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The effects of filter paper strip (FPS) storage time and temperature on antibody detection are poorly understood despite widespread use in wildlife research. We collected sera and FPSs from 23 wild turkeys (Meleagris gallopavo) and 20 northern bobwhites (Colinus virginianus) experimentally infected with West Nile virus (WNV) to compare FPS storage methods with WNV plaque reduction neutralization test (PRNT) sensitivity. FPS storage methods included: immediate elution after drying, and storage at −20°C for 3 mo, −20°C for 6 mo, room temperature (RT) for 3 mo, and RT for 6 mo prior to elution. FPS eluates and sera were co-titrated to determine endpoint antibody titers, which were compared between FPS sera eluted immediately and sera, and among FPS eluates that underwent different storage conditions. Antibody titers were ~4-fold less in FPS sera eluted immediately versus sera, and dropped more frequently below PRNT detection threshold in northern bobwhites, which had ~10-fold lower serum antibody titers than wild turkeys. Antibody titers were lower in FPS samples stored at RT and for 6 mo. WNV serologic surveys may result in falsely low seroprevalence estimates if FPSs are stored at RT for ≥ 3 mo before elution. [ABSTRACT FROM AUTHOR]

Paper-based biosensors are a potential paradigm of sensitivity achieved via microporous spreading/microfluidics, simplicity, and affordability. In this paper, we develop decorated paper with graphene and conductive polymer (herein referred to as graphene conductive polymer paper-based sensor or GCPPS) for sensitive detection of biomolecules. Planetary mixing resulted in uniformly dispersed graphene and conductive polymer ink, which was applied to laser-cut Whatman filter paper substrates. Scanning electron microscopy and Raman spectroscopy showed strong attachment of conductive polymer-functionalized graphene to cellulose fibers. The GCPPS detected dopamine and cytokines, such as tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6) in the ranges of 12.5–400 µM, 0.005–50 ng/mL, and 2 pg/mL–2 µg/mL, respectively, using a minute sample volume of 2 µL. The electrodes showed lower detection limits (LODs) of 3.4 µM, 5.97 pg/mL, and 9.55 pg/mL for dopamine, TNF-α, and IL-6 respectively, which are promising for rapid and easy analysis for biomarkers detection. Additionally, these paper-based biosensors were highly selective (no serpin A1 detection with IL-6 antibody) and were able to detect IL-6 antigen in human serum with high sensitivity and hence, the portable, adaptable, point-of-care, quick, minute sample requirement offered by our fabricated biosensor is advantageous to healthcare applications. [ABSTRACT FROM AUTHOR]

The modification of an easily available resource like paper to circumvent expensive or intensive sample pretreatment could be the answer to sample analysis in resource-poor regions. Therefore, a novel on-paper device combining sample collection with affinity sample pretreatment is introduced here. Universal smart affinity samplers are produced by a simple KIO4-mediated oxidation of cellulose, which functionalizes the paper. This is followed by immobilization of streptavidin. Streptavidin serves as a universal anchor for biotinylated antibodies, enabling simple preparation of tailor-made affinity samplers. The functionality of the device was tested using a model protein (human chorionic gonadotropin, hCG) and biotinylated anti-hCG antibodies for affinity capture. In a laboratory setting, the performance was demonstrated, and a 14-fold increase of target binding compared to binding without bmAb was achieved. The recovery of hCG captured with bmAb-treated samplers was determined to be 33% and comparable to previously described affinity capture approaches. Application of the smart affinity samplers to human serum containing hCG showed an R2 of 0.98 (200–1000 pg mL−1), precision of ≤ 9.1% RSD, and estimated limit of detection of 65 pg mL−1. Although further optimization and validation are necessary prior to application to real samples in clinical settings, the potential of the device for use in determination of low abundant biomarkers in complex samples has been demonstrated. [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]

