoglobulin detection produced against SARS-CoV-2. Unlike other lateral flow-based assays (LFAs) involving the utilization of multiple antibodies, we have reported a label-free paper-based electrochemical platform targeting SARS-CoV-2 antibodies without the specific requirement of an antibody. The presence of SARS-CoV-2 antibodies will interrupt the redox conversion of the redox indicator, resulting in a decreased current response. This electrochemical sensor was proven effective in real clinical sera from patients with satisfactory results. In addition, the proposed format was also extended to antigen detection (the spike protein of SARS-CoV-2), which presents new possibilities for diagnosing COVID-19. • Electrochemical sensor for COVID-19 diagnostic. • Serological assay for SARS-CoV-2 antibodies was demonstrated. • Sensitivity enhancement compared to the traditional LFA devices. • Applicable to real clinical sera with satisfactory results. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to investigate the microagglutination test (MAT) results in serum samples dried on filter paper and stored at different temperatures during 1day, 7days, 30days and 1year to determine the stability of sera antibody against leptospires. Serum samples collected onto filter paper for the detection of leptospires antibody was compared with MAT in a study of 300 serum samples from patients with suspected leptospirosis. Among 300 fresh serum samples analyzed by MAT 156 (52%) were positive and 144 (48%) negative. All the negative fresh serum samples were negative when dried on filter paper (specificity 100%). The sensitivity of MAT performed on dried serum samples was 100%. Storage on filter paper at room temperature and at 4°C for 1 and 7days did not affect the MAT titers. For up to 7days, 98.72% of dried serum samples had titers identical to those of the corresponding serum samples, and 1.18% of dried serum samples showed 1 dilution of difference. After a storage period of one month a prozone phenomenon was observed. After a storage period of one year all serum samples were negative. Serum samples collected onto filter paper are a convenient source of antibodies for serological diagnosis and epidemiological surveys. [Copyright &y& Elsevier]

This paper analyzes the investigation of the distribution of the protein tenascin-C in canine mammary tumors. The method involved immunohistochemistry of tissue slices, performed by the application of an antibody to tenascin-C that specifically can be made visible for microscopic inspection. The first phase of the project is the making of the protocol, the second the deduction of a pattern of tenascin-C distribution in tumors and the third the writing of a paper. Each of the phases is analyzed separately, using the concept of resistance and accommodation. My purpose is to show that in each phase of the process of producing knowledge, the scientist meets resistances which force him to accommodate by changing his conceptual, technical and methodological approaches. In reverse, the details of the non-human agent (protocol, pattern or paper) have to be accommodated to the wishes and expectations of the scientist. Through this interaction a situation of stability of knowledge is reached at the end of each phase. In the protocol phase, resistance is found in the antibody and tissue slices. In the phase of pattern deduction the resistance is in the pathological diagnosis of the tumors and the expectations and hypothesis with which the scientist had entered the project; in the criteria to be used for assigning the slices to a tenascin-C pattern; and in the responses of colleagues and supervisor. In the paper-writing phase the interaction is between the scientist and the scientific community which should take on board the knowledge from the research project. When stabilization of knowledge is obtained in one of the phases, the agents of resistance turn into allies in the next phase, giving support to accommodating the resistances in this later phase. Second, the stabilization of knowledge of the protocol is further enhanced when stabilization of the pattern is achieved; in addition, knowledge of the pattern is more definite when it has become stabilized and closed knowledge within the science community. [Copyright &y& Elsevier]

Abstract: Edwin Bickerstaff and Philip Cloake reported in the 1950's three cases of reversible encephalitis. The concept of antibody associated encephalitis had not been proposed at the time they astutely recognized the importance of disease pattern recognition and postulated a potential immune based mechanism. The syndrome defined by Bickerstaff of progressive, external ophthalmoplegia and ataxia, with disturbance of consciousness or hyperreflexia, has subsequently been associated with anti-GQ1b antibodies. Interestingly one of the three original cases, a young woman who developed seizures, an eye movement disorder and acute psychosis while awaiting ovarian cystectomy, has features that may be more consistent with anti-NMDA receptor encephalitis. [Copyright &y& Elsevier]

