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In 2022, the outbreak of the monkeypox virus occurred in many non-endemic countries, and the World Health Organization (WHO) assessed that this outbreak was "atypical". The establishment of a rapid and effective assay that can be used for the early diagnosis of monkeypox virus infection is crucial for outbreak prevention and control. In this study, the monkeypox virus A29 protein and the homologous vaccinia virus A27 protein and cowpox virus 162 protein were expressed in Escherichia coli BL21 for screening. We synthesized the monkeypox virus A2917–49 peptide as the immunogen and obtained 25 monoclonal antibodies (mAbs) against the A29 protein using mouse hybridoma techniques. Then an immunochromatographic test strip method for detecting A29 was established. The strips utilizing mAb-7C5 and 5D8 showed the best sensitivity and lowest limit of detection: 50 pg mL−1 for purified A29 and specificity tests showed that the strips did not cross-react with other orthopox viruses (vaccinia virus or cowpox virus) as well as common respiratory pathogens (SARS-CoV-2, influenza A and influenza B). Therefore, this method can be used for early and rapid diagnosis of monkeypox virus infection by antigen detection. [ABSTRACT FROM AUTHOR]

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]

[Display omitted] • A µPAD was introduced for quantitative detection of the SARS-CoV-2 antigen employing electrocatalytic activity of the Ni Fe layered double hydroxide. • TO evaluate the applicability of the proposed sensor, nasopharyngeal swabs samples in COVID-19 patient samples collected. • The µPES displayed the attributes of low cost , reliability, selectivity, and high sensitivity for low-level detection of the target. In the offered study, the proposed sensor combined the innovative disposable microfluidic paper-based immunosensor and cost-effective plant-based anti-SARS-CoV-2 monoclonal antibody CR3022, expressed in Nicotiana benthamiana. An electrochemical microfluidic paper-based analytical device (µPAD) was introduced for quantitative detection of the SARS-CoV-2 antigen employing electrocatalytic activity of the Ni Fe layered double hydroxide (NiFe LDH). Its electrocatalytic activity asthe platform of proposed the analytical device enhanced the signal output via redox-recycling of the species the ferri/ferrocyanide [Fe(CN) 6 ]3-/4- in solution and is applied to µPAD response signal. Layered NiFe double hydroxide (LDH) nanosheets with a suitable redox potential were prepared by incorporating Fe into the Ni-based LDH. With the help of Fe, the charge-transfer kinetics for the reduction of Ni3+ to Ni2+ was improved and the formation of unwanted Ni components with higher oxidation state was suppressed. The incorporated Fe as the electron transfer mediator enhanced the process of Ni(OH) 2 /NiOOH redox cycle. Under the optimum conditions, the dependency of impedance signal and SARS-CoV-2 concentration showed a linear region from 10 fg/mL-5 ng/mL with a detection limit of 0.23 fg/mL. This Paper-based immunosensor device also showed long stability and good reproducibility, which can be used for the quantitative assay of the SARS-CoV-2. The results of nasal samples analysis revealed the applicability of the resulting biosensor. [ABSTRACT FROM AUTHOR]

Many studies have been conducted on measuring avian influenza viruses and their hemagglutinin (HA) antigens via electrochemical principles; most of these studies have used gold electrodes on ceramic, glass, or silicon substrates, and/or labeling for signal enhancement. Herein, we present a paper-based immunosensor for label-free measurement of multiple avian influenza virus (H5N1, H7N9, and H9N2) antigens using flexible screen-printed carbon nanotube-polydimethylsiloxane electrodes. These flexible electrodes on a paper substrate can complement the physical weakness of the paper-based sensors when wetted, without affecting flexibility. The relative standard deviation of the peak currents was 1.88% when the electrodes were repeatedly bent and unfolded twenty times with deionized water provided each cycle, showing the stability of the electrodes. For the detection of HA antigens, approximately 10-μl samples (concentration: 100 pg/ml–100 ng/ml) were needed to form the antigen–antibody complexes during 20–30 min incubation, and the immune responses were measured via differential pulse voltammetry. The limits of detections were 55.7 pg/ml (0.95 pM) for H5N1 HA, 99.6 pg/ml (1.69 pM) for H7N9 HA, and 54.0 pg/ml (0.72 pM) for H9N2 HA antigens in phosphate buffered saline, and the sensors showed good selectivity and reproducibility. Such paper-based sensors are economical, flexible, robust, and easy-to-manufacture, with the ability to detect several avian influenza viruses. [ABSTRACT FROM AUTHOR]

A paper-based electrochemical immunosensor was developed for the detection of cancer antigen 125 (CA125) by screen-printing technique. The reduced graphene oxide/thionine/gold nanoparticles (rGO/Thi/AuNPs) nanocomposites were compounded and coated onto the working electrode of immunosensor for CA125 antibody (anti-CA125) immobilization and detection signal amplification. The detection principle was based on the fact that the immunocomplex formed by specify binding of CA125 antibody and antigen could reduce the current response of thionine, which was proportional to the corresponding concentration of CA125 antigen. The immunoassay results showed that the linear range of CA125 was from 0.1 U mL−1 to 200 U mL−1 with the limit of detection (LOD) of 0.01 U mL−1 at signal to noise of 3. Quality control serum samples measured by our proposed immunosensor showed acceptable agreement with traditional ELISA method with the relative error less than 8.05%. The immunosensor exhibited good electrochemical performance with high reproducibility, reliability, stability and accuracy. The proposed immunosensor could be used for the determination of CA125 and had the potential for point-of-care testing (POCT) of other tumor marker. • A novel electrochemical immunosensor based on rGO/Thi/AuNPs nanocomposites was developed for the determination of CA125. • The rGO/Thi/AuNPs nanocomposites were synthesized to improve the sensitivity of immunosensor. • The immunosensor showed acceptable agreement with traditional ELISA method in clinical diagnosis. • It could be used for point-of-care testing of tumor markers and had the potential for disease screening in remote region. [ABSTRACT FROM AUTHOR]

The objective of the present work was to standardize and evaluate indirect plate and dot- enzyme linked immunosorbent assay (ELISA) using purified Paramphistomum epiclitum homologous antigens in the small ruminants. Electrophoretic separation of somatic antigen (PeSAg) in reducing condition on 15% polyacrylamide gel resolved into 16 proteins of the molecular weight ranging from 14 -100 kDa. Two step ethanolic precipitation of supernatant of in-vitro culture of the fluke yielded P. epiclitum excretory-secretory antigen (PeESAg) of molecular weight 28 kDa. The animals (Goats=123; Sheep=91) were broadly kept into post-mortem and faecal examined groups. At many occasion the PeSAg found to cross reacts with other helminths parasites thus minimizing the specificity of the tests and antigens. There was no any direct correlation between the parasites load and ELISA reactivity pattern. The noted prevalence rate after combining the results of post-mortem examination and PeESAg based ELISA (plate and paper/ dot) was 30.08% (37/123) in goats and 28.57% (26/91) in sheep. While using PeESAg, the calculated overall sensitivity% was 92.86 (goats)/ 100 (sheep) in both plate and dot- ELISA, specificity% was 91.58 (goats)/ 91.55 (sheep) in plate ELISA while 88.42 (goats)/ 92.96 (sheep) in dot-ELISA, positive predictive value% was 76.47 (goats)/ 76.92 (sheep) in plate ELISA while 70.27 (goats)/ 80 (sheep) in dot-ELISA and negative predictive value% was 97.75 (goats)/ 100 (sheep) in plate ELISA while 97.67 (goats)/ 100 (sheep) in dot- ELISA, these values were optimum for the field sera sample so the tests and PeESAg can be recommended for the detection P. epiclitum infection in the small ruminants. [ABSTRACT FROM AUTHOR]