Background noise due to nonspecific binding of biomolecules on the assay substrates is one of the most common challenges that limits the sensitivity of microarray-based immunoassays. Background signal intensity usually increases when complex biological fluids are used because they have a combination of molecules and vesicles that can adsorb onto substrate surfaces. Blocking strategies coupled with surface chemistries can reduce such nonspecific binding and improve assay sensitivity. In this paper, we conducted a systematic optimization of blocking strategies on a variety of commonly used substrates for protein measurement in complex biofluids. Four blocking strategies (BSA, non-fat milk, PEG, and a protein-free solution) coupled with four surface chemistries (3-glycidoxypropyltrimethoxysilane (GPS), poly-L-lysine (PLL), aminoalkylsilane (AAS), and nitrocellulose (NC)) were studied for their effect on background, microspot, and net signal intensities. We have also explored the effect that these blocking strategies have when proteins in complex samples (plasma, serum, cell culture media, and EV lysate) are measured. Irregular spot morphology could affect signal extraction using automated software. We found that the microspots with the best morphology were the ones printed on GPS glass surfaces for all immunoassays. On NC membrane, the protein-based blocking strategies yielded the highest net fluorescent intensity with the antigen contained in PBS, plasma, serum, and serum-free cell culture media. Differently, with EV lysate samples, Pierce™ protein-free blocker yielded the best net signal intensity on both GPS and NC surfaces. The choice of blocking strategies highly depends on the substrate. Moreover, the findings discovered in this study are not limited to microarray-based immunoassays but can provide insights for other assay formats. [ABSTRACT FROM AUTHOR]

Identification of specific antibodies in patient plasma is an essential part of many diagnostic procedures and is critical for safe blood transfusion. Current techniques require laboratory infrastructure and long turnaround times which limits access to those nearby tertiary healthcare providers. Addressing this challenge, a novel and rapid paper-based antibody test is reported. We validate antibody detection with reverse blood typing using IgM antibodies and then generalise the validity by adapting to detect SARS CoV-2 (COVID-19) antibodies in patient serum samples. Reagent red blood cells (RBC) are first combined with the patient plasma containing the screened antibody and a droplet of the mixture is then deposited onto paper. The light intensity profile is analyzed to identify test results, which can be detected by eye and/or with image processing to allow full automation. The efficacy of this test to perform reverse blood typing is demonstrated and the performance and sensitivity of this test using different paper types and RBC reagents was investigated using clinical samples. As an example of the flexibility of this approach, we labeled the RBC reagent with an antibody–peptide conjugate to detect SARS CoV-2 (COVID-19) antibodies in patient serum samples. This concept could be generalized to any agglutination-based antibody diagnostics with blood plasma. [ABSTRACT FROM AUTHOR]

The performance of Whatman 3-MM filter papers for the collection, drying, shipment and long-term storage of blood at ambient temperature, and for the detection of African swine fever virus and antibodies was assessed. Conventional and real-time PCR, viral isolation and antibody detection by ELISA were performed on paired samples (blood/tissue versus dried-blood 3-MM filter papers) collected from experimentally infected pigs and from farm pigs in Madagascar and Côte d'Ivoire. 3-MM filter papers were used directly in the conventional and real-time PCR without previous extraction of nucleic acids. Tests that performed better with 3-MM filter papers were in descending order: virus isolation, real-time UPL PCR and conventional PCR. The analytical sensitivity of real-time UPL PCR on filter papers was similar to conventional testing (virus isolation or conventional PCR) on organs or blood. In addition, blood-dried filter papers were tested in ELISA for antibody detection and the observed sensitivity was very close to conventional detection on serum samples and gave comparable results. Filter papers were stored up to 9 months at 20-25°C and for 2 months at 37°C without significant loss of sensitivity for virus genome detection. All tests on 3-MM filter papers had 100% specificity compared to the gold standards. Whatman 3-MM filter papers have the advantage of being cheap and of preserving virus viability for future virus isolation and characterization. In this study, Whatman 3-MM filter papers proved to be a suitable support for the collection, storage and use of blood in remote areas of tropical countries without the need for a cold chain and thus provide new possibilities for antibody testing and virus isolation. [ABSTRACT FROM AUTHOR]

The effect of paper structure on blood typing visualization was quantified and analyzed to engineer low-cost diagnostics. Commercial and experimental papers varying in fibre composition, basis weight, density and porosity were investigated for their ability to separate agglutinated (blood interacted with specific antibodies) from non-agglutinated (blood interacted with non-specific antibodies) red blood cells (RBCs). Antibodies solutions and blood samples were sequentially absorbed on paper, allowed to interact, eluded with a saline solution, and the intensity of the remaining blood spot was quantified by image analysis. The efficiency and clarity of RBC separation was quantified with the relative intensity (R.I.) index defined as the intensity ratio of the non-specific test over the specific system; the lower the R.I., the better is the separation between a positive from a negative test. Thick and dense papers are improper for blood typing as they retain indiscriminately both agglutinated and non-agglutinated RBCs. Thin and porous papers provided the best performance. The R.I. index (the lower the better) increased fairly linearly with paper density and thickness but inversely proportionally with paper pore size. The type of fibres played a minor role. The paper structure is critical in the design of blood typing assay. However, it is only one element of the diagnostic system to engineer with the interactions RBC-antibody-paper. [ABSTRACT FROM AUTHOR]