For many years, space human factors research has focused on the impact of microgravity on human physiology and the health risks astronauts face during space flights of various durations. Our review paper reveals that microgravity leads to both reversible and non-reversible effects on astronauts' oral health. In addition to oral health concerns, astronauts often experience muscle degeneration during space missions, which is closely tied to bone loss. Despite our knowledge of high bone resorption and rapid mineral and calcium loss in microgravity, the exact underlying mechanisms remain unclear. However, to fully understand these processes and develop effective strategies to counteract the consequences of microgravity, more research is needed. This includes a deeper exploration of how these molecules and other biomarkers work, identification of early mechanotransduction sensors, and a comprehensive understanding of the physiological effects of weightlessness. Current solutions primarily involve administering antioxidants, regular exercise, and appropriate nutrition. Additionally, innovative approaches such as experimental drug therapies using anti -myostatin antibodies and r-irisin have gained attention. Further large-scale, long-term studies are required to understand the effects of microgravity on the oral cavity and prevent its adverse consequences. • Impact of Microgravity on Dental Health: Discuss the physiological changes experienced by astronauts in microgravity environments, including fluid redistribution, decreased mechanical loading, and altered bone metabolism, and their implications for oral health. • Weight-Bearing vs. Non-Weight-Bearing Bones: Examines the relationship between microgravity and bone loss, with a focus on distinguishing its effects on weight-bearing and non-weight-bearing bones. • Comprehensive Impact Assessment: Calls for comprehensive research on how microgravity affects various bones in both animals and humans, including analyses of bone structure, density, and strength. • Bone Loss and Tooth Mobility: Examine how microgravity-induced bone loss affects the integrity of the jawbone, leading to increased tooth mobility and potential dental complications such as loosened crowns, dental implants, or even tooth loss. • Differential Recovery: Investigates how weight-bearing and non-weight-bearing bones recover following bone loss in microgravity conditions and the underlying mechanisms at play. • Addressing Inconsistencies: Highlights the need to resolve inconsistencies in existing literature regarding the influence of microgravity on osteogenic proliferation and differentiation. • Osteocytes and Osteoclasts in Microgravity: Emphasises the importance of understanding the roles of osteocytes and osteoclasts in microgravity-induced bone loss to identify potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Lung transplantation. like other transplants, carries a risk of graft rejection due to genetic differences between the donor and the recipient. In this paper, we focus on antibody-mediated rejection, which can cause acute and more importantly chronic graft dysfunction and subsequently shortened allograft survival. We present the case of a 46-year-old patient who, two months after lung transplantation (LTx), developed AMR manifested by the deterioration of graft function and de novo production of donor-specific antibodies (DSA): DQ3 (DQ7, DQ8. DQ9). As the patient was after left single LTx and heavily oxygen dependent a transbronchial biopsy was deemed to be high risk and it was decided to determine the clinical significance of the detected antibodies by their ability to bind complement. The test confirmed that the detected DSAs have the ability cause cytotoxicity of the transplanted organ. After treatment with methotrexate, intravenous immunoglobulin G (IVIg) and alemtuzumab, the patient's condition improved and a complete decrease in DSA was obtained. However, after a year, the production of antibodies increased sharply. Treatment with IVIg, cyclophosphamide and plasmapheresis slightly improved the patient's condition, reducing the MFI DSA values by half, but leaving them at high levels. Based on this clinical case, we discuss problems with making a diagnosis, choosing the right AMR treatment and monitoring the patient's condition during treatment. We also indicate a poor prognosis in the case of the production of DSA antibodies at tlie DQ locus. [ABSTRACT FROM AUTHOR]

Antibody-drug conjugates unite the specificity and long circulation time of an antibody with the toxicity of a chemical cytostatic or otherwise active drug using appropriate chemical linkers to reduce systemic toxicity and increase therapeutic index. This combination of a large biological molecule and a small molecule creates an increase in complexity. Multiple production processes are required to produce the native antibody, the drug and the linker, followed by conjugation of afore mentioned entities to form the final antibody-drug conjugate. The connected processes further increase the number of points of control, resulting in necessity of additional specifications and intensified analytical characterization. By combining scientific understanding of the production processes with risk-based approaches, quality can be demonstrated at those points where control is required and redundant comparability studies, specifications or product characterization are avoided. Over the product development lifecycle, this will allow process qualification to focus on those areas critical to quality and prevent redundant studies. The structure of the module 3 common technical document for an ADC needs to reflect each of the production processes and the combined overall approach to quality. Historically, regulatory authorities have provided varied expectations on its structure. This paper provides an overview of essential information to be included and shows that multiple approaches work as long as adequate cross-referencing is included. [ABSTRACT FROM AUTHOR]

• This review paper underscores how recent advances in artificial intelligence, particularly deep learning models, have revolutionized antibody design by accurately predicting antibody structures from amino acid sequences, addressing historical challenges in antibody structure prediction. • This paper discusses how AI techniques have significantly improved the accuracy of predicting antibody-antigen interactions, enhancing our understanding of antibody recognition. It emphasizes the importance of combining computational tools with experimental methods for more precise predictions. • This review paper sheds light on how AI methods, especially deep learning, have streamlined the process of antibody affinity maturation by predicting mutation effects. It emphasizes the need for considering environmental factors and molecular water in mutation prediction models. Given their high affinity and specificity for a range of macromolecules, antibodies are widely used in the treatment of autoimmune diseases, cancers, inflammatory diseases, and Alzheimer's disease (AD). Traditional experimental methods are time-consuming, expensive, and labor-intensive. Recent advances in artificial intelligence (AI) technologies provide complementary methods that can reduce the time and costs required for antibody design by minimizing failures and increasing the success rate of experimental tests. In this review, we scrutinize the plethora of AI-driven methodologies that have been deployed over the past 4 years for modeling antibody structures, predicting antibody–antigen interactions, optimizing antibody affinity, and generating novel antibody candidates. We also briefly address the challenges faced in integrating AI-based models with traditional antibody discovery pipelines and highlight the potential future directions in this burgeoning field. [ABSTRACT FROM AUTHOR]