Context.--In 2018 the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee designed and implemented a new plasma cell neoplasia flow cytometry proficiency testing program--PCNEO--to allow clinical flow cytometry laboratories to monitor and assess their performance compared with a peer group. Objective.--To report the results from the first 4 years of the PCNEO program. Design.--Program participants were sent 2 sets of challenges per year, each including 1 wet challenge and 2 dry challenges, with associated clinical and laboratory findings. The wet challenges were composed of myeloma cell line specimens (with or without dilution in preserved whole blood) for flow cytometric analysis. The dry (paper) challenges were composed of clinical case summaries and images of flow cytometric test results from various flow cytometry laboratories of committee members. Results.--A total of 116 to 145 laboratories from 17 countries enrolled in the proficiency testing program. For the wet challenges, almost all participants (97%--100%; cumulative, 98.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of undiluted myeloma cell lines. Slightly fewer participants (89.0%--97.4%; cumulative, 95.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of diluted myeloma cell lines (10% or 50% dilutions into peripheral blood) intended to better represent a typical clinical sample. There was generally agreement among 80% or more of participants for positive or negative staining for CD38, CD138, CD19, CD20, and surface and cytoplasmic K and λ light chains. Similarly, 84% to 100% of participants were able to correctly identify the presence of neoplastic plasma cell populations in paper challenges, including the presence of small, neoplastic plasma cell populations (0.01%--5.0% clonal plasma cells) and the presence of nonneoplastic plasma cell populations (correctly identified by 91%--96% of participants). Conclusions.--Participant performance in the new proficiency testing program was excellent overall, with the vast majority of participants able to perform flow cytometric analysis and identify neoplastic plasma cell populations and to identify small plasma cell clones or expanded populations of reactive plasma cells in dry challenge flow cytometry results. This program will allow laboratories to verify the accuracy of their testing program and test interpretations for the assessment of patients suspected of having a plasma cell neoplasm. [ABSTRACT FROM AUTHOR]

Early stage diagnosis of breast cancer by monitoring the proteins in human biological fluids is the best method and the first section toward efficient therapy, delaying metastasis and hindrance mortality. In this study, graphite ink was synthesized and combined with CA 15-3 antibody in order to develop a novel immunosensor for detection of CA 15-3, breast cancer biomarker. Conductivity of bioink was examined by designing various conductive patterns on paper using a rollerball pen. Electrochemical behavior of engineered immunosensor was evaluated through employing sensitive diagnostic technique, DPV (differential pulse voltammetry). Under optimal conditions, the linear concentration range and low limit of quantification (LLOQ) of designed immunosensor was 15–250 U/mL and 15 U/mL, respectively. The process was successfully applied to assay of breast cancer biomarker (CA15-3) in unprocessed human plasma specimens. Unlabelled Image [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]

The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that ∼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (∼95 °C, 10–25min) and we could perform at least five rounds (the following antibodies used: Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications. [Display omitted] • We discover that heat-induced antigen retrieval technologies can strip the NC blots repeatedly up to 5 times. • Modified stripping protocol is composed of 0.1 M Tris, 0.01 M EDTA and 0.01% Tween-20 (pH 9.0–9.5). • The stripping buffer is easier and more efficient for probing different proteins. [ABSTRACT FROM AUTHOR]

The current approaches of diagnostic platforms for detecting SARS-CoV-2 infections mostly relied on adapting the existing technology. In this work, a simple and low-cost electrochemical sensing platform for detecting SAR-CoV-2 antigen was established. The proposed sensor combined the innovative disposable paper-based immunosensor and cost-effective plant-based anti-SARS-CoV-2 monoclonal antibody CR3022, expressed in Nicotiana benthamiana. The cellulose nanocrystal was modified on screen-printed graphene electrode to provide the abundant COOH functional groups on electrode surface, leading to the high ability for antibody immobilization. The quantification of the presence receptor binding domain (RBD) spike protein of SARS-CoV-2 was performed using differential pulse voltammetry by monitoring the changing current of [Fe(CN) 6 ]3-/4- redox solution. The current change of [Fe(CN) 6 ]3-/4- before and after the presence of target RBD could be clearly distinguished, providing a linear relationship with RBD concentration in the range from 0.1 pg/mL to 500 ng/mL with the minimum limit of detection of 2.0 fg/mL. The proposed platform was successfully applied to detect RBD in nasopharyngeal swab samples with satisfactory results. Furthermore, the paper-based immunosensor was extended to quantify the RBD level in spiked saliva samples, demonstrating the broadly applicability of this system. This electrochemical paper-based immunosensor has the potential to be employed as a point-of-care testing for COVID-19 diagnosis. [Display omitted] • The electrochemical paper-based device integrated with a label-free immunoassay was developed for detecting SARS-CoV-2. • A novel plant-based mAb CR3022 offers a remarkable simplicity and cost-effectiveness in terms of Ab production. • CNC containing the abundant COOH functional groups has the potential to be used as the immobilizing material. • The proposed immunosensor was successfully applied to detect SARS-CoV-2 from nasopharyngeal swabs and saliva samples. [ABSTRACT FROM AUTHOR]

In this work, we report the development of a general strategy for enhancing the efficiency of target capture in immunoassays, using a bifunctional fusion protein construct which incorporates a substrate-anchoring moiety for the high-abundance immobilization of an antigen-binding domain. This approach was informed by the development of a pseudo first-order rate constant model, and tested in a paper-based assay format using a fusion construct consisting of an rcSso7d binding module and a cellulose-binding domain. These rcSso7d-CBD fusion proteins were solubly expressed and purified from bacteria in high molar yields, and enable oriented, high-density adsorption of the rcSso7d binding species to unmodified cellulose within a 30-second incubation period. These findings were validated using two distinct, antigen-specific rcSso7d variants, which were isolated from a yeast surface display library via flow cytometry. Up to 1.6 micromoles of rcSso7d-CBD was found to adsorb per gram of cellulose, yielding a volume-averaged binder concentration of up to 760 μM within the resulting active material. At this molar abundance, the target antigen is captured from solution with nearly 100% efficiency, maximizing the attainable sensitivity for any given diagnostic system. [ABSTRACT FROM AUTHOR]

There is currently a lot of interest in the construction of point-of-care devices stemming from paper-based origami biosensors. These devices demonstrate how paper's foldability permits the construction of sensitive, selective, user-friendly, intelligent, and maintainable analytical devices for the detection of several ailments. Herein, the first example of the electrochemical aptasensor-based polyvalent dengue viral antigen detection using the origami paper-folding method is presented. Coupling it with an aptamer leads to the development of a new notation known as OBAs, or origami-based aptasensor, that presents a multitude of advantages to the developed platform, such as assisting in safeguarding the sample from air-dust particles, providing confidentiality, and providing a closed chamber to the electrodes. In this paper, gold-decorated nanocomposites of zinc and graphene oxide (Au/ZnO/GO) were synthesized via the chemical method, and characterization was conducted by Scanning Electron Microscope, Transmission Electron Microscope, UV-Vis, and XRD which reveals the successful formation of nanocomposites, mainly helping to enhance the signal and specificity of the sensor by employing aptamers, since isolation and purification procedures are not required. The biosensor that is being demonstrated here is affordable, simple, and efficient. The reported biosensor is an OBA detection of polyvalent antigens of the dengue virus in human serum, presenting a good range from 0.0001 to 0.1 mg/mL with a limit of detection of 0.0001 mg/mL. The reported single-folding ori-aptasensor demonstrates exceptional sensitivity, specificity, and performance in human serum assays, and can also be used for the POC testing of various viral infections in remote areas and underdeveloped countries, as well as being potentially effective during outbreaks. Highlights: (1) First report on origami-based aptasensors for the detection of polyvalent antigens of DENV; (2) In-house construction of low-cost origami-based setup; (3) Gold-decorated zinc/graphene nanocomposite characterization was confirmed via FESEM/UV-Vis/FTIR; (4) Cross-reactivity of dengue-aptamer has been deduced; (5) Electrochemical validation was conducted through CV. [ABSTRACT FROM AUTHOR]

