Pre-existing humoral immunity and CD4+ T cell response correlate with cross-reactivity against SARS-CoV-2 Omicron subvariants after heterologous prime-boost vaccination.

BACKGROUND: Information regarding the heterologous prime-boost COVID vaccination has been fully elucidated. The study aimed to evaluate both humoral, cellular immunity and cross-reactivity against variants after heterologous vaccination. METHODS: We recruited healthcare workers previously primed with Oxford/AstraZeneca ChAdOx1-S vaccines and boosted with Moderna mRNA-1273 vaccine boost to evaluate the immunological response. Assay used: anti-spike RBD antibody, surrogate virus neutralizing antibody and interferon-γ release assay. RESULTS: All participants exhibited higher humoral and cellular immune response after the booster regardless of prior antibody level, but those with higher antibody level demonstrated stronger booster response, especially against omicron BA.1 and BA.2 variants. The pre-booster IFN-γ release by CD4+ T cells correlates with post-booster neutralizing antibody against BA.1 and BA.2 variant after adjustment with age and gender. CONCLUSIONS: A heterologous mRNA boost is highly immunogenic. The pre-existing neutralizing antibody level and CD4+ T cells response correlates with post-booster neutralization reactivity against the Omicron variant.

Rapid Escherichia coli Cloned DNA Detection in Serum Using an Electrical Double Layer-Gated Field-Effect Transistor-Based DNA Sensor.

In this study, a rapid diagnosis platform was developed for the detection of Escherichia coli O157:H7. An electrical double layer (EDL)-gated field-effect transistor-based biosensor (BioFET) as a point-of-care testing device is demonstrated with its high sensitivity, portability, high selectivity, quick response, and ease of use. The specially designed ssDNA probe was immobilized on the extended gate electrode to bind the target complementary DNA segment of E. coli, resulting in a sharp drain current change within minutes. The limit of detection for target DNA is validated to a concentration of 1 fM in buffer solution and serum. Meanwhile, the results of a Kelvin probe force microscope were shown to have reduced surface potential of the DNA immobilized sensors before and after the cDNA detection, which is consistent with the decreased drain current of the BioFET. A 1.2 kb E. coli duplex DNA synthesized in plasmid was sonicated and detected in serum samples with the sensor array. Gel electrophoresis was used to confirm the efficiency of sonication by elucidating the length of DNA. Those results show that the EDL-gated BioFET system is a promising platform for rapid identification of pathogens for future clinical needs.

Peritoneal Fluid Analysis of Advanced Ovarian Cancers after Hyperthermic Intraperitoneal Chemotherapy.

This study investigated miRNA and cytokine expression changes in peritoneal fluid samples of patients with advanced ovarian cancer (OVCA) after receiving hyperthermic intraperitoneal chemotherapy (HIPEC) during cytoreduction surgery (CRS). We collected samples prior to HIPEC, immediately after HIPEC, and 24/48/72 h after CRS from a total of 6 patients. Cytokine levels were assessed using a multiplex cytokine array, and a miRNA PanelChip Analysis System was used for miRNA detection. Following HIPEC, miR-320a-3p, and miR-663-a were found to be immediately down-regulated but increased after 24 h. Further, significant upregulation post-HIPEC and sustained increases in expression were detected in six other miRNAs, including miR-1290, miR-1972, miR-1254, miR-483-5p, miR-574-3p, and miR-574-5p. We also found significantly increased expression of cytokines, including MCP-1, IL-6, IL-6sR, TIMP-1, RANTES, and G-CSF. The changing expression pattern throughout the study duration included a negative correlation in miR-320a-3p and miR-663-a to cytokines including RANTES, TIMP-1, and IL-6 but a positive correlation in miRNAs to cytokines including MCP-1, IL-6sR, and G-CSF. Our study found miRNAs and cytokines in the peritoneal fluid of OVCA patients demonstrated different expression characteristics following CRS and HIPEC. Both changes in expression demonstrated correlations, but the role of HIPEC remains unknown, prompting the need for research in the future.

Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

Paper-Based Exosomal MicroRNA-21 Detection for Wound Monitoring: A Proof of Concept and Clinical Validation Trial Study.