• Heterologous mRNA boost following GLS-5310 DNA vaccination was well tolerated. • Neutralizing antibody responses were enhanced 110 fold to GMT of 1244.8. • Binding antibody responses were enhanced 1,187-fold to GMT of 405,920. • Boosted antibody responses similar to other vaccine combinations. • Boosted T cell responses were many-fold higher than other vaccine combinations. Heterologous boost regimens are being increasingly considered against SARS-CoV-2. We report results for the 32 of 45 participants in the Phase 1 CoV2-001 clinical trial (Kim et al., Int J Iinfect Dis 2023, 128:112–120) who elected to receive an EUA-approved SARS-CoV-2 mRNA vaccine 6 to 8 months following a two-dose primary vaccination with the GLS-5310 bi-cistronic DNA vaccine given intradermally and followed by application of suction using the GeneDerm device. Receipt of EUA-approved mRNA vaccines after GLS-5310 vaccination was well-tolerated, with no reported adverse events. Immune responses were enhanced such that binding antibody titers, neutralizing antibody titers, and T-cell responses increased 1,187-fold, 110-fold, and 2.9-fold, respectively. This paper is the first description of the immune responses following heterologous vaccination with a DNA primary series and mRNA boost. [ABSTRACT FROM AUTHOR]

Mathematical models have been considered as a robust tool to support biological and medical studies of the coronavirus disease 2019 (COVID-19). This new disease is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This paper develop a within-host SARS-CoV-2 dynamics model with logistic growth for healthy epithelial cells, humoral (antibody) immune response and general SARS-CoV-2-target incidence rate. The model is incorporated with four mixed (distributed/discrete) time delays, delay in the formation of latent infected epithelial cells, delay in the formation of active infected epithelial cells, delay in the activation of latent infected epithelial cells, and maturation delay of new SARS-CoV-2 particles. The model is formulated as a system of delay differential equations (DDEs). We establish that the model's solutions are non-negative and ultimately bounded. We deduce that the model has three equilibria and their existence and stability are perfectly determined by two threshold parameters. We prove the global stability of the model's equilibria by utilizing the Lyapunov method and applying the LaSalle's invariance principle. To support and illustrate our theoretical findings we present numerical simulations for the model with a special form of the general incidence rate function. The effect of time delays on the SARS-CoV-2 dynamics is addressed. We observe that increasing time delays values can have the same impact as drug therapies in suppressing viral progression. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate enzyme linked immunosorbent assay (ELISA) as a testing strategy for detection of antibodies against measles virus from microquantities of blood soaked onto filter paper. We studied 165 healthy children in the age group of 1 to 2 years, attending the outpatient department of pediatrics. Two sets of samples were collected from each child. One by venipuncture and the other on Whatman filter paper-3 discs of 20 mm size by finger or heel prick so that each strip is completely soaked with blood on both sides. These were tested for measles virus antibodies by ELISA using Melotest measles IgG commercial ELISA kit manufactured by Melotec S. A. (Barcelona, Spain). The resulting sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the filter paper (FP) ELISA compared to serum ELISA was 100, 90, 97.8, and 100%, respectively. The correlation coefficient r = 0.93% (p < 0.001) and the agreement between the two techniques was 98% as calculated by the Kappa statistical method. The present study has found filter paper testing by ELISA to be a promising qualitative technique for detection of immunity against measles. [Copyright &y& Elsevier]

[Display omitted] • SARS-CoV-2 detection strategies using biosensors. • Advances in 2D material-based SARS-CoV-2 detection. • Physicochemical properties of 2D materials beyond graphene in biorecognition. • Perspectives in viral disease diagnostics. The SARS-CoV-2 pandemic has resulted in a devastating effect on human health in the last three years. While tremendous effort has been devoted to the development of effective treatment and vaccines against SARS-CoV-2 and controlling the spread of it, collective health challenges have been encountered along with the concurrent serious economic impacts. Since the beginning of the pandemic, various detection methods like PCR-based methods, isothermal nucleic acid amplification-based (INAA) methods, serological methods or antibody tests, and evaluation of X-ray chest results have been exploited to diagnose SARS-CoV-2. PCR-based detection methods in these are considered gold standards in the current stage despite their drawbacks, including being high-cost and time-consuming procedures. Furthermore, the results obtained from the PCR tests are susceptible to sample collection methods and time. When the sample is not collected properly, obtaining a false result may be likely. The use of specialized lab equipment and the need for trained people for the experiments pose additional challenges in PCR-based testing methods. Also, similar problems are observed in other molecular and serological methods. Therefore, biosensor technologies are becoming advantageous with their quick response, high specificity and precision, and low-cost characteristics for SARS-CoV-2 detection. In this paper, we critically review the advances in the development of sensors for the detection of SARS-CoV-2 using two-dimensional (2D) materials. Since 2D materials including graphene and graphene-related materials, transition metal carbides, carbonitrides, and nitrides (MXenes), and transition metal dichalcogenides (TMDs) play key roles in the development of novel and high-performance electrochemical (bio)sensors, this review pushes the sensor technologies against SARS-CoV-2 detection forward and highlights the current trends. First, the basics of SARS-CoV-2 detection are described. Then the structure and the physicochemical properties of the 2D materials are explained, which is followed by the development of SARS-CoV-2 sensors by exploiting the exceptional properties of the 2D materials. This critical review covers most of the published papers in detail from the beginning of the outbreak. [ABSTRACT FROM AUTHOR]

α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples. [Display omitted] • The first report ever on nanobodies against α-hemolysin of Staphylococcus aureus. • RANbody (Nanobody-vHRP fusion protein) with excellent recognition and catalytic activity was generated. • Sandwich ELISA based on RANbody eliminates the need for a second antibody, and saves detection time and reagents. • The sandwich ELISA was used successfully to evaluate α-hemolysin in food samples. [ABSTRACT FROM AUTHOR]