During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP—KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3—to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3–25 nM) exhibited the lowest limits of detection (ca. μg mL−1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual's immune response can impact the interpretation of assay results. [ABSTRACT FROM AUTHOR]

Infectious diseases are among the leading causes of mortality worldwide. A new coronavirus named severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) was identified in Wuhan, China in 2019, and the World Health Organization (WHO) declared its outbreak, coronavirus disease 2019 (COVID-19), as a global pandemic in 2020. COVID-19 can spread quickly from person to person. One of the most challenging issues is to identify the infected individuals and prevent potential spread of SARS-CoV-2. Recently, anti-SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody tests using immunochromatographic methods have been used as a complement to current detection methods and have provided information of the approximate course of COVID-19 infection. However, blood sampling causes pain and poses risks of infection at the needle puncture site. In this study, a novel patch sensor integrating porous microneedles and an immunochromatographic assay (PMNIA) was developed for the rapid detection of anti-SARS-CoV-2 IgM/IgG in dermal interstitial fluid (ISF), which is a rich source of protein biomarkers, such as antibodies. Biodegradable porous microneedles (MNs) made of polylactic acid were fabricated to extract ISF from human skin by capillary effect. The extracted ISF was vertically transported and flowed into the affixed immunoassay biosensor, where specific antibodies could be detected colorimetrically on-site. Anti-SARS-CoV-2 IgM/IgG antibodies were simultaneously detected within 3 min in vitro. Moreover, the limit of detection of anti-SARS-CoV-2 IgM and IgG concentrations was as low as 3 and 7 ng/mL, respectively. The developed device integrating porous MNs and immunochromatographic biosensors is expected to enable minimally invasive, simple, and rapid anti-SARS-CoV-2 IgM/IgG antibody testing. Furthermore, the compact size of the MN and biosensor-integrated device is advantageous for its widespread use. The proposed device has great potential for rapid screening of various infectious diseases in addition to COVID-19 as an effective complementary method with other diagnostic tests. [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]

An enzyme-linked immunosorbent assay method was developed here for benzophenone (BZP) detection in package paper samples based on polyclonal antibody. 4-Aminobenzophenone (ABZP) was used as hapten and conjugated with bovine serum albumin for antibody production. Cross-reactivity results showed that the antibody can be recognised BZP, ABZP and Michler's ketone very well (IC50 value was 31.2±7.8 ng/mL, 35.9±8.3 ng/mL and 43.7±8.2 ng/mL, respectively). After optimisation of the effects from pH, ionic strength and organic solvents on immunoassay, we established a rapid method and used for BZP detection in fortified package paper samples with good recovery data (98.5 and 99.7%) at 100 and 600 ng/g spiked level.) [ABSTRACT FROM AUTHOR]

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng μL−1 (0.112 IU mL−1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development. [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]

The paper mulberry (Broussonetia papyrifera) leaf is rich in alkaloids and flavonoids, which has high medicinal and feeding value. We aimed to analyze the effects of B. papyrifera leaf extract on growth performance, antioxidant capacity, immune functions, and fecal microflora of weaned piglets. Thirty healthy, 28-day-old piglets were randomly assigned to three groups and fed with a basal diet supplemented with 0, 150, and 300 g/t B. papyrifera leaf extract for 42 days (control group, group I, and group II) separately. The result revealed that the final weight of piglets in group II was higher than the other groups, and the diarrhea rate in this group was 62.9% lower than in the control group. The feed conversion ratio in group I was significantly lower than the other two groups. Higher blood urine nitrogen concentration was noted in group II, higher glutathione peroxidase and catalase in group II, higher superoxide dismutase in the control group, and higher immune globulins (Ig) IgG, IgA, and IgM in group II. There was no significant difference in community richness and community diversity among the three groups of fecal samples. The relative level of Roseburia was higher in groups I and II, while Lactobacillus was higher in the control group. In conclusion, supplementation with B. papyrifera leaf extract at a certain dosage can increase growth performance and antioxidant capacity of weaned piglets, reduce the occurrence of diarrhea, enhance immune functions and disease resistance, and affect the composition of fecal microflora. [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]