Emerging evidence has shown that microRNAs play pivotal roles in wound healing. MicroRNA-21 (miR-21) was previously found to upregulate in order to fulfill an anti-inflammation role for wounds. Exosomal miRNAs have been identified and explored as essential markers for diagnostic medicine. However, the role of exosomal miR-21 in wounds has yet to be well studied. In order to facilitate the early management of poorly healing wounds, we developed an easy-to-use, rapid, paper-based microfluidic-exosomal miR-21 extraction device to determine wound prognosis in a timely manner. We isolated and then quantitatively examined exosomal miR-21 in wound fluids from normal tissues and acute and chronic wounds. Eight improving wounds displayed lower levels of exosomal miR-21 expression after wound debridement. However, four instances of increased exosomal miR-21 expression levels were notably associated with patients with poor healing wounds despite aggressive wound debridement, indicating a predictive role of tissue exosomal miR-21 for wound outcome. Paper-based nucleic acid extraction device provides a rapid and user-friendly approach for evaluating exosomal miR-21 in wound fluids as a means of monitoring wounds. Our data suggest that tissue exosomal miR-21 is a reliable marker for determining current wound status.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

The Pathomechanism, Antioxidant Biomarkers, and Treatment of Oxidative Stress-Related Eye Diseases.

Oxidative stress is an important pathomechanism found in numerous ocular degenerative diseases. To provide a better understanding of the mechanism and treatment of oxidant/antioxidant imbalance-induced ocular diseases, this article summarizes and provides updates on the relevant research. We review the oxidative damage (e.g., lipid peroxidation, DNA lesions, autophagy, and apoptosis) that occurs in different areas of the eye (e.g., cornea, anterior chamber, lens, retina, and optic nerve). We then introduce the antioxidant mechanisms present in the eye, as well as the ocular diseases that occur as a result of antioxidant imbalances (e.g., keratoconus, cataracts, age-related macular degeneration, and glaucoma), the relevant antioxidant biomarkers, and the potential of predictive diagnostics. Finally, we discuss natural antioxidant therapies for oxidative stress-related ocular diseases.

Immune Responses to COVID-19 Vaccines in Patients with Chronic Kidney Disease and Lead Exposure.

Literature data regarding the response rate to COVID-19 vaccination in chronic kidney disease (CKD) patients remain inconclusive. Furthermore, studies have reported a relationship between lead exposure and susceptibility to viral infections. This study examined immune responses to COVID-19 vaccines in patients with CKD and lead exposure. Between October and December 2021, 50 lead-exposed CKD patients received two doses of vaccination against COVID-19 at Chang Gung Memorial Hospital. Patients were stratified into two groups based on the median blood lead level (BLL): upper (≥1.30 µg/dL, n = 24) and lower (<1.30 µg/dL, n = 26) 50th percentile. The patients were aged 65.9 ± 11.8 years. CKD stages 1, 2, 3, 4 and 5 accounted for 26.0%, 20.0%, 22.0%, 8.0% and 24.0% of the patients, respectively. Patients in the lower 50th percentile of BLL had a lower proportion of CKD stage 5 than patients in the upper 50th percentile BLL group (p = 0.047). The patients in the lower 50th percentile BLL group also received a higher proportion of messenger RNA vaccines and a lower proportion of adenovirus-vectored vaccines than the patients in the upper 50th percentile BLL group (p = 0.031). Notably, the neutralizing antibody titers were higher in the lower 50th percentile than in the upper 50th percentile BLL group. Furthermore, the circulating levels of granulocyte-colony stimulating factor, interleukin-8, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α were higher in the upper 50th percentile than in the lower 50th percentile BLL group. Therefore, it was concluded that lead-exposed CKD patients are characterized by an impaired immune response to COVID-19 vaccination with diminished neutralizing antibodies and augmented inflammatory reactions.

Successful Phakic Intraocular Lens Implantation with the Usage of Topical Ascorbic Acid in Patient with Reduced Corneal Endothelial Cell Density.

We aimed to describe the use of topical ascorbic acid (AA) in a patient with reduced endothelial cells density (ECD) who was scheduled for phakic intraocular lens (pIOL) implantation. A 28-year-old woman presenting with dry eye and reduced ECD would like to have her high myopia (spherical equivalence >−15.0 D) corrected. The procedure of laser refractive surgery or even pIOL was not indicated for the reduced ECD of 1865/mm2 in the right eye and 2188/mm2 in the left eye, as well as level 3 dry eye. Fortunately, the ECD increased to 3144/mm2 in the right eye and 2538/mm2 in the left eye after topical AA was prescribed for one year preoperatively and one month postoperatively, with concomitant improvement of dry eye to level 1. Finally, bilateral pIOL implantation was performed smoothly and no sign of corneal decompensation was found postoperatively. Three months postoperatively, the ECD showed a satisfactory level of 2983/mm2 in the right eye and 3003/mm2 in the left eye. In conclusion, topical AA instillation might increase and maintain the density of central human corneal endothelial cells (HCECs) even after pIOL implantation.