Background Patients with end-stage renal failure (ESRD) require hemodialysis. According to this point that CMV infection is related to mortality in immunocompromised, and damages in hemodialysis, this study is designed to survey the seroprevalence of CMV and primary infection in hemodialysis (HD) patients. Material and methods Current cross-sectional studies were found by online reviewing the national and international databases (Web of Science, Pubmed, Scopus, Science Direct, Google scholar), and suitable studies were selected. NOS checklist were used for evaluation of the qualities of all papers. Assessment of heterogeneity among the studies of primary studies was performed using Chi-squared test (Cochran's Q) and I2 index (significance level of 50%). The statistical analysis were performed using Comprehensive Meta-Analysis (CMA) V.2. For assessing publication bias, the Egger test was used by subjective judgment in each study. Also the effect of potential factor on heterogeneity of studies was performed by Meta-regression test and the impact of each study on the overall estimate was assessed by sensitivity analysis. Results This meta- analysis included 23 primary studies investigating seroprevalence of Cytomegalovirus antibodies and primary infection among hemodialysis patients. Finding showed that seroprevalence of CMV IgG and IgM antibodies in hemodialysis patients at 95% confidence interval using random effect model was 88.7% (81.7–93.2%) and 10.9% (5.9–19.2%) respectively. Also the frequency of CMV infection among these studies were reported 41% (18.8–67.6%). Conclusion We found that frequency of CMV IgG was higher and it was detected in HD patients. The result of our study showed that most patients were exposure to CMV during dialysis may reactivate by various stimulation, including immunosuppression and inflammation, so screening of patients should be performed to prevent future consequences such as kidney transplant rejection during of dialysis. [ABSTRACT FROM AUTHOR]

The development of a rapid, affordable, and sensitive diagnostic kit for point-of-care is important in most healthcare settings. In this follow-up paper to our previous work on quantification of dengue NS1 protein via impedimetric measurement, our present technology aims to provide quantification by utilizing proprietary stabilized and improved electroactive immunonanoparticles that bind to the target biomarker and subsequently move along toward the biofunctionalized screen-printed gold electrodes (SPGE) to generate an amperometric signal. The SPGE functions simultaneously as a signal transducer and a solid-state support for a sandwich ELISA-like immunoassay. The successful immunocomplex formation is then recorded electrochemically using a potentiostat, whereby the signal was contributed by the presence of a more hydrophilic redox label than ferrocene, namely radical TEMPO (TEMPO ), on the formulated nanoparticles. In this paper, a bifunctional ligand, thiolated polyethylene glycol (PEG-thiol) polymer, was used to stabilize 20 nm gold colloidal nanoparticles (AuNPs) in the formulation. PEG was incorporated to not only prevent the salt-mediated AuNPs aggregations, but also provide an anchor for antibody and redox species conjugation. To-date, we have successfully miniaturized a 3D-printed prototype device able to sensitively detect and quantify dengue NS1 protein with only 0.6 μL human clinical serum samples diluted in a volume ratio of 1:100 (PBS diluent) in less than 30 min with a simple cyclic voltammetry analysis. The positive and negative samples were also tested with ELISA, a gold standard validation method, which means that the development of the prototype is a promising start as a point-of-care diagnostic technology. [ABSTRACT FROM AUTHOR]

Vaccine reactogenicity is well documented at the clinical level but the mechanism involved at the local or systemic level are still poorly understood. Muscular tissue where most vaccines are administered is the first place of interaction between the vaccine formulation and the host's immune cells. So far, this site of vaccine administration is not well documented from a mechanistic standpoint. The study of early molecular events at the injection site is crucial to understand the local response to vaccines. In this paper, we report a standardized workflow, from the injection of vaccine formulations in rabbit muscle, to the analysis by desorption electrospray ionization and histology staining to understand the role of lipids involved in the inflammation and its resolution on striated muscular tissue. The analysis of lipid mediators was optimized at the site of needle insertion to allow the spatial comparison of cellular infiltrates at the injection site. We showed that lipids were distributed across the spatial tissue morphology in a time-dependent manner. The MS imaging applied to vaccinology could pave the way to a better understanding of vaccine reactogenicity and mechanism of action. [Display omitted] [ABSTRACT FROM AUTHOR]

Bio-affinity drug screening strategies have gained popularity in pre-clinical and clinical drug discovery for natural products, small molecules, and antibodies due to their superior selectivity, large number of compounds to be screened, and ability to reduce the time and cost of the drug discovery process. [Display omitted] • Bioaffinity strategies have high efficiency for clinical drug discovery. • Bioaffinity strategies are suitable for screening different drug types. • Continuous and latest bioaffinity drug screening strategies have been reported. Bioaffinity drug screening strategies have gained popularity in preclinical and clinical drug discovery for natural products, small molecules and antibodies owing to their superior selectivity, the large number of compounds to be screened and their ability to minimize the time and expenses of the drug discovery process. This paper provides a systematic summary of the principles of commonly used bioaffinity-based screening methods, elaborates on the success of bioaffinity in clinical drug development and summarizes the active compounds, preclinical drugs and marketed drugs obtained through affinity screening methods. Owing to the high demand for new drugs, bioaffinity-guided screening techniques will play a greater part in clinical drug development. [ABSTRACT FROM AUTHOR]