This study investigated the stability of the primary blood typing antibodies (Anti-A, Anti-B and Anti-D IgM) on paper. This knowledge is critical to manufacture a new type of paper-based blood typing device where blood group antibodies must be kept active on paper for extended periods. Two strategies were explored. The first involved mixing additives such as polyvinylpyrrolidone (PVP), dextran and glycerol, with antibodies before sorption onto paper. While all the additives tested improved the antibody stability on paper, their protection for storage at room temperature was limited; dextran provided the longest protection, followed by PVP and then glycerol. The second strategy relied on freeze-drying to stabilize the antibodies in paper. Freeze dried antibodies sorbed into paper could be stored for long periods at ambient conditions without significantly loss of their activity. The thermal stability of antibodies in paper was also improved by freeze-drying. Our work shows that the use of additives and freeze-drying are effective approaches to retain the activities of IgM blood group antibodies on paper. These approaches will be further explored for the large scale development of a new generation of clinical and home-care blood testing devices. [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]

Many studies are being conducted worldwide, giving a more complete picture of the composition and properties of bovine colostrum, which allows it to be used in various diseases in clinical practice. An analysis of modern literature shows that bovine colostrum is rich in immunoglobulins, antimicrobial, and growth factors. Bovine colostrum has a positive effect in treating diarrhea in patients with immunodeficiency syndrome, in treating intestinal disorders caused by nonsteroidal anti-inflammatory drugs, in postoperative responses and the treatment of upper respiratory tract infections. The aim of this research was to reveal the regularities of changes in microbiological and physicochemical parameters of colostrum after its defatting and freeze-drying. We used 4 samples of bovine colostrum in the first 6 hours after calving. The samples were degreased using a Hermle Z32HK centrifuge (Germany). Colostrum is stored in a household refrigerator at sub-zero temperatures. Freeze drying was carried out on equipment Erstvak EV-DF10A, (Russia). The microbiological parameters of the studied samples of colostrum were determined: Number of mesophilic aerobic and facultative anaerobic microorganisms (QMAFAnM) - according to GOST 32901-2014; Molds and yeasts - according to GOST 33566-2015; Bacteria of the group of Escherichia coli (BGKP) - according to GOST 32901; Staphylococcus aureus - according to GOST 30347; and Proteus spp. - according to GOST 28560-90. Investigated physical and chemical indicators were: Mass fraction of non-protein nitrogenous substance - according to GOST 23327-98; Mass fraction of protein - according to GOST 23327-98; and Mass fraction of fat - according to GOST 5867-90. To obtain defatted colostrum, native colostrum was heated to 40 0C and defatted by centrifugation in a Hermle Z32HK centrifuge (Germany) at 3,000 rpm for 20 minutes. Colostrum drying was carried out at condenser temperature (-60 0C), product temperature (25 0C), vacuum (1 Pa) on Erstvak EV-DF10A, (Russia). Samples of true colostrum obtained in the bactericidal phase from healthy cows of the second and third calving's did not contain pathogenic or opportunistic microflora. In samples of colostrum and preparations from colostrum after defrosting and drying, there is no pathogenic microflora, the titer of molds and yeasts in the samples meets the requirements for raw materials for specialized dairy products. In accordance with the obtained experimental physicochemical indicators of samples of native, low-fat, dry low-fat colostrum; colostrum whey, dry colostrum whey, the following conclusions can be drawn: - Colostrum is collected in the bactericidal phase, which is confirmed by physicochemical indicators obtained on an automatic FOSS analyzer; - The colostrum of the first milking of cows is of better quality and immunocompetent in terms of physicochemical properties and composition. - In the colostrum of first-calf heifers and cows obtained 6 hours after calving, there is a decrease in the readings of density, acidity, total protein, immunoglobulins and fat compared to the colostrum of the first milking, regardless of the age of the animals. - Thus, colostrum is a unique secret of the mammary gland and, due to its composition, is characterized by the following biological properties: - ensures the creation of passive immunity; has a bactericidal effect due to the content of lysozyme, which dissolves the membranes of microorganisms; inhibits the development of pathogenic microorganisms due to high acidity; and has a high nutritional value and excellent dietary properties. [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]