This paper describes a novel strategy for fabricating the movable valve on paper-based microfluidic devices to manipulate capillary-driven fluids. The movable valve fabrication is first realized using hollow rivets as the holding center to control the paper channel in different layer movement that results in the channel's connection or disconnection. The relatively simple valve fabrication procedure is robust, versatile, and compatible with microfluidic paper-based analytical devices (μPADs) with differing levels of complexity. It is remarkable that the movable valve can be convenient and free to control fluid without the timing setting, advantages that make it user-friendly for untrained users to carry out the complex multistep operations. For the performance of the movable valve to be verified, several different designs of μPADs were tested and obtained with satisfactory results. In addition, in the proof-of-concept enzyme-linked immunosorbent assay experiments, we demonstrate the use of these valves in μPADs for the successful analysis of samples of carcino-embryonic antigen, showing good sensitivity and reproducibility. We hope this technique will open new avenues for the fabrication of paper-based valves in an easily adoptable and widely available way on μPADs and provide potential point-of-care applications in the future. [ABSTRACT FROM AUTHOR]

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Both the ABO and Rhesus (Rh) blood groups play crucial roles in blood transfusion medicine. Herein, we report a simple and low-cost paper-based analytical device (PAD) for phenotyping red blood cell (RBC) antigens. Using this Rh typing format, 5 Rh antigens on RBCs can be simultaneously detected and macroscopically visualized within 12 min. The proposed Rh phenotyping relies on the presence or absence of hemagglutination in the sample zones after immobilizing the antibodies targeting each Rh antigen. The PAD was optimized in terms of filter paper type, antibodies, and distance of the visualization zone. In this study, the optimal conditions were Whatman filter paper Grade 4; anti-D, –C, -E, -c, and -e antibodies; RBC suspension of 30%; and a visualization zone of 1 cm above the sample zone. The accuracy of simultaneously phenotyping the five Rh RBC antigens in the blood samples (n = 4692) was 99.19%, comparable with the accuracy of the gold-standard tube method used by blood bank laboratories in several regions of Thailand. Furthermore, decision making based on this method can be assisted by deep learning. After implementing a two-stage objective detection algorithm (YOLO v4-tiny) and classification model (DenseNet-201), the ambiguous images (n = 48) were interpreted with 100% accuracy. The PAD integrated with customized-region convolutional neural networks can reduce the interpretation discrepancies in RBC antigen phenotyping in any laboratory. [Display omitted] • Five Rh antigens typing PADs combined with deep learning algorithm for an assisted human-decision. • Rh antigens typing PADs were tested with real blood samples and compared with the standard tube method. • Dataset of photographs following Rh PADs testing were trained and evaluated by the in-house deep learning platform CiRA CORE. • Misinterpretation caused by weak or ambiguous reactions was solved by the CiRA CORE platform. [ABSTRACT FROM AUTHOR]

The initial Phase-I single centre, single dose, randomized, double-blind, cross-over study was planned to assess the pharmacokinetic and pharmacodynamic bioequivalence of the trastuzumab biosimilar (MYL-1401O) compared to the reference Herceptin®. Their respective immunomodulation profile presented in this paper involved healthy males receiving a single infusion of both monoclonals, separated by a washout period. Sixty parameters were assessed in total, including serum cytokines, peripheral mononuclear cell (PBMC) subsets, cell activation and response to recall antigens and mitogen, pre- and post- infusion, as well as a cytokine release assay (CRA) at baseline. Trastuzumab infusion induced a transient and weak peak of serum IL-6 at 6 h, and a modulation of mononuclear cell subset profile and activation level, notably CD16 + cells. Except for CD8 + T cells, there were no significant differences between Herceptin® and MYL-1401O. In CRA, PBMC stimulated with MYL-1401O or Herceptin® similarly secreted IL-6, TNF-α, IL-1β, GM-CSF, IFN-γ, and IL-10, but no or low level of IL-2. Interestingly, some observed adverse events correlated with IL-2 and IFN-γ in CRA. MYL-1401O exhibited a very similar immunomodulation profile to Herceptin®, strongly supporting its bioequivalence. This approach may thus be included in a proof-of-concept study. CRA may be used as a predictive assay for the evaluation of clinical monoclonals. [ABSTRACT FROM AUTHOR]

Abstract: Paper based colorimetric biosensing platform utilizing cross-linked siloxane 3-aminopropyltriethoxysilane (APTMS) as probe was developed for the detection of a broad range of targets including H2O2, glucose and protein biomarker. APTMS was extensively used for the modification of filter papers to develop paper based analytical devices. We discovered when APTMS was cross-linked with glutaraldehyde (GA), the resulting complex (APTMS–GA) displays brick-red color, and a visual color change was observed when the complex reacted with H2O2. By integrating the APTMS–GA complex with filter paper, the modified paper enables quantitative detection of H2O2 through the monitoring of the color intensity change of the paper via software Image J. Then, with the immobilization of glucose oxidase (GOx) onto the modified paper, glucose can be detected through the detection of enzymatically generated H2O2. For protein biomarker prostate specific antigen (PSA) assay, we immobilized capture, not captured anti-PSA antibody (Ab1) onto the paper surface and using GOx modified gold nanorod (GNR) as detection anti-PSA antibody (Ab2) label. The detection of PSA was also achieved via the liberated H2O2 when the GOx label reacted with glucose. The results demonstrated the possibility of this paper based sensor for the detection of different analytes with wide linear range. The low cost and simplicity of this paper based sensor could be developed for “point-of-care” analysis and find wide application in different areas. [Copyright &y& Elsevier]

The aim of this document was to provide a critical review of the current knowledge on hypersensitivity pneumonitis caused by the occupational environment and to propose practical guidance for the diagnosis and management of this condition. Occupational hypersensitivity pneumonitis ( OHP) is an immunologic lung disease resulting from lymphocytic and frequently granulomatous inflammation of the peripheral airways, alveoli, and surrounding interstitial tissue which develops as the result of a non-IgE-mediated allergic reaction to a variety of organic materials or low molecular weight agents that are present in the workplace. The offending agents can be classified into six broad categories that include bacteria, fungi, animal proteins, plant proteins, low molecular weight chemicals, and metals. The diagnosis of OHP requires a multidisciplinary approach and relies on a combination of diagnostic tests to ascertain the work relatedness of the disease. Both the clinical and the occupational history are keys to the diagnosis and often will lead to the initial suspicion. Diagnostic criteria adapted to OHP are proposed. The cornerstone of treatment is early removal from exposure to the eliciting antigen, although the disease may show an adverse outcome even after avoidance of exposure to the causal agent. [ABSTRACT FROM AUTHOR]

Highlights: [•] AuNPs on paper were functionalized to develop SERS biodiagnostic platform. [•] Reproducible SERS spectra of antibody–antigen were achieved. [•] Modification of antibody structure upon antigen binding resulted in Raman shifts. [•] SERS intensity increased as a function of antigen concentration. [ABSTRACT FROM AUTHOR]

Poly(carboxybetaine) (PCB) functionalized cellulose paper was used as a paper-based microfluidic device. The results showed that the PCB modified paper sensor was able to achieve (a) more rapid and sensitive glucose detection from undiluted human serum compared to bare cellulose and (b) specific antigen detection via covalently immobilized antibodies. [ABSTRACT FROM AUTHOR]

