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The sandwich format immunoassay is generally more sensitive and specific than more common assay formats, including direct, indirect, or competitive. A sandwich assay, however, requires two receptors to bind non-competitively to the target analyte. Typically, pairs of antibodies (Abs) or antibody fragments (Fabs) that are capable of forming a sandwiching with the target are identified through a slow, guess-and-check method with panels of candidate binding partners. Additionally, sandwich assays that are reliant on commercial antibodies can suffer from changes to reagent quality outside the researchers' control. This report presents a reimagined and simplified phage display selection protocol that directly identifies sandwich binding peptides and Fabs. The approach yielded two sandwich pairs, one peptide-peptide and one Fab-peptide sandwich for the cancer and Parkinson's disease biomarker DJ-1. Requiring just a few weeks to identify, the sandwich pairs delivered apparent affinity that is comparable to other commercial peptide and antibody sandwiches. The results reported here could expand the availability of sandwich binding partners for a wide range of clinical biomarker assays.

Abstract: Unlocking the potential of personalized medicine in point‐of‐care settings requires a new generation of biomarker and proteomic assays. Ideally, assays could inexpensively perform hundreds of quantitative protein measurements in parallel at the bedsides of patients. This goal greatly exceeds current capabilities. Furthermore, biomarker assays are often challenging to translate from benchtop to clinic due to difficulties achieving and assessing the necessary selectivity, sensitivity, and reproducibility. To address these challenges, we developed an efficient (70 %) immunoassay method. Specifically, the immunoblot membrane is dotted with the sample and then developed in a vortex fluidic device (VFD) reactor. All assay steps—blocking, binding, and washing—leverage the unique thin‐film microfluidics of the VFD. The approach can accelerate direct, indirect, and sandwich immunoblot assays. The applications demonstrated include assays relevant to both the laboratory and the clinic.

A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic interference (AIN), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a prior infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous putative antigen, a sequence derived from the neuraminidase protein of H3N2 influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIN from a previous infection could underlie some cases of COVID-19 disease severity.

UNLABELLED: A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic imprinting (AIM), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a previous infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous potential antigen, a sequence derived from the neuraminidase protein of H3N2 Influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIM from a previous infection could underlie some cases of COVID-19 disease severity. IMPORTANCE: Infections with SARS-COV-2 result in diverse disease outcomes, ranging from asymptomatic to fatal. The mechanisms underlying different disease outcomes remain largely unexplained. Previously, our laboratory identified a strong association between the presence of an antibody and increased disease severity in a subset of COVID-19 patients. Here, we report that this severity-associated antibody cross-reacts with viral proteins from an influenza A viral strain from 2014. Therefore, we speculate that antibodies generated against previous infections, like the 2014 influenza A, play a significant role in directing some peoples’ immune responses against SARS-COV-2. Such understanding of the sources and drivers of COVID-19 disease severity can help early identification and pre-emptive treatment.

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, enzyme-linked immunosorbent assay (ELISA) and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the intensive care unit (ICU), requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within 6 days post-symptom onset and sometimes within 1 day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patient's comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 likelihood ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention.IMPORTANCE The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients. Diagnosis of COVID-19 is readily accomplished through a multitude of reliable testing platforms; however, prognostic prediction remains elusive. To this end, we identified a short epitope from the SARS-CoV-2 nucleocapsid protein and also a disease risk factor score based upon comorbidities and age. The presence of antibodies specifically binding to this epitope plus a score cutoff can predict severe COVID-19 outcomes with 96.7% specificity.

The author list contained an error: Gregory A. Weiss was not identified as a corresponding author. Both of the corresponding authors, Gregory A. Weiss and Reginald M. Penner, are indicated in this Addition and Correction.