Abstract: A novel ultra‐low‐cost biochemical analysis platform to quantify protein dissociation binding constants and kinetics using paper microfluidics is reported. This approach marries video imaging with one of humankind's oldest materials: paper, requiring no large, expensive laboratory equipment, complex microfluidics or external power. Temporal measurements of nanoparticle–antibody conjugates binding on paper is found to follow the Langmuir Adsorption Model. This is exploited to measure a series of antibody–antigen dissociation constants on paper, showing excellent agreement with a gold‐standard benchtop interferometer. The concept is demonstrated with a camera and low‐end smartphone, 500‐fold cheaper than the reference method, and can be multiplexed to measure ten reactions in parallel. These findings will help to widen access to quantitative analytical biochemistry, for diverse applications spanning disease diagnostics, drug discovery, and environmental analysis in resource‐limited settings. [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]

Objectives The immune trypanolysis test ( TL) is an accurate sero-diagnostic tool increasingly implemented for sleeping sickness medical surveillance, but it is restricted to the reference laboratories. To facilitate storage and transport of the test specimen, we developed a protocol for the examination of blood spotted on filter paper ( TL-fp) that can be stored and shipped at ambient temperature. We compared its performance with the classical TL on plasma ( TL-pl) that needs to be kept frozen until use. Methods The study was conducted in active foci of the Republic of Guinea. In total, 438 specimens from treated and untreated sleeping sickness patients and serological suspects were tested with both methods. Result TL-fp gave significantly less positive results than TL-pl, but all the confirmed sleeping sickness cases were positive with the TL-fp protocol. Conclusion TL-fp appears to offer a good compromise between feasibility and sensitivity to detect currently infected subjects who play a role in the transmission of Trypanosoma brucei gambiense and is useful for contributing to the elimination of gambiense sleeping sickness. [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]

Background: Food allergy (FA) affects approximately 8% of children and may be immunoglobulin E (IgE)-mediated or non-IgE-mediated. It is recognized clinically that children with both subtypes of FA may present with features of pediatric feeding disorder (PFD); however, there is currently a limited detail of presenting characteristics. Objective: The objective of this study was to synthesize the current evidence regarding the feeding characteristics of children with FA, with a focus on the feeding skills and psychosocial domains of PFD. Method: This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews guidelines. Papers published between 2001 and 2022 describing feeding characteristics in the feeding skills and psychosocial domains in children with FA/history of FA were included. Papers that focused solely on characteristics from the medical or nutritional domains were excluded. Descriptive information regarding demographics, methodology, allergy profile and history, and the characteristics of PFD observed was extracted using a preconceived data extraction form. Results: Overall, 40 papers contained descriptions of feeding characteristics of children with non-IgE-mediated FA (n = 22) and IgE-mediated FA (n = 11), while four were nonspecific. In the psychosocial domain, food refusal/aversion, anxiety with eating, and poor intake were the most frequently reported, regardless of FA subtype. Less information was reported regarding feeding skills, although slowness in eating, immature diet, and delays in oral sensory-motor skills were described. Conclusions: Children with FA/history of FA may present with a range of characteristics that map across the feeding skill and psychosocial domains of PFD. Systematic research is needed to fully describe the feeding characteristics of children with FA. [ABSTRACT FROM AUTHOR]

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]

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]

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]

Morphine was used as a model analyte to examine the possibility of using cellulose, physically modified by papermaking and converting techniques, as a capillary matrix in a lateral flow type of diagnostic assay. This research was directed toward low-cost, disposable, and portable paper-based diagnostics, with the aim of addressing the analytical performance of paper as a substrate in the analysis for drugs of abuse. Antibody Fab fragments were used as sensing molecules, and gold nanoparticle detection was employed. Inkjet printing was used to pattern sensing biomolecules as detection zones on paper. To validate the usefulness of paper as a diagnostic platform, the principle of a direct sandwich assay, based on immunocomplex formation between morphine and the anti-morphine Fab fragment and detection of the formed immunocomplex by another Fab fragment, was implemented. Results were compared with that achieved by using nitrocellulose as a reference material. Possible interfering from the sample matrix on assay quality was investigated with spiked oral fluid samples. Under optimized conditions, a visually assessed limit of detection for the sandwich assay was 1 ng/mL, indicating that the paper-based test devices developed in this work can perform screening for drugs of abuse and can fulfill the requirement for a sensitive assay in diagnostically relevant ranges. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: Reagent storage has been a long-standing challenge for diagnostics, especially those designed for low-resource settings and point-of-care applications. In general, the stability of a reagent relies on careful temperature control, often by refrigeration, which is costly and often unavailable in these remote settings. Poor reagent integrity can negatively affect the reproducibility and reliability of an assay. Given the recent interest in paper-based devices designed for quantitative analysis in point-of-care settings, a better understanding of reagent stability on filter paper is critical for proper device use and its longevity. In this article, we present an independent method to examine the stability of reconstituted antibodies that were stored on filter paper using flow cytometry. We validated the method by measuring the activity as measured by the mean fluorescence intensity (MFI) of antibodies stored with known stabilizers. Furthermore, we demonstrated the potential of our method to screen the influence of other paper treatments and storage processes on antibody stability, which may be applicable to the storage of reagents on paper in general. [Copyright &y& Elsevier]