Electrochemical paper-based analytical devices (ePADs) are useful analytical devices that serve as point-of-care testing (POCT) devices for various clinical biomarkers in view of their simplicity, portability, and low-cost format. However, multistep reagent manipulation usually restricts the performance of the device for end users. Herein, we developed a sequential ePAD for sequential immunosensing fluid delivery by integrating dual flow behaviors (fast-flow/delayed) within a single paper platform for the simultaneous detection of hepatitis B surface antigen (HBsAg) and hepatitis C core antigen (HCVcAg). In the present work, a fast-flow channel was used for the automated washing of unbound antigens, while a delayed channel was created to store a redox reagent for further electrochemical analysis with a single buffer loading (the analysis time can be completed within 500 s). Hence, the undesirable complex procedure of multi-step reagent manipulation is scarcely needed by the user. The detection limit of the proposed ePAD was as low as 18.2 pg mL−1 for HBsAg and 1.19 pg mL−1 for HCVcAg. In addition, this proposed ePAD was also proven to be effective in real clinical sera from patients to verify its biological applicability. The ePAD sensor shows high promise as an easy-to-use, portable, and extendable sensor for other multiplex biological assays. [Display omitted] • ePAD for simultaneous HBsAg and HCVcAg was created. • First time employment of dual flow behaviors on ePAD for hepatitis markers sensing. • ePAD shows an easy to use, portability, and broad sensing applicability. • The sensor was applied to detect hepatitis markers in real clinical sera. [ABSTRACT FROM AUTHOR]

A laboratory-based lateral flow (LF) test that utilizes up-converting reporter particles (UCP) for ultrasensitive quantification of Schistosoma circulating anodic antigen (CAA) in urine is a well-accepted test to identify active infection. However, this UCP-LF CAA test requires sample pre-treatment steps not compatible with field applications. Flow, a new low-cost disposable, allows integration of large-volume pre-concentration of urine analytes and LF detection into a single field-deployable device. We assessed a prototype Flow-Schistosoma (Flow-S) device with an integrated UCP-LF CAA test strip, omitting all laboratory-based steps, to enable diagnosis of active Schistosoma infection in the field using urine. Flow-S is designed for large-volume (5–20 mL) urine, applying passive paper-based filtration and antibody-based CAA concentration. Samples tested for schistosome infection were collected from women of reproductive age living in a Tanzania region where S. haematobium infection is endemic. Fifteen negative and fifteen positive urine samples, selected based on CAA levels quantified in paired serum, were analyzed with the prototype Flow-S. The current Flow-S prototype, with an analytical lower detection limit of 1 pg CAA/mL, produced results correlated with the laboratory-based UCP-LF CAA test. Urine precipitates occurred in frozen banked samples and affected accurate quantification; however, this should not occur in fresh urine. Based on the findings of this study, Flow-S appears suitable to replace the urine pre-treatment required for the laboratory-based UCP-LF CAA test, thus allowing true field-based applications with fresh urine samples. The urine precipitates observed with frozen samples, though less important given the goal of testing fresh urines, warrant additional investigation to evaluate methods for mitigation. Flow-S devices permit testing of pooled urine samples with applications for population stratified testing. A field test with fresh urine samples, a further optimized Flow-S device, and larger statistical power has been scheduled. [ABSTRACT FROM AUTHOR]

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]

The lateral flow test has become the standard bioassay format in low-resource settings because it is rapid, easy to use, and low in cost, uses reagents stored in dry form, and is equipment-free. However, lateral flow tests are often limited to a single chemical delivery step and not capable of the multistep processing characteristic of high performance laboratory-based assays. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions; this allows incorporation of multistep chemical processing, while still retaining the advantages of conventional lateral flow tests. Here, we demonstrate this format for an easy-to-use, signal-amplified sandwich format immunoassay for the malaria protein PJHRP2. The card contains reagents stored in dry form such that the user need only add sample and water. The multiple flows in the device are activated in a single user step of folding the card dosed; the configuration of the paper network automatically delivers the appropriate volumes of (i) sample plus antibody conjugated to a gold partide label, (ii) a rinse buffer, and (iii) a signal amplification reagent to the capture region. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low. resource settings in the developed and developing worlds. [ABSTRACT FROM AUTHOR]

Background: In vitro stimulation of whole blood or isolated blood cells with specific antigens is used for several purposes. Immediately following incubation with antigens, samples have to be centrifuged to stop the reactions by remaining cells and the supernatant refrigerated or analysed directly to preserve the analytes of interest, which makes samples difficult to prepare outside laboratories. We have tested whether spotting whole blood on filter paper after activation can be used in one of the tests for Mycobacterium tuberculosis infection (MTI), the QuantiFERON®-TB Gold In Tube test (QFT), where the spotting technique can make it suitable for use in locations without facilities like a centrifuge and a refrigerator. Materials and methods: Samples from 22 individuals undergoing screening for MTI and 10 healthy controls were incubated, centrifuged and IFN-γ measured by Enzyme-linked immunosorbent assay (ELISA), as described in the kit insert. In parallel, activated blood was spotted on filter paper (Schleicher & Schuell) and dried. The dried blood spot samples were analysed for 21 inflammatory markers with an in-house assay based on Luminex technology. Results: Our multiplex measurements of inflammatory markers in samples from suspected MTI patients confirmed the IFN-γ findings in the QFT. IL-2, GM-CSF, IL-5, and IL-1β were also found as useful markers for MTI. We were not able to distinguish between active tuberculosis and latent MTI. Conclusion: Applying blood on filter paper after incubation makes in vitro stimulation tests feasible in locations where heat and electricity is unavailable. [ABSTRACT FROM AUTHOR]

Demonstration of successful elimination of lymphatic filariasis (LF) in endemic countries requires sensitive diagnostics for accurate definitions of endpoints and future surveillance. There has been interest in complementing available diagnostics with antibody serology testing in children, since negative serology would correspond with cessation of LF transmission. The Filariasis CELISA detects antifilarial IgG4 and has favourable results with serum samples but field application requires an easier sampling method. Ninety-four paired plasma and filter paper samples were assayed with promising results. The filter paper method resultedin a sensitivity of 92% and a specificity of 77% when compared to the paired plasma. One hundred and one filter paper samples were assessed for storage effects. Following 10-month storage at -20°C there was a significant reduction in reactivity (P < .001). Overall the results indicated that filter paper sampling would be a favourable sensitive and specific alternative for blood collection in surveys. [ABSTRACT FROM AUTHOR]

Background: A simple filter paper method was developed for, the transport and storage of monoclonal antibodies (Mabs) at room temperature or -20°C after spotting on filter paper, for subsequent sero-typing of outer membrane antigens of N.meningitidis by dot-blot ELISA. Methods: Monoclonal antibodies (Mabs) were spotted within a 0.5-1 cm diameter area of Whatman grade 903 paper, which were stored individually at room temperature or at -20°C. These MAbs were stored and analyzed after periods of one week, 4 weeks, 12 months, or 13 years in the case of frozen Mab aliquots, or after 4 weeks at -20°C or at room temperature (RT) in the case of Mabs dried on filter paper strips. Assays were performed in parallel using dot-blot ELISA. In addition to the MAbs specific for serotyping class 1, 2 or 3, we used a larger number of Mabs for polysaccharides, lipooligosaccharides (LOS), class 5 and cross-reactive antigens for native outer membrane of N.meningitidis. The Mabs dried on filter paper were eluted with phosphate-buffered saline (PBS) containing 0.2% gelatin. Results: Mabs of the isotypes IgG and IgM dried on filter papers were not affected by duration of storage. The detection by serotyping Mabs was generally consistent for dried filter paper MAb samples stored frozen for over 1 year at -20°C, and although decreased reactive antibody titers were found after storage, this did not interfere with the specificity of the Mabs used after 13 years as dry spots on filter paper. Conclusion: The use of filter paper is an inexpensive and convenient method for collecting, storing, and transporting Mab samples for serotyping studies. In addition, the samples occupy little space and can be readily transported without freezing. The efficiency of using immunoglobulin G (IgG) or M (IgM) eluted was found to be consistent with measurement of IgG or IgM titers in most corresponding, ascites Mabs stored frozen for over 1 year. The application of meningococcal typing methods and designations depend on the question being asked. [ABSTRACT FROM AUTHOR]