Small-volume detection: platform developments for clinically-relevant applications.

Biochemical analysis of human body fluids is a frequent and fruitful strategy for disease diagnosis. Point-of-care (POC) diagnostics offers the tantalizing possibility of providing rapid diagnostic results in non-laboratory settings. Successful diagnostic testing using body fluids has been reported on in the literature; however, small-volume detection devices, which offer remarkable advantages such as portability, inexpensiveness, capacity for mass production, and tiny sample volume requirements have not been thoroughly discussed. Here, we review progress in this research field, with a focus on developments since 2015. In this review article, we provide a summary of articles that have detailed the development of small-volume detection strategies using clinical samples over the course of the last 5 years. Topics covered include small-volume detection strategies in ophthalmology, dermatology or plastic surgery, otolaryngology, and cerebrospinal fluid analysis. In ophthalmology, advances in technology could be applied to examine tear or anterior chamber (AC) fluid for glucose, lactoferrin, interferon, or VEGF. These approaches could impact detection and care for diseases including diabetic mellitus, dry-eye disease, and age-related maculopathy. Early detection and easy monitoring are critical approaches for improving overall care and outcome. In dermatology or plastic surgery, small-volume detection strategies have been applied for passive or interactive wound dressing, wound healing monitoring, and blister fluid analysis for autoimmune disease diagnosis. In otolaryngology, the analysis of nasal secretions and mucosa could be used to differentiate between allergic responses and infectious diseases. Cerebrospinal fluid analysis could be applied in neurodegenerative diseases, central neural system infection and tumor diagnosis. Other small-volume fluids that have been analyzed for diagnostic and monitoring purposes include semen and cervico-vaginal fluids. We include more details regarding each of these fluids, associated collection and detection devices, and approaches in our review.

Lysophosphatidic acid improves corneal endothelial density in tissue culture by stimulating stromal secretion of interleukin-1ß.

The short supply of donor corneas is exacerbated by the unsuitability of donors with insufficient endothelial cell density. Few studies have investigated promoting corneal endothelial cell proliferation to increase the endothelial cell density. We hypothesize that pre-transplantation treatment of proliferative tissue-cultivated corneas may increase corneal endothelial cell density. We observed that the airlift cultures were superior to immersion cultures with respect to both transparency and thickness. In this tissue culture system, we observed that lysophosphatidic acid increased the rabbit corneal endothelial cell density, number of BrdU-positive cells and improve wound healing. We also observed an indirect effect of lysophosphatidic acid on corneal endothelial cell proliferation mediated by the stimulation of interleukin-1ß secretion from stromal cells. Human corneal tissues treated with lysophosphatidic acid or interleukin-1ß contained significantly more Ki-67-positive cells than untreated group. The lysophosphatidic acid- or interleukin-1ß-treated cultured tissue remained hexagon-shaped, with ZO-1 expression and no evidence of the endothelial-mesenchymal transition. Our novel protocol of tissue culture may be applicable for eye banks to optimize corneal grafting.

Rapid detection of biofilm with modified alcian blue staining: In-vitro protocol improvement and validation with clinical cases.