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]

Here we show that a simple paper-based electrochemical sensor, fabricated by paper folding, is able to detect a 30-base nucleotide sequence characteristic of DNA from the hepatitis B virus (HBV) with a detection limit of 85 pM. This device is based on design principles we have reported HBV previously for detecting proteins via a metalloimmunoassay. It has four desirable attributes. First, its design combines simple origami (paper folding) assembly, the open structure of a hollow-channel paper analytical device to accommodate micrometer-scale particles, and a convenient slip layer for timing incubation steps. Second, two stages of amplification are achieved: silver nanoparticle labels provide a maximum amplification factor of 250 000 and magnetic microbeads, which are mobile solid-phase supports for the capture probes, are concentrated at a detection electrode and provide an additional ~25-fold amplification. Third, there are no enzymes or antibodies used in the assay, thereby increasing its speed, stability, and robustness. Fourth, only a single sample incubation step is required before detection is initiated. [ABSTRACT FROM AUTHOR]

Lateral flow type detection is becoming interesting not only in regions with a poor medical infrastructure but also for practitioners in day-to-day clinical work or for veterinary control in case of possible epidemics. In this work, we describe the first steps of development of a multi-channel strip with potential internal calibration of multiparametric and colorimetric lateral flow assays for the simultaneous detection of the lipopolysaccharides (LPS) of Salmonella typhimurium ( S. typhimurium) and Salmonella enteritidis ( S. enteritidis). We structured four channels in the nitrocellulose membrane with a Yb:KGW solid-state femtosecond laser ('cold' ablation process) to form distinct tracks of porous material and used gold nanoparticles for the labeling of the antibodies. In addition, calibration curves of the spot intensities of both serovars are presented, and it was shown that no cross reactivity between the different capture antibodies and LPS occurred. Finally, we detected LPS of both Salmonella serovars simultaneously. The color changes (spot intensities of the reaction zones) were evaluated using the open-source image-processing program ImageJ. [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]

Aim: The present study was designed to investigate the feasibility of the filter paper sampling of blood for the detection of IBDV antibodies in chicken sera using a commercial ELISA kit. Materials and Methods: Optimum dilution for filter paper extracts that would give a result equivalent to that of the corresponding serum sample in a commercial ELISA was determined. Correlation between the filter paper extracts based ELISA and conventional serum based ELISA was also examined and the reproducibility of the filter paper extracts based ELISA was tested. Results: A very good correlation (r = 0.9; n = 15; p<0.01) was observed between the results obtained with filter paper extracts based ELISA and serum based ELISA. The antibody titre determined by two systems were very close and did not exceed more than a two-fold dilution with the exception of samples having very low antibody levels, where relatively higher background reaction was observed with filter paper extracts based ELISA. The filter paper extracts based ELISA appeared to be quite reproducible with a coefficient of variation less than 10%. Conclusion: Filter paper based ELISA could be a useful alternative to serum dependant ELISA assays for sero-profiling chicken flocks for IBDV antibodies. [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]