COVID-19 has evolved into a global pandemic. Early and rapid detection is crucial to control of the SARS-CoV-2 transmission. While representing the gold standard for early diagnosis, nucleic acid tests for SARS-CoV-2 are often complicated and time-consuming. Serological rapid antibody tests are characterized by high rates of false-negative diagnoses, especially during early infection. Here, we developed a novel nanozyme-based chemiluminescence paper assay for rapid and sensitive detection of SARS-CoV-2 spike antigen, which integrates nanozyme and enzymatic chemiluminescence immunoassay with the lateral flow strip. The core of our paper test is a robust Co–Fe@hemin-peroxidase nanozyme that catalyzes chemiluminescence comparable with natural peroxidase HRP and thus amplifies immune reaction signal. The detection limit for recombinant spike antigen of SARS-CoV-2 was 0.1 ng/mL, with a linear range of 0.2-100 ng/mL. Moreover, the sensitivity of test for pseudovirus could reach 360 TCID 50 /mL, which was comparable with ELISA method. The strip recognized SARS-CoV-2 antigen specifically, and there was no cross reaction with other coronaviruses or influenza A subtypes. This testing can be completed within 16 min, much shorter compared to the usual 1-2 h required for currently used nucleic acid tests. Furthermore, signal detection is feasible using the camera of a standard smartphone. Ingredients for nanozyme synthesis are simple and readily available, considerably lowering the overall cost. In conclusion, our paper test provides a high-sensitive point-of-care testing (POCT) approach for SARS-CoV-2 antigen detection, which should greatly facilitate early screening of SARS-CoV-2 infections, and considerably lower the financial burden on national healthcare resources. • Point-of-care antigen tests for screening of SARS-CoV-2 infection were established. • Chemiluminescence improves sensitivity of paper test and benefits early screening. • Nanozyme with high peroxidase activity and stability catalyzes chemiluminescence. • Nanozyme chemiluminescence paper test brings high efficiency and low cost. [ABSTRACT FROM AUTHOR]

A disposable paper-based bipolar electrode (BPE) was reported for the first time for the sensitive electrochemiluminescence detection of a prostate specific antigen (PSA). [ABSTRACT FROM AUTHOR]

Thirty patients with haematological malignancies received peripheral blood stem cells from human leucocyte antigen (HLA)-identical sibling donors after non-myeloablative conditioning with fludarabine and total body irradiation. Twenty-seven patients received the transplant as an outpatient procedure. All patients engrafted. The probability of acute graft- versus-host disease (GVHD) grades II–IV and extensive chronic GVHD was 57% and 80%, respectively. Patients alive on day +365 experienced a median of 44 d (range 4–151) of hospitalization during the first year. In the entire cohort, GVHD accounted for 22%, infections for 18%, thrombotic thrombocytopenic purpura (TTP) for 16% and engraftment syndrome for 14% of the time in hospital. The 1-year risk of TTP was 26%. Acute GVHD was a risk factor for the development of TTP ( P = 0·008). With a median follow-up of 602 d, the 2-year estimates for overall survival, progression-free survival, non-relapse mortality and relapse related mortality were 68%, 43%, 22% and 13%, respectively. This transplantation regimen is feasible and induces long-term remissions in heavily pretreated patients. The procedure can be performed in the outpatient setting, but complications could result in a substantial number of admissions during the first year. [ABSTRACT FROM AUTHOR]

Without transplantation from a human leucocyte antigen-identical family donor, refractory severe aplastic anaemia (SAA) has an unfavourable prognosis. Conventional transplantation from a matched unrelated donor carries a high rate of mortality. We transplanted large numbers of highly purified CD34+ cells from matched unrelated ( n = 4), mismatched unrelated ( n = 4) and mismatched related ( n = 1) donors into nine children with refractory SAA. The grafts consisted of granulocyte colony-stimulating factor-mobilized peripheral positively selected CD34+ cells. A median of 15·1 × 106/kg CD34+ stem cells and 11 × 103/kg CD3+ T-lymphocytes were infused. No additional pharmacological graft versus host disease (GVHD) prophylaxis was given. At a median follow-up of 47 (range 37–72) months, eight patients (89%) were in complete remission with >90% donor chimaerism and no evidence of GVHD. One patient died on day +238 as a consequence of GVHD. The use of highly purified mobilized CD34+ stem cells warrants further clinical exploration in children with refractory SAA. [ABSTRACT FROM AUTHOR]

Recent studies have shown that human myeloid leukaemia cells can differentiate into dendritic cell (DC)-like cells (leukaemia-DCs) when cultured with a combination of cytokines. In the present study, we examined whether the transduction of leukaemia-DCs with OX40 ligand (OX40L), a member of the tumour necrosis factor (TNF) family, resulted in augmentation of their antigen presenting activity. Bicistronic retroviral vectors expressing both human OX40L and enhanced green fluorescent protein (EGFP) or EGFP alone were generated and used for transduction. Fresh leukaemic cells from five patients with acute myeloid leukaemia (AML) were isolated and retrovirally transduced with OX40L during the culture with a combination of cytokines from stem cell factor, fms-like tyrosine kinase (Flt)-3 ligand, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and TNF- α. After 7 d, the majority of cells showed DC-like morphology, and expressed higher levels of CD80, CD86 and HLA-DR than fresh leukaemic cells. The transduction efficiency was 8·5–27·2%. Leukaemia-DCs transduced with OX40L elicited higher proliferative response of allogeneic CD4+ T cells than fresh leukaemic cells, non-transduced, or mock-transduced leukaemia-DCs. Co-culture of allogeneic CD4+ T cells with OX40L-transduced leukaemia-DCs was superior in the generation of interferon (IFN)- γ producing CD4+ T cells and in production of IFN- γ. Furthermore, OX40L-transduced leukaemia-DCs could elicit significant proliferative response of human leucocyte antigen-matched T cells from the donor in allogeneic stem cell transplantation. These results indicate that retroviral transduction of leukaemia-DCs with OX40L augments their antigen presenting cell activity and thus renders them more suitable for tumour vaccines or ex vivo stimulation of leukaemia-specific T cells. [ABSTRACT FROM AUTHOR]

The 48 G>C transversion in exon 1 of the RHCE gene leads to Trp16Cys, usually present in the conventional RHCE Ce, while Trp16 is associated with RHCE ce. The presence of Cys16 in RHCE ce is associated with the R0 (Dce) haplotype in Africans, leading to a weak ‘e’ antigen expression on red blood cells (RBCs). VS is a common red cell antigen in individuals of African descent and results from a single point mutation in exon 5 of the RHCE (733C>G), leading to Leu245Val substitution; VS positivity is also associated with weak expression of ‘e’. This study investigated the association of Cys16 and/or VS with the RHCE ce alleles in a cohort of sickle cell disease (SCD) patients phenotyped as R0r or R0R0 and rr. DNA samples from 58 SCD patients were tested for the 48 G>C transversion, encoding Cys16, by allele-specific polymerase chain reaction (PCR). We also amplified exon 5 of the RHCE by PCR and subjected the amplified product to restriction fragment length polymorphism analysis, using BfaI, in order to determine the VS status. Further cDNA analysis was performed on three samples to verify whether the mutations were located on the same or on different alleles. Fifty-six of the 58 SCD patients studied (97%) were heterozygous for 48G/48C (Cys16). Of these, 18 (32%) were also heterozygous for 733C/G (245Val). All of these 18 samples showed weak ‘e’ expression on RBCs when tested with at least one monoclonal antibody to e antigen. cDNA sequencing of three of 18 patient samples showed that the genes encoding Cys16 and Val245 (VS) were on different alleles. We found a high incidence of Cys16 associated with the RHCE ce in our SCD cohort. A high percentage of these patients were also found to be heterozygous for VS. cDNA analysis showed that, in at least three samples, the two mutations were on different alleles, with consequent weakening of expression of the e antigen on RBCs. [ABSTRACT FROM AUTHOR]