Advances in cell culture technology have enabled the production of antibody titers upwards of 30 g/L. These highly productive cell culture systems can potentially lead to productivity bottlenecks in downstream purification due to lower column loadings, especially in the primary capture chromatography step. Alternative chromatography solutions to help remedy this bottleneck include the utilization of continuous processing systems such as periodic counter-current chromatography (PCC). Recent studies have provided methods to optimize and improve the design of PCC for cell culture titers up to about 3 g/L. This paper defines a continuous loading strategy for PCC that is independent of cell culture background and encompasses cell culture titers up to about 31 g/L. Initial experimentation showed a challenge with determining a difference in change in UV280 nm signal (ie. ΔUV) between cell culture feed and monoclonal antibody (mAb) concentration. Further investigation revealed UV280 nm absorbance of the cell culture feedstock without antibody was outside of the linear range of detection for a given cell pathlength. Additional experimentation showed the difference in ΔUV for various cell culture feeds can be either theoretically predicted by Beer’s Law given a known absorbance of the media background and impurities or experimentally determined using various UV280 nm cell pathlengths. Based on these results, a 0.35 mm pathlength at UV280 nm was chosen for dynamic control to overcome the background signal. The pore diffusion model showed good agreement with the experimental frontal analysis data, which resulted in definition of a ΔUV setpoint range between 20 and 70% for 3C-PCC experiments. Product quality of the elution pools was acceptable between various cell culture feeds and titers up to about 41 g/L. Results indicated the following ΔUV setpoints to achieve robust dynamic control and maintain 3C-PCC yield: ∼20-45% for titers greater than 10 g/L depending on UV absorbance of the HCCF and ∼45-70% for titers of up to 10 g/L independent of UV absorbance of the HCCF. The strategy and results presented in this paper show column loading in a continuous chromatography step can be dynamically controlled independent of the cell culture feedstock and titer, and allow for enhanced process control built into the downstream continuous operations. [ABSTRACT FROM AUTHOR]

Vaccines against bovine babesiosis must, ideally, induce a humoral immune response characterized by neutralizing antibodies against conserved epitopes and a cellular Th1 immune response. In Babesia bovis , proteins such as AMA-1, MSA-2c, and RAP-1 have been characterized and antibodies against these proteins have shown a neutralizing effect, demonstrating the implication of B and T-cell epitopes in the immune response. There is evidence of the existence of B and T-cell epitopes in these proteins, however, it remains to be defined, the presence of conserved peptides in strains from around the world containing B and T-cell epitopes, and their role in the generation of a long-lasting immunity. The aim in this paper was to identify peptides of Babesia bovis AMA-1, MSA-2c, and RAP-1 that elicit a neutralizing and long-lasting Th1 immune response. Peptides containing B-cell epitopes of AMA-1, MSA-2c and RAP-1, were identified. The immune response generated by each peptide was characterized in cattle. All peptides tested induced antibodies that recognized intraerythrocytic parasites, however, only 5 peptides generated neutralizing antibodies in vitro : P2AMA-1 (6.28%), P3MSA-2c (10.27%), P4MSA-2c (10.42%), P1RAP-1 (32.45%), and P4RAP-1 (36.98%). When these neutralizing antibodies were evaluated as a pool, the inhibition percentage of invasion increased to 52.37%. When the T cellular response was evaluated, two peptides: P3MSA2c and P2AMA1 induced a higher percentage (>70%) of activated CD4 +/CD45RO+ T cells than unstimulated cells. Additionally, both peptides induced the production of gamma interferon (IFN−) in PBMCs from vaccinated cattle after one year proving the implication of a long-lasting Th1 immune response. In conclusion, we identified conserved peptides containing B and T-cell epitopes in antigens of B. bovis that elicit a Th1 immune response and showed evidence that peptides from the same protein elicit different immune responses, which has implication for vaccine development in bovine babesiosis. [ABSTRACT FROM AUTHOR]

Antibody drug conjugates (ADCs) are an emerging therapeutic modality for targeted cancer treatment. They represent the unique amalgamation of chemotherapy and immunotherapy. ADCs comprise of monoclonal antibodies linked with drugs (payloads) through a chemical linker designed to deliver the cytotoxic moiety to the cancer cells. The present paper is a review of recent clinical advances of each component of ADCs (antibody/linker/payload) and how the individual component influences the activity of ADCs. The review discusses opportunities for improving ADCs efficiency and ways to have a better antibody-based molecular platform, which could substantially increase chemotherapy outcomes. This review casts an outlook on how ADCs enhancement in terms of their pharmacokinetics, therapeutic indexes and safety profiles can overcome the prevailing challenges like drug resistance in cancer treatment. A novel strategy of augmenting antibodies with nanoparticles anticipates a huge success in terms of targeted delivery of drugs in several diseases. Antibody conjugated nanoparticles (ACNPs) are a very promising strategy for the cutting-edge development of chemo/immunotherapies for efficient delivery of payloads at the targeted cancer cells. The avenues of a high drug to antibody ratio (DAR) owing to the selection of broad chemotherapy payloads, regulating drug release eliciting higher avidity of ACNPs over ADCs will be the modern immunotherapeutics. ACNPs carry immense potential to mark a paradigm shift in cancer chemotherapy that may be a substitute for ADCs. [Display omitted] • Antibody drug conjugates are mAbs linked with cytotoxic payloads. • ADCs-ACNPs are the recent advances in immuno-chemo-oncology. • ADCs are therapeutic modalities embedded with high mAb specificity and efficacy. • Progress to the perfection of how ADCs deliver payloads onto the tumor is presented. • The emerging therapeutic options for cancer treatment are discussed. [ABSTRACT FROM AUTHOR]