Background: The method of collection as well as the packaging conditions in which samples are submitted to laboratories play a critical role on the acquisition of reliable results on diagnostic tests. Alternative methods however have been proposed, as the adsorption of blood or serum in filter paper. In this work, it was evaluated the viability of using serum or whole blood samples from bovines collected in filter paper for serological testing against bovine herpesvirus type 1 (BoHV-1). Materials, Methods & Results: One hundred and seven whole blood and serum samples were collected by standard methods. Serum neutralization test was used as a golden standard method for evaluation of the serum samples. The same samples of both whole blood and sera were also adsorbed on filter paper strips for further comparisons. Optimal conditions for serum and blood elution from filter paper were determined. Adsorbed samples on filter paper disks were eluted in PBS and subsequently diluted further with PBS 5% skimmed milk. The eluates were tested for antibodies to BoHV-1 in an indirect ELISA (iELISA) and matched with the results obtained by serum neutralization of standard serum samples. Comparison between results obtained by serum neutralization of standard serum samples and the ones from the iELISA of serum eluted from paper disks resulted in sensitivity, specificity, positive and negative predictive values of 95, 94, 80, 99%, respectively, and a correlation coefficient (κ) of 0.83. Comparison between the results of serum neutralization of standard serum samples and the ones from the iELISA of blood eluted from paper disks resulted in sensitivity, specificity, positive and negative predictive values of 91, 97, 87, 98% , respectively, and a correlation coefficient (κ) of 0.86. Standard serum samples were also tested in the iELISA and the results compared with those of iELISA from samples eluted from filter paper. Comparison of the results from iELISA between serum samples and serum eluted from filter paper resulted in sensitivity, specificity, positive and negative predictive values of 91, 81, 69, 95%, respectively, and a correlation coefficient (κ) of 0.66. The comparison of iELISA results between standard serum and blood samples eluted from filter paper resulted, in sensitivity, specificity, positive and negative predictive values of 78, 92, 82, 89%, respectively, and a correlation coefficient (κ) of 0.70. Fifty samples collected on filter paper were retested eight months later in order to determine whether those would retain its viability; both sensitivity and specificity remained unaltered. Discussion: Sampling on filter paper has been successfully described for antibody detection in a number of diseases such as Aujeszky's disease virus and Newcastle disease virus. In this work, it has been demonstrated that both blood and sera collected in filter paper can be used for submission of samples aiming detection of antibodies to BoHV-1 in an iELISA, without significant loss of sensitivity and specificity. Submission of samples on filter paper is a practical and economical alternative as no special conditions are required for storaging and transporting. This method also enables the collection of samples from distant places assuring its quality for serological tests. [ABSTRACT FROM AUTHOR]

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]

The pulp and paper industry together with lignocellulosic biofuel production provides plentiful streams of lignin and lignin-derived molecules (LDMs) that currently remain underutilized. The heterogeneity and complexity of lignin along with the lack of convenient tools significantly hamper its utilization. Selective separation of these LDMs from streams using specific tools would allow the recovery of aromatic compounds, as well as facilitate biological processes aiming at lignin valorization. To this end, here we report the isolation and characterization of single-chain variable fragment (scFv) antibodies against ferulate, coumarate, and caffeate, which are the molecular representatives of LDMs. Binders for the target LDMs were enriched by interrogating a synthetic scFv library with the phage display technique. As a result, scFv binders specific against each of the target molecules were obtained with affinities in the micromolar range. The selectivity of scFvs towards specific LDMs was proved by recovering caffeate from simulated LDM solution, Kraft lignin, and rice straw hydrolysate samples. Further proof of concept studies with model compounds demonstrated the applicability of antibody-based binders as a detection tool for monitoring microbial LDM conversion. Overall, this study demonstrates the potential of scFv binders as a specific toolset for lignin compound recovery and analysis. [ABSTRACT FROM AUTHOR]

This study introduced a barcode-like design into a paper-based blood typing device by integrating with smartphone-based technology. The concept of presenting a paper-based blood typing assay in a barcode-like pattern significantly enhanced the adaptability of the assay to the smartphone technology. The fabrication of this device involved the use of a printing technique to define hydrophilic bar channels which were, respectively, treated with Anti-A, -B, and -D antibodies. These channels were then used to perform blood typing assays by introducing a blood sample. Blood type can be visually identified from eluting lengths in bar channels. A smartphone-based analytical application was designed to read the bar channels, analogous to scanning a barcode, interpret this information, and then report results to users. The proposed paper-based blood typing device is rapidly read by smartphones and easy for the user to operate. We envisage that the adaptation of paper-based devices to the widely accepted smartphone technology will increase the capability of paper-based diagnostics with rapid assay result interpretation, data storage, and transmission. [ABSTRACT FROM AUTHOR]

Background and Objectives: Serological diagnosis of hydatidosis is usually performed by detecting the circulating antibodies in serum by ELISA. The present study was carried out to standardize and evaluate procedure of the ELISA using elute from dried blood spots (DBS) on filter paper and blood stored at different temperatures and at different durations for its further application under field conditions. Materials and Methods: Dried blood spots were collected from fifty study subjects and fifty control subjects and evaluated for the detection of IgG antibodies against hydatid. Samples were stored at room temperature and 4°C and tested by ELISA at 0, 15 and 30 days. Results: The present study shows that elute of DBS on filter paper can be stored at room temperature for a maximum of 30 days without a decrease in antibody titer as compared to serum samples tested by ELISA. Conclusions: The collection of blood sample on filter paper may serve useful purpose in resource limited countries for carrying out sero-epidemiological surveys at a cost effective level. [ABSTRACT FROM AUTHOR]