We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese woman who suffered occasional epistaxis. The patient had low levels of both FVII coagulant activity (FVII:C) and antigen (FVII:Ag) (5·0% and 7·3% of normal controls respectively). DNA sequence analysis of the FVII gene showed that the patient was homozygous for a mutation that resulted in a Cys for Gly354 substitution, a novel missense mutation in the catalytic domain. Haplotype analysis showed that this missense mutation was inherited from her consanguineous parents. Transient expression experiments showed that secreted FVII Cys354, FVII:C and FVII:Ag levels in conditioned media were reduced to 4% and 5%, respectively, of levels secreted from wild-type FVII. However, the intracellular FVII Cys354 was 67% that of normal recombinant protein. Immunohistochemical analysis showed that intracellular FVII:Ag from FVII 354Cys was present diffusely throughout the cytoplasm. Substitution of FVII 354Gly with amino acids other than Cys (Arg, Asp, Ser and Phe), did not produce a phenotype similar to FVII Cys354Gly. Molecular modelling indicated that FVII Gly354 was located outside the FVII heavy chain, and that Cys135 in the EGF2 domain, Cys262 in the catalytic domain and Cys127 all exist within 10 Å of Gly354. Therefore, we propose that the introduction of an additional free cysteine residue in the FVII molecule results in the formation of illegitimate disulphide bonds and a mis-folded domain, leading to defective secretion. [ABSTRACT FROM AUTHOR]

Despite a successful outcome of the anti-D prophylaxis programme, alloimmunization still occurs. The aim of this study was to estimate the frequency of Rh-specific B lymphocytes in the peripheral blood of nine Rh-alloimmunized individuals at different time intervals after parturition. The donors’ B lymphocytes were transformed with Epstein–Barr virus (EBV) and cultured at different cell densities over a feeder of human fetal fibroblasts. Culture supernatants were screened for human immunoglobulin by enzyme-linked immunosorbent assay (ELISA) and for anti-Rh antibody by using a direct haemagglutination technique. The percentage of CD19+ B lymphocytes in peripheral blood was determined by flow cytometry, and the frequency of Rh-specific B lymphocytes was estimated by limiting-dilution assay (LDA). The frequency of Rh-specific B lymphocytes varied from 1 : 150 to 1 : 27 850 in different donors. There was a decrease in this frequency and level of anti-Rh antibody with increase in time interval between bleeding and last exposure to the antigen. Furthermore, a positive correlation was observed between the titre of Rh-specific antibody and frequency of Rh-specific B cells in each of three subjects bled at multiple time-points postdelivery. The magnitude of the specific antibody response to Rh antigens varies greatly in Rh-alloimmunized women, which partly reflects the difference in frequency of specific B cells in these individuals. [ABSTRACT FROM AUTHOR]

The aim of the study was to perform fetal RHD genotyping in maternal plasma using a fluorescent polymerase chain reaction (PCR) technique. Duplex PCR, amplifying RHD and SRY in the same tube, was undertaken. The effect of varying storage temperatures on the concentration of fetal DNA was investigated in a separate study involving 10 RhD-negative pregnant women. Primers and probes for the RHD gene's exon 7 and the sex-determining region, Y, were designed, and monoplex and duplex PCR were performed. Blood samples from 10 RhD-negative women were split into four and treated in four different ways before measuring the concentration of fetal DNA by quantitative PCR. DNA extracted from the plasma of 114 RhD-negative pregnant women was tested for the presence of fetal RHD. The discrepancy between genotyping and serological RhD typing of the babies postpartum was 8% when counting one positive replicate as a positive result. Duplex PCR, amplifying RHD and SRY in the same tube, showed a reduced sensitivity for amplification of the SRY gene segment. There was a statistically significant reduction of fetal DNA in blood samples stored at room temperature for 48 h compared with the same sample stored at a temperature of < 10 °C for the same length of time. This method is not suitable for routine analysis because of the lack of a positive control for RHD-negative female fetuses and a decrease in PCR sensitivity when performing duplex PCR. Fetal DNA in maternal plasma is better preserved when the blood sample is kept cool. [ABSTRACT FROM AUTHOR]

A combination of blotting of bacterial colonies on nitrocellulose paper discs and immunoenzymatic detection of bound antigens with specific monoclonal antibodies was used to detect and serotype Actinobacillus actinomycetemcomitans directly off the initial agar culture dish. The A. actinomycetemcomitans antigens, representative of specific colonies, were identified immunoenzymatically using monoclonal antibody specific for a species-specific antigen. The serotype of the bacteria in colonies was identified by dividing the blotting paper into sections and immunoenzymatically identifying the serotype antigens with serotype-specific monoclonal antibodies. This procedure provides for simple, rapid, sensitive and accurate identification and characterization of A. actinomycetemcomitans isolates from the initial isolation agar plates. [ABSTRACT FROM AUTHOR]

Novel antibodies have been generated by immunizing with bacterially expressed fragments of the repetitive motif of the Ki-67 gene. One such antibody, MIB1, recognizes a fixation and embedding resistant epitope on the Ki-67 protein if sections are previously microwaved in a citrate buffer. We have investigated the utility of this antibody as a marker of cell proliferation in archival material. The microwave technique is simple but requires careful monitoring since different tissues and fixatives require different irradiation times. Strong nuclear immunoreactivity was detected with all fixatives studied. Cytoplasmic staining was not identified. In a wide range of normal tissues the distribution and number of MIB1 immunoreactive cells matched that of cryostat sections stained with Ki-67. In nude mouse xenografts in which the growth fraction had been defined using a fraction of labelled mitosis method, the labelling index with MIB1 matched that previously determined for Ki-67 and correlated well with the growth fraction. Other markers of proliferation (e.g. proliferating cell nulcear antigen) have been shown to be expressed in DNA repair, thus we investigated expression of MIB1 immunoreactivity in situations of DNA repair in vivo—ultraviolet irradiated human skin. MIB1 staining correlated with semi-conservative DNA synthesis rather than excision repair DNA synthesis. Finally, the morphological and cell cycle distribution of MIB1 expression is identical to that of Ki-67. Thus, MIB1 represents a new anti-Ki-67 antibody which appears to be a robust marker of cell proliferation easily applicable to archival material. [ABSTRACT FROM AUTHOR]