The blood microvascular endothelium consists of a heterogeneous population of cells with regionally distinct morphologies and transcriptional signatures in different tissues and organs. In addition to providing an anti-thrombogenic surface for blood flow, endothelial cells perform a multitude of additional regulatory tasks involving organogenesis, metabolism, angiogenesis, inflammation, repair and organ homeostasis. To communicate with surrounding cells and accomplish their many functions, endothelial cells secrete angiocrine factors including growth factors, chemokines, cytokines, extracellular matrix components, and proteolytic enzymes. Nonendothelial parenchymal and stromal cells in turn regulate endothelial growth, differentiation and survival during embryonal development and in the adult by paracrine mechanisms. Driven by advances in molecular biology, animal genetics, single cell transcriptomics and microscopic imaging, knowledge of organotypic vasculatures has expanded rapidly in recent years. The kidney vasculature, in particular, has been the focus of intensive investigation and represents a primary example of how endothelial heterogeneity and crosstalk with nonendothelial cells contribute to the development and function of a vital organ. In this paper, we review the morphology, function, and development of the kidney vasculature, with an emphasis on blood microvascular endothelial heterogeneity, and provide examples of endothelial and nonendothelial-derived factors that are critically involved in kidney development, growth, response to injury, and homeostasis. To communicate with surrounding cells and accomplish their many functions, endothelial cells secrete angiocrine factors including growth factors, chemokines, cytokines, extracellular matrix components, and proteolytic enzymes. Nonendothelial parenchymal and stromal cells in turn regulate endothelial growth, differentiation, and survival during embryonal development and in the adult by paracrine mechanisms. Driven by advances in molecular biology, animal genetics, single cell transcriptomics and microscopic imaging, knowledge of organotypic vasculatures has expanded rapidly in recent years. The kidney vasculature has been the focus of intensive investigation and represents a primary example of how endothelial heterogeneity and crosstalk with nonendothelial cells contribute to the development and function of a vital organ. In this paper, we review the morphology, function, and development of the kidney vasculature, with an emphasis on blood microvascular endothelial heterogeneity, and provide examples of endothelial and nonendothelial-derived factors that are critically involved in kidney development, growth, response to injury, and homeostasis. A.Section of human kidney stained with anti-PLVAP antibody by immunofluorescence shows diffusely positive peritubular capillaries (PTC, arrows) and negative glomeruli (G). Nuclei are counterstained in blue with DAPI. B. Higher magnification view of PLVAP-positive PTC next to next negatively stained glomerulus. Magnification bars: A, B: 50 μm. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Au-Galinstan nanodendrites based disposable electrochemical sensor was fabricated to detect KIM-1. • The limit of detection of the sensor was 64 pg ml−1 for the 0.1–1000 ng ml−1 linear range. • The senor showed high selectivity compared to other biomolecules. • Excellent recovery rates were achieved in diluted human serum (1:50) containing KIM-1. In this work, we developed a novel Gold (Au)-Galinstan (Ga-In-Sn) nanodendrites modified label-free paper-based electrochemical immunosensor to detect one of the most prominent renal injury biomarkers known as Kidney injury molecule-1 (KIM-1). The Au-Galnstan dendritic nanostructure was obtained by a simple galvanic replacement method supported by polyethylene glycol (PEG) assisted co-reduction, where pH played an essential role in developing the dendritic morphology. Various physicochemical characterizations confirmed the successful formation of the nanodendrites in-depth. The large surface area, excellent synergistic charge transfer kinetics, and catalytic properties of the Au-Galinstan nanodendrites (Nds) modified paper electrode facilitated the oriented immobilization of the anti-KIM-1 antibodies involving Staphylococcal protein A (SPA). Under optimized conditions, the proposed immunosensor obtained an adequately low limit of detection (LOD) of 64 pg ml−1 in a wide linear range (0.1 ng ml−1 –1000 ng ml−1). The highly stable and easily reproducible sensor selectively identified KIM-1 in the presence of interferents. The high recovery rate obtained by the sensor in KIM-1 spiked human serum confirmed its applicability in a real clinical sample. Hence, the proposed immunosensor introduces an emerging platform to provide a simple, cost-effective, stable, and reliable detection of kidney injury-related markers. [ABSTRACT FROM AUTHOR]

This paper describes how two different chip-based liquid sensors have been used for experimental Fast DSC measurements on human serum. The paper starts with describing the first chip-based liquid sensor, based on the XI-400 chip. Repeatability (measurements with the same chip) and reproducibility (measurements with different chips) measurements are described when a linear or a second-degree polynomial baseline is subtracted from the raw data. Human serum with deviating Immunoglobulin levels was measured. The second half of the paper describes the second generation chip based-liquid sensor, based on the XI-468B chip. The new more sensitive chip is described and used for measurements with ten serum samples from healthy individuals and five serum samples from patients diagnosed with CCRCC kidney cancer. Differences are found between the serum from healthy individuals and the serum from the CCRCC kidney cancer patients. [ABSTRACT FROM AUTHOR]