A paper-based lateral flow immunoassay for pathogen detection that avoids the use of secondary antibodies and is revealed by the photoluminescence quenching ability of graphene oxide is reported. Escherichia coli has been selected as a model pathogen. The proposed device is able to display a highly specific and sensitive performance with a limit of detection of 10 CFU mL-1 in standard buffer and 100 CFU mL-1 in bottled water and milk. This low-cost disposable and easy-to-use device will prove valuable for portable and automated diagnostics applications. [ABSTRACT FROM AUTHOR]

Bullous pemphigoid (BP), a common autoimmune blistering disease, is increasing in incidence and conveys a high mortality. Detection of autoantibodies targeting the noncollagenous 16A (NCI6A) domain of type XVII collagen using enzyme-linked immunosorbent assay (ELISA) has demonstrated high sensitivity and specificity for diagnosing BP. We have developed a rapid, low-cost, and widely applicable ELISA-based system to detect the NC16A autoimmune antibody and then diagnose and monitor BP disease activity using a piece of filter paper, a wax-printer, and NC16A antigens. Both sera and/or blister fluids from 14 untreated BP patients were analyzed. The control group included healthy volunteers and patients with other blistering disorders such as pemphigus vulgaris. In our established paper-based ELISA (P-ELISA) system, only 2 μL of serum or blister fluid and 70 min were required to detect anti-NC16A autoimmune antibodies. The relative color intensity was significantiy higher in the BP group than in the control groups when using either serum (P < 0.05) or blister fluid (P < 0.001) specimens from BP patients. The results of P- ELISA were moderately correlated with the titer of the commercial ELISA kit (MBL, Japan) (rho = 0.5680, P = 0.0011). This newly developed system allows for rapid and convenient diagnosis and/or monitoring of BP disease activity. [ABSTRACT FROM AUTHOR]

Microfluidic paper-based analytical devices (μPADs) fabricated by wax-printing are suitable platforms for the development of simple and affordable molecular diagnostic assays for infectious diseases, especially in resource-limited settings. Paper devices can be modified for biological assays by adding appropriate reagents to the test areas. For this purpose, the use of affinity immobilization strategies can be a good solution for bioactive paper fabrication. This paper describes a methodology to capture labeled-DNA strands and hybrids on paper via the anchoring of antibodies with a fusion protein that combines a family 3 carbohydrate binding module (CBM) from Clostridium thermocellum, with high affinity to cellulose, and the ZZ fragment of the staphyloccocal protein A, which recognizes IgG antibodies via their Fc portion. Antibodies immobilized via CBM-ZZ were able to capture appropriately labeled (biotin, fluorescein) DNA strands and DNA hybrids. The ability of an antibody specific to biotin to discriminate complementary from noncomplementary, biotin-labeled targets was demonstrated in both spot and microchannel assays. Hybridization was detected by fluorescence emission of the fluorescein-Iabeled DNA probe. The efficiency of the capture of labeled-DNA by antibodies immobilized on paper via the CBM-ZZ construct was significantly higher when compared with a physical adsorption method where antibodies were simply spotted on paper without the intermediation of other molecules. The experimental proof of concept of wax-printed μPADs functionalized with CBM-ZZ for DNA detection at room temperature presented in this study constitutes an important step toward the development of easy to use and affordable molecular diagnostic tests. [ABSTRACT FROM AUTHOR]

An inkjet-printed microfluidic paper-based analytical device (μPAD) for the detection of lactoferrin has been developed. The analyte concentration dependent fluorescence emission, caused by the sensitization of pre-deposited terbium (Tb3+) upon complexation with lactoferrin on the paper device, is captured using a digital camera. The dynamic response range (0.5–3 mg mL−1) and the limit of detection (0.30 mg mL−1) of the μPAD are suitable for the analysis of normal human tears and the detection of eye disorders. Finally, lactoferrin concentrations in human tear samples were analyzed by the μPADs and the assay results corresponded within 6% error to those obtained by an immunoassay (ELISA). The μPADs provide a simple, rapid and accurate method for lactoferrin detection in tear fluid. Results are obtained within 15 min of a single application of 2.5 μL of sample. To the best of our knowledge, this is the first report of a device for lactoferrin quantification relying neither on an immunoassay nor on high cost analytical instrumentation. [ABSTRACT FROM AUTHOR]