PCNA is a nuclear protein that is synthesized in late G[sub 1] and S phases of the cell cycle and is, therefore, correlated with the cell proliferative state. A new monoclonal antibody (PC10) to genetically engineered PCNA has been shown to label proliferating cells in formalin-fixed paraffin-embedded normal human tissues. Previous studies in lymphomas, using various markers of cell proliferation, have shown a strong correlation between indices of cell proliferation and histological grade. These studies have shown that within each histological subtype there is often a wide range of proliferative indices and that these may be of some prognostic significance. Thirty-one gastrointestinal lymphomas were studied. Our results show that there is a good correlation between PC10 index and histological grade of tumour (0.01 > P > 0.001 ) and also a significant relationship between PC 10 index and S + G[sub 2] + M phase fraction as measured by flow cytometric analysis (r² = 0.62; P< 0.01). Twenty-three cases were available for survival analysis. In these cases a high PC10 score correlated with poor survival (P=0.04). Based on this series, it appears that there is a significant relationship between PC 10 index and histological grade, and between PC 10 index and S + G[sub 2] + M phase as measured by flow cytometric analysis. In addition, our results suggest that a high PC10 index is an adverse prognostic factor in primary gastrointestinal lymphoma. [ABSTRACT FROM AUTHOR]

The lesions induced by reactive oxygen species in both nuclear and mitochondrial genomes include altered bases, abasic (AP) sites, and single-strand breaks, all repaired primarily via the base excision repair (BER) pathway. Although the basic BER process (consisting of five sequential steps) could be reconstituted in vitro with only four enzymes, it is now evident that repair of oxidative damage, at least in mammalian cell nuclei, is more complex, and involves a number of additional proteins, including transcription- and replication-associated factors. These proteins may be required in sequential repair steps in concert with other cellular changes, starting with nuclear targeting of the early repair enzymes in response to oxidative stress, facilitation of lesion recognition, and access by chromatin unfolding via histone acetylation, and formation of metastable complexes of repair enzymes and other accessory proteins. Distinct, specific subclasses of protein complexes may be formed for repair of oxidative lesions in the nucleus in transcribed vs. nontranscribed sequences in chromatin, in quiescent vs. cycling cells, and in nascent vs. parental DNA strands in replicating cells. Characterizing the proteins for each repair subpathway, their signaling-dependent modifications and interactions in the nuclear as well as mitochondrial repair complexes, will be a major focus of future research in oxidative damage repair. [Copyright &y& Elsevier]

An intrusion detection method using rough-fuzzy set and parallel quantum genetic algorithm (RFS-QGAID) is proposed in this paper. The RFS-QGAID is applied to solve the serious problems of determining the optimal antibodies subsets used to detect an anomaly. To obtain a simplified antibodies collection for high dimensional Log data sets, RFS is applied to delete the redundant antibody features and obtain the optimal antibodies features combination. Then, the optimal attitudes are entered into the QGA classifier for learning and training in the following stage. At last, the detected Log antigens are fed into RFS-QGAID, and we can classify the intrusion types. With RFS-QGAID, we give the simulations, the results on real Log data sets show that: the higher detection accuracy of RFS-QGAID is higher detection accuracy, but the false negative rate is lower for small samples sets, the adaptive performance is higher than other detection algorithms. [ABSTRACT FROM AUTHOR]

In omics sciences, many compounds are measured simultaneously in a sample in a single run. Such analytical performance opens up prospects for improving cellular cancer vaccines and other cell-based immunotherapeutics. This article provides an overview of proteomics technology, known as cell proteomic footprinting. The molecular phenotype of cells is highly variable, and their antigenic profile is affected by many factors, including cell isolation from the tissue, cell cultivation conditions, and storage procedures. This makes the therapeutic properties of cells, including those used in vaccines, unpredictable. Cell proteomic footprinting makes it possible to obtain controlled cell products. Namely, this technology facilitates the cell authentication and quality control of cells regarding their molecular phenotype, which is directly connected with the antigenic properties of cell products. Protocols for cell proteomic footprinting with their crucial moments, footprint processing, and recommendations for the implementation of this technology are described in this paper. The provided footprints in this paper and program source code for their processing contribute to the fast implementation of this technology in the development and manufacturing of cell-based immunotherapeutics. [ABSTRACT FROM AUTHOR]

Standardization of allergen provocation tests of the airways requires standardization of each of the steps involved, including standardization of the extracts used. The value of international standards is emphasized and so is the determination of a clinically relevant potency. The allergen extracts used for bronchial provocation tests must be compared with qualitatively and quantitatively clinically relevant standards using complementary immunochemical and biological methods. Ideally the same extracts should be used for diagnosis (skin test, specific IgE determinations, provocations) and treatment (immuno- therapy). The importance of the biological compared with the non-biological methods is that the biological methods are established in allergenic patients in order to select a clinically relevant potency of the allergen in question, and this potency ought to be confirmed in clinical trials. Once established the biological potency of an in-house preparation can be reproduced by supplementary in vitro methods. Very few commercial allergen preparations are available which fulfil the above-mentioned criteria. [ABSTRACT FROM AUTHOR]

Simple Summary: In this paper, we report on the inhibition of glioblastoma (GBM) cell growth and migration by the putative S1PR1 modulator ACT-2009905, using appropriate in vitro models. This work is based on our previously published finding that S1PR1 expression is strongly up-regulated in human glioblastoma samples and the fact that there is an association between S1PR1 with patients' survival times. We now show that pharmacological modulation by the putative S1PR1 modulator ACT-209905 inhibits GBM cell growth and migration. Furthermore, we investigated the influence of co-culture of GBM cells with THP-1 cells as a model for human monocytes, showing pro-tumoral effects and arguing for a complex interplay between GBM cells, immune cells and underlying signaling pathways. We believe that this manuscript fits the interests of the readership of the journal Cancers as it addresses the impact of alternative therapeutic options using ACT-209905 for improving GBM therapy. Glioblastoma (GBM) is still a deadly tumor due to its highly infiltrative growth behavior and its resistance to therapy. Evidence is accumulating that sphingosine-1-phosphate (S1P) acts as an important tumor-promoting molecule that is involved in the activation of the S1P receptor subtype 1 (S1PR1). Therefore, we investigated the effect of ACT-209905 (a putative S1PR1 modulator) on the growth of human (primary cells, LN-18) and murine (GL261) GBM cells. The viability and migration of GBM cells were both reduced by ACT-209905. Furthermore, co-culture with monocytic THP-1 cells or conditioned medium enhanced the viability and migration of GBM cells, suggesting that THP-1 cells secrete factors which stimulate GBM cell growth. ACT-209905 inhibited the THP-1-induced enhancement of GBM cell growth and migration. Immunoblot analyses showed that ACT-209905 reduced the activation of growth-promoting kinases (p38, AKT1 and ERK1/2), whereas THP-1 cells and conditioned medium caused an activation of these kinases. In addition, ACT-209905 diminished the surface expression of pro-migratory molecules and reduced CD62P-positive GBM cells. In contrast, THP-1 cells increased the ICAM-1 and P-Selectin content of GBM cells which was reversed by ACT-209905. In conclusion, our study suggests the role of S1PR1 signaling in the growth of GBM cells and gives a partial explanation for the pro-tumorigenic effects that macrophages might have on GBM cells. [ABSTRACT FROM AUTHOR]