• RSV neutralization assays are complex biological assays used for measurement of functional antibody responses. • A broad variety of RSV neutralization assay formats are currently in use. • Use of the 1st International Standard for Antiserum to RSV is necessary to harmonize neutralization assay output across formats. • This paper outlines good practices for key assay components and data analysis to promote the quality and consistency of RSV neutralization assay results. Respiratory syncytial virus (RSV) is a leading cause of respiratory illness among children and infants worldwide, yet no licensed vaccine exists to reduce the risk of disease. At least 16 RSV vaccine candidates are currently in clinical development and many are designed to induce robust virus neutralizing immune responses. RSV neutralizing antibody (nAb)-mediated interventions such as intravenous immunoglobulin (IVIG) and palivizumab provide passive protection against serious lower respiratory tract disease due to RSV, validating nAbs as a correlate of protection. To identify correlates of protection for vaccine candidates that have demonstrated their protective efficacy, an investigator can use assays designed to measure nAb responses. However, there is no standard method of measurement; individual laboratories have developed their own methods to measure the ability of nAbs to reduce the infectivity of a defined virus dose in a variety of cell lines, leading to establishment of the broad variety of RSV neutralization assay formats currently in use. Standardizing the RSV neutralization assay is an essential step toward better assessment of nAb responses to vaccine candidates. Use of a common reference standard by all makes comparing the immunogenicity of different vaccine candidates feasible. In the context of vaccine development, the WHO 1st International Standard for Antiserum to RSV (RSV IS) has been shown to be suitable for harmonizing results across laboratories and assay formats used to measure nAb titers to RSV/A and RSV/B in human sera. This review describes the broad variety of RSV virus neutralization assay formats currently in use and the importance of the RSV IS for harmonization of results across formats and across laboratories. It also outlines good practices for key assay components and data analysis to promote the quality and consistency of measuring RSV nAb titers in serum specimens. [ABSTRACT FROM AUTHOR]

Amino groups on the antibody surface (amino terminus and Lys) are very interesting conjugation targets due to their substantial quantities and selectivity toward various reactive groups. Oriented immobilization of antibodies via amino moieties on the Fc region instead of the antigen-binding fragment (Fab) is highly appreciated to conserve antigen-binding capacity. In this paper, targeting amino moieties on distinct regions, three antibody immobilization strategies were compared with the recognition ability of corresponding adsorbents. Our results demonstrate that oriented immobilization of antibodies onto heterofunctional chelate-epoxy support selectively involving Lys residues placed at the bottom of the Fc region, thus preserved the highest antigen recognition capacity (over 75% functionality). For homofunctional aldehyde support, immobilization at pH 10 demonstrates 50% remaining functionality due to the random orientation of tethered antibodies; while only 10% functionality remained when N-terminus were specifically conjugated at pH 8.5. With the rationalization of moieties density onto heterofunctional support, 2-fold recognition capacity was exhibited over randomly immobilization for antigens with higher size (β -galactosidase, 425 kDa vs. horseradish peroxidase, 40 kDa). Meanwhile, at least 97% of antigens with a varied concentration in diluted human serum were efficiently captured by the optimized chelate-epoxy support. Therefore, our antibody immobilization protocol proved the potential to be utilized as a promising candidate to capture voluminous antigens (large proteins and cells) in real samples. • IgGs were immobilized through NH 2 groups with different orientation on the support. • Immobilization through amino termini is very easy but highly inefficient. • Random immobilization is easy and efficient. • Immobilization through the bottom of the Fc region is the most efficient. • The best derivative adsorbs near 1.6 μmols of horseradish peroxidase per μmol of anti-HRP. [ABSTRACT FROM AUTHOR]

• Modifications in Fc and VH3 binding region of ProA ligand can allow mild elution pH. • Jetted A50 HipH can elute Fc-containing proteins at or above pH 4.6. • Mild elution pH can be achieved with high yield and >50 % HMW reduction. • pH-sensitive proteins can elute within stable pH region and stay stable over time. • Jetted A50 HipH is a flexible platform ProA resin for stable and labile proteins. Protein A affinity chromatography is widely used as a capture step for monoclonal antibodies (mAb) and molecules that possess an Fc-domain, such as fusion proteins and bispecific antibodies. However, the use of low pH (3.0–4.0) to elute the molecule and achieve acceptable yield (>85 %) can lead to product degradation (e.g. fragmentation, aggregation) for molecules sensitive to low pH. In this paper, we describe a comprehensive evaluation of two protein A resins with ligands designed to elute at a milder pH as a result of modified sequences in their Fc and VH3 binding regions. One of the evaluated resins has been made commercially available by Purolite and named Praesto Jetted A50 HipH. Results demonstrated that Jetted A50 HipH could elute the Fc-fusion protein and most mAbs evaluated with an elution pH at or above 4.6. Elution and wash optimization determined run conditions for high recovery (>90 % monomer yield), reduction of high molecular weight (HMW) species (>50 %), and significant host cell protein (HCP) clearance at the mildest elution pH possible. For a pH-stable mAb and a pH-sensitive fusion protein, cell culture material was purified with optimized conditions and demonstrated the mild elution pH resins' ability to purify product with acceptable yield, comparable or better impurity clearance, and significantly milder native eluate pH compared to traditional resins. The benefits of the mild elution pH resins were clearly exemplified for the pH-sensitive protein, where a milder elution buffer and native eluate pH resulted in only 2 % HMW in the eluate that remained stable over 48 h. In contrast, a traditional protein A resin requiring low pH elution led to eluate HMW levels of 8 %, which increased to 16 % over the same hold time. Additionally, these resins have high dynamic binding capacity and allow the use of traditional HCP washes. Therefore, Jetted A50 HipH is an ideal candidate for a platform protein A resin and provides flexibility for pH-sensitive proteins and stable mAbs, while preserving product quality, recovery, and seamless integration into a downstream process. [ABSTRACT FROM AUTHOR]