DJ-1, a 20.7 kDa protein, is overexpressed in people who have bladder cancer (BC). Its elevated concentration in urine allows it to serve as a marker for BC. However, no biosensor for the detection of DJ-1 has been demonstrated. Here, we describe a virus bioresistor (VBR) capable of detecting DJ-1 in urine at a concentration of 10 pM in 1 min. The VBR consists of a pair of millimeter-scale gold electrodes that measure the electrical impedance of an ultrathin (≈ 150-200 nm), two-layer polymeric channel. The top layer of this channel (90-105 nm in thickness) consists of an electrodeposited virus-PEDOT (PEDOT is poly(3,4-ethylenedioxythiophene)) composite containing embedded M13 virus particles that are engineered to recognize and bind to the target protein of interest, DJ-1. The bottom layer consists of spin-coated PEDOT-PSS (poly(styrenesulfonate)). Together, these two layers constitute a current divider. We demonstrate here that reducing the thickness of the bottom PEDOT-PSS layer increases its resistance and concentrates the resistance drop of the channel in the top virus-PEDOT layer, thereby increasing the sensitivity of the VBR and enabling the detection of DJ-1. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise (S/N > 100) and excellent sensor-to-sensor reproducibility characterized by coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a concentration of 30 pM, near the 10 pM limit of detection (LOD), encompassing four orders of magnitude in concentration.

Background: The present study aimed to evaluate the potential synergy between pharmaceutical formulations containing Bixa orellana L. (granulated—CHR OR and injectable nanodispersion—CHR IN) in conjunction with a cannabidiol (CBD)-rich extract of Cannabis sativa L. (CSE) on experimental pain models in Wistar rats. Methods: Chemical analysis was performed using gas chromatography (GC-MS). The pain tests employed were acetic acid-induced writhing (injection i.p. of 0.9% acetic acid), formalin (solution 1%), hot plate (55 ± 0.5 °C), and cold-water tail withdrawal tests. Results: Chemical analyses by chromatography confirmed that the oil from B. orellana is rich in δ-tocotrienol (72.0 ± 1.0%), while the oil from Cannabis sativa highlighted the presence of cannabidiol (CBD). The results from the experimental pain tests indicated that the combined administration of formulations containing Bixa orellana and C. sativa, such as the granulated CHR OR (400 mg/kg, orally) with CSE (40 mg/kg, orally) or the nanodispersion CHR IN (10 mg/kg, intramuscularly) with CSE (40 mg/kg, orally), demonstrated significant results (p < 0.001) in pain reduction. Although the formulations containing Bixa orellana extract showed statistical significance in the tests when used in isolation, their effects were inferior compared to the combined use with CSE or the isolated use of CSE. These findings suggest that combining formulations containing extracts of these plant species may represent a viable therapeutic option, considering the synergistic action in reducing pain under the experimental conditions employed. Conclusions: these results imply that combining the phytocomplexes present in B. orellana and C. sativa may be a promising approach for pain treatment. [ABSTRACT FROM AUTHOR]

Background/objectives: The growing use of mobile health (mHealth) applications needs reliable tools to assess their usability and user experience in clinical practice to improve the digital health (eHealth) interventions and ensure engagement, as higher engagement is often linked to increased efficacy of healthcare interventions. This study aimed to validate the patient Satisfaction and Usability with APPs questionnaire (pSUAPP), a multidimensional tool designed for the comprehensive assessment of mHealth applications, particularly for the integrated follow-up of patients with chronic diseases. Methods: A validation study was conducted between August and December 2022 with 85 participants from two hospitals in Spain, who completed the pSUAPP questionnaire, comprising 27 Likert-like items across four dimensions: first contact, registration, features and overall experience, and 1 open question. The questionnaire was validated by a panel of 11 experts and further assessed for psychometric properties. Results: The mean pSUAPP score was 79.0 (SD = 12.0), indicating high usability and positive user experience, with the highest scores in the 'features' dimension. The pSUAPP demonstrated moderate correlation with the System Usability Scale (SUS) and high reliability (Cronbach's alpha and omega t > 0.9). A reproducibility analysis showed negligible changes between repeated measures. Conclusions: The pSUAPP questionnaire was found to be a robust tool for evaluating mHealth app usability and user experience, with potential application across various clinical settings. [ABSTRACT FROM AUTHOR]