For chronic wounds, biofilm infection is a critical issue because it can tip the scales toward an unhealing state. Biofilm-based wound therapy has been extensively advocated. However, point-of-care biofilm diagnosis still largely relies on clinical judgment. In this study, we aimed to develop a rapid tool for diagnosing wound biofilm presence by alcian blue staining. First, we sought to optimize alcian blue staining using a colorimetric-based approach to detect the biofilm, specifically targeting polysaccharides in the extracellular polymeric substances. Among examined transfer membranes and cationic detergents at various concentrations, we selected a positively charged nylon transfer membrane for sample loading, and 1% cetyl trimethyl ammonium chloride (CTAC) as the blocking solution. After sample loading and blocking, the membrane was immersed in alcian blue solution for staining, followed by immersion in 1% CTAC to decrease background noise. Each step required only 30 seconds, and the whole procedure was completed within a few minutes. In the second part of this study, we enrolled 31 patients with chronic wounds to investigate the predictive validity of biofilm detection for unhealed wounds at a 1-month follow-up visit. Among the 18 cases with positive wound biofilm staining, 15 wounds (83.3%) were not healed at the 1-month follow-up visit. Only three unhealed wounds (30%) produced in negative staining cases. This finding indicates that biofilm infection is associated with poor healing outcome for chronic wounds. Moreover, our staining results correlated well with the clinical microbiological culture assessment (83.9% consistency; 95.2% sensitivity, and 60% specificity). In conclusion, the modified alcian blue staining protocol used here represents a rapid and sensitive procedure for detecting biofilm in chronic wounds. This technique provides a practical point-of-care approach for detection of wound biofilm, the implementation of which may improve clinical outcomes for chronic wound patients. Additional studies are required to validate this method.

Accelerated corneal endothelial cell loss in two patients with granulomatosis with polyangiitis following phacoemulsification.

BACKGROUND: Generally, the loss rate of human endothelial cells (HCEC) in routine cataract surgery is 8.5%. When the corneal endothelial cells density (ECD) drops, the HCEC may decompensate to keep cornea dehydration which leads to corneal edema. Granulomatosis with polyangiitis (GPA) is an uncommon autoimmune disease involving multiple organs including eyes such as conjunctivitis, scleritis, uveitis, and corneal ulcer. In this study, we report two cases of GPA whose corneal ECD decreased significantly after phacoemulsification cataract surgery. CASE PRESENTATION: In the first case of 69-year-old male with GPA, the ECD dropped 39.6% (OD) four months after phacoemulsification and 38.1% (OS) six months postoperatively respectively. At the final follow-up, the residual ECD was only 55% in the right eye in the 49th month, and 56% remained in the left eye in the 39th month. In the second case of 54-year old female, left ECD dropped 63.9% at the 4th month after surgery and 69.6% ECD remained at the 15th month postoperatively while similar ECD of right eye before and after left eye surgery. CONCLUSION: Extensive preoperative ophthalmic evaluation and meticulous postoperative inflammation control should be applied to prevent severe loss of HCEC in GPA patients.

Up-regulation of miR-455-5p by the TGF-ß-SMAD signalling axis promotes the proliferation of oral squamous cancer cells by targeting UBE2B.

MicroRNAs (miRNAs) are involved in the tumourigenesis of various cancers by regulating their downstream targets. To identify the changes of miRNAs in oral squamous cell carcinoma (OSCC), we investigated the expression profiles of miRNAs in 40 pairs of OSCC specimens and their matched non-tumour epithelial tissues. Our data revealed higher miR-455-5p expression in the tumour tissues than in the normal tissues; the expression was also higher in oral cancer cell lines than in normal keratinocyte cell lines. MiR-455-5p knockdown reduced both the anchorage-independent growth and the proliferative ability of oral cancer cells, and these factors increased in miR-455-5p-overexpressing cells. Furthermore, by analysing the array data of patients with cancer and cell lines, we identified ubiquitin-conjugating enzyme E2B (UBE2B) as a target of miR-455-5p, and further validated this using 3'-untranslated region luciferase reporter assays and western blot analysis. We also demonstrated that UBE2B suppression rescued the impaired growth ability of miR-455-5p-knockdown cells. Furthermore, we observed that miR-455-5p expression was regulated, at least in part, by the transforming growth factor-ß (TGF-ß) pathway through the binding of SMAD3 to specific promoter regions. Notably, miR-455-5p expression was associated with the nodal status, stage, and overall survival in our patients, suggesting that miR-455-5p is a potential marker for predicting the prognosis of patients with oral cancer. In conclusion, we reveal that miR-455-5p expression is regulated by the TGF-ß-dependent pathway, which subsequently leads to UBE2B down-regulation and contributes to oral cancer tumourigenesis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Analytical Devices Based on Direct Synthesis of DNA on Paper.

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

Development of a Sampling Collection Device with Diagnostic Procedures.