The antinuclear antibody (ANA) test plays the role of screening, diagnosis, treatment and prognosis for autoimmune diseases. However, the ANA test is time-consuming, and strongly depends on the interpretation of experienced medical examiners. With the rise of artificial intelligence, a lot of convolutional neural networks (CNN) have been used to recognize the features of ANA images, such as GoogLeNet, ResNet, and MobileNet. By extracting the disease-related features from the entire ANA image, these models achieve acceptable accuracy, but none of them can replace the manual ANA test completely. This is because they ignore the key features of ANA images. Accordingly, this paper aims to design an object detection model that can directly identify the key features of ANA images, i.e., the Metaphase cells. This process is critical in the manual ANA test performed by the medical examiners. Based on the YOLO model, our design first applies various image preprocessing and data augmentation techniques to enhance the critical features in the training data. We modify the original YOLO model, in which the residual block is replaced with SENet module to improve the classification performance by emphasizing critical features and reducing the influence of low-level features. Moreover, we use the different activation function to prevent the modified model from partial judgment. The experimental results show that our design can effectively improve the performance of ANA metaphase identification. Compared to the related CNN models, our design can improve F1-score and mAP by range 5%–14% and 11%–13%, respectively. In particular, the Kappa value of our design is 0.82 that is almost perfect agreement with the ANA experts. [ABSTRACT FROM AUTHOR]

A lateral flow assay (LFA) platform is a powerful tool for point-of-care testing (POCT), especially for self-testing. Although the LFA platform provides a simple and disposable tool for Coronavirus disease of 2019 (COVID-19) antigen (Ag) and antibody (Ab) screening tests, the lower sensitivity for low virus titers has been a bottleneck for practical applications. Herein, we report the combination of a microfluidic paper-based nanoelectrokinetic (NEK) preconcentrator and an LFA platform for enhancing the sensitivity and limit of detection (LOD). Biomarkers were electrokinetically preconcentrated onto a specific layer using the NEK preconcentrator, which was then coupled with LFA diagnostic devices for enhanced performance. Using this nanoelectrokinetic-assisted LFA (NEK-LFA) platform for self-testing, the severe acute respiratory syndrome coronavirus 2 Immunoglobulin G (SARS-CoV-2 IgG) sample was preconcentrated from serum samples. After preconcentration, the LOD of the LFA was enhanced by 32-fold, with an increase in analytical sensitivity (16.4%), which may offer a new opportunity for POCT and self-testing, especially in the COVID-19 pandemic and endemic global context. • Lateral flow assay (LFA) is a simple, disposable tool for COVID-19 Ag and Ab tests. • A microfluidic paper-based large-volume preconcentrator (LVP) is developed. • The detection performance improves considerably upon coupling the LVP with LFA. • The detection limit enhances by 32-folds after biomarker preconcentration. • The strategy offers a new opportunity for point-of-care testing and self-testing. [ABSTRACT FROM AUTHOR]

Different schedules for pediatric use of the 13-valent pneumococcal conjugate vaccine (PCV-13) are recommended in different countries and the U.S. Advisory Committee on Immunization Practices (ACIP) has considered potential of changing from 3 primary doses plus a booster (3p + 1) to two primary doses plus a booster (2p + 1) for protection against Streptococcus pneumoniae. In this paper, we report results of IgG antibody measured by ELISA and opsonophagocytic assay (OPA) after 2p, 3p, at child age 15 months of pre-booster and 18 months (post-booster) in serum samples opportunistically available from a prior study that focused on PCV effectiveness against AOM. A total of ~ 100 sera for each of the 4 study time points (390 sera tested) from 169 children were tested. Geometric mean concentrations (GMCs) and percentage of children exceeding the presumed protective antibody thresholds measured by ELISA and OPA were calculated. 2p doses produced lower antibody levels measured by ELISA but not OPA until the booster dose for serotypes 6A, 6B, 5 and 23F only. Booster dosing at 15 months resulted in significant increases in antibody. There was no difference in the percentage of children with ≥ correlate of protection (COP) for OPA for 2p vs 3p doses except for serotype 23F. A 2p + 0 or 3p + 0 schedule would likely result in many children failing to sustain protective levels of antibody into the second year of life. We conclude that protection from invasive pneumococcal infection in early childhood would be similar for most serotypes in PCV13 using a 2p + 1 or 3p + 1 but not a 2p + 0 or 3p + 0 schedule. [ABSTRACT FROM AUTHOR]