Pre-exposure prophylaxis (PrEP) is an effective HIV prevention strategy that consists in the use of antiretroviral drugs by seronegative people at risk of HIV. Negative perceptions, inadequate understanding, and access barriers have been associated with decreased medication adherence. Manaus is the largest city in the Brazilian Amazon, where the incidence of HIV/AIDS is high, and the rates of adherence to the antiretroviral treatment for HIV and PrEP are low. In this qualitative study among PrEP users, mostly MSM, we explored perceptions, knowledge, and access barriers. We conducted 21 in-depth interviews with an intentionally sampled group of participants who had used PrEP at least once in their lifetime, selected through the snowball technique, between April and July 2022. A thematic analysis was conducted with a predominantly inductive approach. We highlight three relevant themes: (i) access to information about PrEP and its influences on users, (ii) access, monitoring, and barriers encountered, and (iii) facilitators for PrEP adherence and sexual behaviors. One of the negative perceptions identified in the study involves a misunderstanding of the association between PrEP users and the HIV/AIDS status. Participants revealed that some non-PrEP users suspect that individuals claiming PrEP usage are concealing an HIV-positive status to engage in unprotected sex. Lack of information by health professionals regarding HIV prevention methods poses significant barriers to PrEP access and adherence. Participants emphasized social media's crucial role in PrEP awareness. The results suggest a need to increase digital outreach regarding PrEP, decentralize PrEP services, and provide comprehensive healthcare training to improve the effectiveness of the preventive measure. [ABSTRACT FROM AUTHOR]

Background: Diabetes Mellitus (DM) is a syndrome that interferes with energy metabolism and is caused by a decrease and dysfunction of insulin, leading to chronic hyperglycemia. DM associated with dyslipidemia is a severe health risk, increasing the chance of cardiovascular events, such as acute myocardial infarction and stroke. The Cannabis sativa L. fixed oil (CSO) is composed of unsaturated fatty acids and can be crucial in treating metabolic alterations. In addition, the nanoemulsion of C. sativa oil (NCS) has advantages in optimizing treatments. Objectives: This study aimed to evaluate the effects of treatments with CSO and its nanoemulsion (NCS) on induced diabetes and dyslipidemia in Wistar rats. Materials and Methods: CSO's physical-chemical and chromatographic characterization was performed, followed by the preparation of an NE containing 6% CSO. DM was induced in Wistar rats by intraperitoneal injection of streptozotocin (STZ) at a 55 mg/kg dose. Four days later, animals with blood glucose levels exceeding 300 mg/dL were considered diabetic. The rats were then divided into five groups (n = 5) and treated orally. The groups included a normoglycemic control group (NOR), a diabetic control group, a group treated with metformin (100 mg/kg), a group treated with CSO (400 mg/kg), and a group treated with nanoemulsion (NCS 200 mg/kg). Subsequently, the pancreas's clinical, biochemical, and histopathological parameters were evaluated. Results: In the chemical profile of CSO, it was observed the majority composition of palmitoleic (14.58%), oleic (12.50%), linoleic (42.40%), and linolenic (8.55%) acids. The results demonstrated that the induction of DM by STZ could reproduce the typical symptoms and clinical signs of DM. It was observed that treatments with CSO and NCS showed a significant improvement (p < 0.001) in polydipsia, polyuria, and loss of body mass, as well as a significant reduction (p < 0.001) of glucose levels in urine and blood and serum lipids. Histopathology of the pancreas revealed that treatment with CSO and NCS showed an increase in the number of cells in the islets of Langerhans and a decrease in regions devoid of cells, indicating possible cell regeneration. Moreover, insulin levels were significantly increased (p < 0.05) in the preferred groups. With dyslipidemia induced by Triton (Tyloxapol), it was observed that the treatment with CSO and NCS significantly decreased the levels of triglycerides (p < 0.05) and cholesterol (p < 0.001), as well as low-density lipoproteins (LDL) (p < 0.01). Conclusion: Treatment with CSO and NCS under the conditions of this study demonstrated an anti-diabetic effect and the ability to act in the reduction of triglyceride, cholesterol, and LDL levels. In this respect, treatments with CSO and NCS act in the control of DM, as well as in the prevention of cardiovascular diseases. [ABSTRACT FROM AUTHOR]