Cervicovaginal fluid plays an important role in the detection of many female genital diseases, but the lack of suitable collection devices in the market severely challenges test success rate. Appropriate clinical sampling devices for cervicovaginal fluid collection would help physicians detect diseases and disease states more rapidly, efficiently, and accurately. The objective of this study was to develop a readily usable sampling collection device that would eliminate macromolecular interference and accurately provide specimens for further studies. This study was designed to develop an effective device to collect cervicovaginal fluid from women with symptoms of endometrial lesions, women appearing in the clinic for a routine Papanicolaou smear, and/or women seeking a routine gynecologic checkup. Paper-based assay, ELISA, and qNano were used to provide accurate diagnoses. A total of 103 patients successfully used the developed device to collect cervicovaginal fluid. Some of the collected specimens were used to detect glycogen, lactate, and pH for determining pathogen infection. Other specimen samples were tested for the presence of female genital cancer by comparing interleukin 6 concentration and microvesicle concentration. We proposed a noninvasive screening test for the diagnosis of female genital diseases using a dual-material collection device. The outer, nonwoven fabric portion of this device was designed to filter macromolecules, and the inner cotton portion was designed to absorb cervicovaginal fluid.

The vascularized periosteum flap as novel tissue engineering model for repair of cartilage defects.

Periosteum is a promising tissue engineering scaffold in research of cartilage repair; so far however, periosteum transfers have not been realized successfully because of insufficient nourishment of the graft. In a translational approach we, for the first time, designed a vascularized periosteum flap as 'independent' biomaterial with its own blood supply to address this problem and to reconstruct circumscript cartilage defects. In six 3-month-old New Zealand rabbits, a critical size cartilage defect of the medial femur condyle was created and covered by a vascularized periosteum flap pedicled on the saphenous vessels. After 28 days, formation of newly built cartilage was assessed macroscopically, histologically and qualitatively via biomechanical compression testing, as well as on molecular biological level via immunohistochemistry. All wounds healed completely, all joints were stable and had full range of motion. All flaps survived and were perfused through their pulsating pedicles. They showed a stable attachment to the bone, although partially incomplete adherence. Hyaline cartilage with typical columnar cell distribution and positive Collagen II staining was formed in the transferred flaps. Biomechanical testing revealed a significantly higher maximum load than the positive control, but a low elasticity. This study proved that vascularization of the periosteum flap is the essential step for flap survival and enables the flap to transform into cartilage. Reconstruction of circumscript cartilage defects seems to be possible. Although these are the first results out of a pilot project, this technique, we believe, can have a wide range of potential applications and high relevance in the clinical field.

Mechanical coupling of cytoskeletal elasticity and force generation is crucial for understanding the migrating nature of keloid fibroblasts.

One of the key features of keloid is its fibroblasts migrating beyond the original wound border. During migration, cells not only undergo molecular changes but also mechanical modulation. This process is led by actin filaments serving as the backbone of intra-cellular force and transduces external mechanical signal via focal adhesion complex into the cell. Here, we focus on determining the mechanical changes of actin filaments and the spatial distribution of forces in response to changing chemical stimulations and during cell migration. Atomic force microscopy and micropost array detector are used to determine and compare the magnitude and distribution of filament elasticity and force generation in fibroblasts and keloid fibroblasts. We found both filament elasticity and force generation show spatial distribution in a polarized and migrating cell. Such spatial distribution is disrupted when mechano-signalling is perturbed by focal adhesion kinase inhibitor and in keloid fibroblasts. The demonstration of keloid pathology at the nanoscale highlights the coupling of cytoskeletal function with physical characters at the subcellular level and provides new research directions for migration-related disease such as keloid.

Mechanotransduction in intervertebral discs.

Mechanotransduction plays a critical role in intracellular functioning--it allows cells to translate external physical forces into internal biochemical activities, thereby affecting processes ranging from proliferation and apoptosis to gene expression and protein synthesis in a complex web of interactions and reactions. Accordingly, aberrant mechanotransduction can either lead to, or be a result of, a variety of diseases or degenerative states. In this review, we provide an overview of mechanotransduction in the context of intervertebral discs, with a focus on the latest methods of investigating mechanotransduction and the most recent findings regarding the means and effects of mechanotransduction in healthy and degenerative discs. We also provide some discussion of potential directions for future research and treatments.

Paper-based ELISA for the detection of autoimmune antibodies in body fluid-the case of bullous pemphigoid.

Bullous pemphigoid (BP), a common autoimmune blistering disease, is increasing in incidence and conveys a high mortality. Detection of autoantibodies targeting the noncollagenous 16A (NC16A) 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 significantly 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.