Background: Vaccination is essential for the prevention of infectious diseases and has helped to reduce disease-related mortality, such as pneumonia. However, traditional vaccine development is time-consuming and risky. Reverse vaccinology (RV) is a promising alternative to developing vaccines based on the in silico discovery of antigens, often termed 'potential vaccine candidates' (PVCs), using a pathogen's proteome. RV prediction technologies, such as VaxiJen (founded in 2007), are used to take the first step toward vaccine development. VaxiJen is used by researchers to identify PVCs for various diseases. A 10-year review of these PVCs was published in 2017. There has since been no review of viral PVCs predicted by VaxiJen from 2017 to 2021. The proposed scoping review aims to address this gap. Methods: This protocol is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) 2015 checklist. The review will employ Arksey and O'Malley's five-stage methodological framework, which was later enhanced by Levac et al. and the Joanna Briggs Institute (JBI). The PRISMA extension for Scoping Reviews (PRISMA-ScR) reporting guideline will be utilized with this framework. PubMed, Scopus, Web of Science, EBSCOhost, and ProQuest One Academic will be searched using the term 'vaxijen'. The inclusion criteria will be English-only full-text original articles published in peer-reviewed journals and unpublished papers from 2017 to 2021. Rayyan will be used to deduplicate, screen titles and abstracts of articles. The articles' full texts will be examined. The data will be extracted using Microsoft Excel. Using a data charting form, data will be sifted and organized by key categories and themes. Discussion: This protocol was submitted for publication and went through an extensive peer review process. The review has implications for novel vaccine development against various viruses. The key limitation of this study is language bias due to the selection of English-only papers because of limited resources. This study will not require ethical clearance since it will use secondary data and will not include patients. Nevertheless, this research is part of a larger project that was submitted for ethical consideration to the Biomedical Research Ethics Committee of the University of KwaZulu-Natal in South Africa. This study's findings will be published in a peer-reviewed journal and provided to relevant stakeholders. Systematic review registration: Open Science Framework (OSF): https://osf.io/ht8wr [ABSTRACT FROM AUTHOR]

Simple Summary: This paper aims at showing the impact of novel imaging technologies and modern radiotherapy techniques on the management of cancer, with a particular focus on prostate adenocarcinoma. The manuscript explores the value of diagnostic imaging before treatment, the role of radiomics in predicting outcomes, the benefit of novel imaging in radiotherapy planning and the influence of advanced technologies in systemic treatment and in the management of other non-oncological conditions in order to tailor the best therapeutical strategies. Introduction: Targeted dose-escalation and reduction of dose to adjacent organs at risk have been the main goal of radiotherapy in the last decade. Prostate cancer benefited the most from this process. In recent years, the development of Intensity Modulated Radiation Therapy (IMRT) and Stereotactic Body Radiotherapy (SBRT) radically changed clinical practice, also thanks to the availability of modern imaging techniques. The aim of this paper is to explore the relationship between diagnostic imaging and prostate cancer radiotherapy techniques. Materials and Methods: Aiming to provide an overview of the integration between modern imaging and radiotherapy techniques, we performed a non-systematic search of papers exploring the predictive value of imaging before treatment, the role of radiomics in predicting treatment outcomes, implementation of novel imaging in RT planning and influence of imaging integration on use of RT in current clinical practice. Three independent authors (GF, IM and ID) performed an independent review focusing on these issues. Key references were derived from a PubMed query. Hand searching and clinicaltrials.gov were also used, and grey literature was searched for further papers of interest. The final choice of papers included was discussed between all co-authors. Results: This paper contains a narrative report and a critical discussion of the role of new modern techniques in predicting outcomes before treatment, in radiotherapy planning and in the integration with systemic therapy in the management of prostate cancer. Also, the role of radiomics in a tailored treatment approach is explored. Conclusions: Integration between diagnostic imaging and radiotherapy is of great importance for the modern treatment of prostate cancer. Future clinical trials should be aimed at exploring the real clinical benefit of complex workflows in clinical practice. [ABSTRACT FROM AUTHOR]

Pt nanoparticles deposited on single-walled carbon nanotubes (PtSWCNTs), synthesized via the deposition precipitation (DP) method, were introduced as a substrate for immobilizing antibodies on an electrode surface and then enhancing the electrochemical sensitivity. A PtSWCNT-modified paper-based screen-printed graphene electrode was successfully developed to diagnose hepatitis C virus (HCV) infection. The hepatitis C virus core antigen (HCV-cAg) level was determined by differential pulse voltammetry (DPV) using [Fe(CN)6]3−/4− as a redox solution. In the presence of HCV-cAg, the DPV current response decreased with increasing HCV-cAg concentration. Under the optimal conditions, the change in current response provides a good linear correlation with the logarithm of HCV-cAg concentration in the range 0.05 to 1000 pg mL−1 (RSD < 5%), and the limit of detection was 0.015 pg mL−1 (or 0.71 fmol L−1). Furthermore, the proposed immunosensor has been utilized to quantify HCV-cAg in human serum samples with reliable results compared with standard immunoassays (% relative error < 10%). This sensor offers a simple, sensitive, selective, disposable, and inexpensive means for determination of HCV-cAg in human serum samples. The paper-based label-free immunosensor is versatile and feasible for clinical diagnosis. [ABSTRACT FROM AUTHOR]

Simple Summary: The frequency of metastatic melanoma, an extremely deadly malignancy, is rapidly increasing worldwide. Recently, messenger RNA (mRNA) injections have emerged as a promising treatment option. While mRNA therapies have demonstrated significant promise, the stability of the naked form remains a barrier, as naked mRNAs are vulnerable to common ribonucleases, and are unable to effectively penetrate plasma membranes and escape from endosomes. Thus, for this paper, we used a hyper-branched cyclodextrin-based polymer (Ppoly) as a carrier to enhance mRNA delivery for melanoma cancer. The in vitro results demonstrated that Ppoly was able to deliver the EGFP-mRNA effectively in both 2D and 3D melanoma cell lines compared to naked mRNA; in addition, Ppoly did not show any cytotoxicity. The anti-tumour effect of intratumourally injected OVA-mRNA loaded on Ppoly results showed a significant decrease in both tumour size and weight compared to other formulations by inducing an efficient adaptive immune response and OVA-specific CD8+ T cells in both spleen and tumour tissues compared to other groups. mRNA technology has demonstrated potential for use as an effective cancer immunotherapy. However, inefficient in vivo mRNA delivery and the requirements for immune co-stimulation present major hurdles to achieving anti-tumour therapeutic efficacy. Therefore, we used a cationic hyper-branched cyclodextrin-based polymer to increase mRNA delivery in both in vitro and in vivo melanoma cancer. We found that the transfection efficacy of the mRNA-EGFP-loaded Ppoly system was significantly higher than that of lipofectamine and free mRNA in both 2D and 3D melanoma cancer cells; also, this delivery system did not show cytotoxicity. In addition, the biodistribution results revealed time-dependent and significantly higher mEGFP expression in complexes with Ppoly compared to free mRNA. We then checked the anti-tumour effect of intratumourally injected free mRNA–OVA, a foreign antigen, and loaded Ppoly; the results showed a considerable decrease in both tumour size and weight in the group treated with OVA-mRNA in loaded Ppoly compared to other formulations with an efficient adaptive immune response by dramatically increasing most leukocyte subtypes and OVA-specific CD8+ T cells in both the spleen and tumour tissues. Collectively, our findings suggest that the local delivery of cationic cyclodextrin-based polymer complexes containing foreign mRNA antigens might be a good and reliable concept for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

This paper outlines methods used to identify novel antigens for use in the development of serological assays. Specifically, we applied these methods to a neurogenic parasitic nematode of cervids called Parelaphostrongylus tenuis. This parasite is of particular concern in both wild and domestic ungulates as it causes significant neurological signs and definitive diagnosis is only possible post-mortem, necessitating the development of serologic assays for antemortem diagnosis. Proteins extracted from P. tenuis organisms were affinity isolated using antibodies enriched from seropositive moose (Alces alces). The proteins were analyzed using mass spectrometry and liquid chromatography to obtain amino acid sequences that were then cross-referenced to open reading frames predicted from an assembled transcriptome. An antigen of interest was assessed for immunogenic epitopes and subsequently synthesized into 10-mer synthetic overlapping peptides representing these regions. These synthetic peptides were then assessed for reactivity against positive and negative moose sera and demonstrated potential use as a serological assay in diagnostic laboratories. Known negative moose sera revealed significantly lower optical density when compared to the positive samples (p < 0.05). This method serves as a pipeline for the construction of diagnostic assays of pathogens in both human and veterinary medicine. [ABSTRACT FROM AUTHOR]