Detection and Functional Evaluation of the P2X7 Receptor in hiPSC Derived Neurons and Microglia-Like Cells

A large body of evidence suggests the involvement of the ATP-gated purinergic receptor P2X7 (P2X7R) in neurodegenerative diseases, including Alzheimer’s disease. While it is well-described to be present and functional on microglia cells contributing to inflammatory responses, some reports suggest a neuronal expression of the receptor as well. Here, we present experimental results showing P2X7 receptors to be expressed on human hiPSC-derived microglia-like cells, hiPSC-derived neuronal progenitors and hiPSC-derived matured neuronal cells. By applying cell surface protein detection assays, we show that P2X7R is not localized on the cell membrane, despite being detected in neuronal cells and thus may not be available for directly mediating neurotoxicity. On hiPSC-derived microglia-like cells, a clear membranous expression was detected. Additionally, we have not observed differences in P2X7R functions between control and familial Alzheimer’s disease patient-derived neuronal cells. Functional assays employing a P2X7R antagonist JNJ 47965567 confirm these findings by showing P2X7R-dependent modulation of microglia-like cells viability upon treatment with P2X7R agonists ATP and BzATP, while the same effect was absent from neuronal cells. Since the majority of P2X7R research was done on rodent models, our work on human hiPSC-derived cells presents a valuable contribution to the field, extending the work on animal models to the human cellular system and toward clinical translation.

Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies

High-quality affinity probes are critical for sensitive and specific protein detection, in particular to detect protein biomarkers at early phases of disease development. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. In particular, clonal reagents offer opportunities for site-directed attachment of exactly one modification per affinity reagent at a site designed not to interfere with target binding to help standardize assays. The proximity extension assays (PEA) is a widely used protein assay where pairs of protein-binding reagents are modified with oligonucleotides (oligos), so that their proximal binding to a target protein generates a reporter DNA strand for DNA-assisted readout. The assays have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Here we explore nanobodies (Nb) as an alternative to polyclonal antibodies pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via Sortase A (SrtA) enzyme coupling. The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

Development of a Textile Based Protein Sensor for Monitoring the Healing Progress of a Wound

This article focuses on the design and fabrication of flexible textile-based protein sensors to be embedded in wound dressings. Chronic wounds require continuous monitoring to prevent further complications and to determine the best course of treatment in the case of infection. As proteins are essential for the progression of wound healing, they can be used as an indicator of wound status. Through measuring protein concentrations, the sensor can assess and monitor the wound condition continuously as a function of time. The protein sensor consists of electrodes that are directly screen printed using both silver and carbon composite inks on polyester nonwoven fabric which was deliberately selected as this is one of the common backing fabrics currently used in wound dressings. Three sensor designs were investigated to determine if any were suitable for protein detection. These sensors were experimentally evaluated and compared to each other by using albumin protein in phosphate buffered saline (PBS). A comprehensive set of cyclic voltammetry measurements were used to determine the optimal sensor design to provide the measurement of protein in solution. The best sensor was comprised of only silver conductive ink present to form the tracks outside the interface zone and a carbon only layer in the working and counter electrodes at the interface zone. This design prevents the formation of silver dioxide and protects the sensor from rapid decay, which allows for the recording of consecutive measurements using the same sensor. The chosen printed protein sensor was able to detect BSA at varying concentrations ranging from 30-0.3 mg/ml with a sensitivity of 0.0026µA/M.

Distance-based β-amyloid protein detection on PADs for scanning and subsequent follow up of Alzheimer’s disease in human urine sample

We report on the first development of a simple distance-based β-amyloid (Aβ) protein quantification using paper-based devices (dPADs) to screen for Alzheimer’s disease (AD) and to subsequently follow up on...

Colorimetric assay based on iron (III) ion triggering the aggregation of poly(tannic acid) coated Au nanocomposite for protein detection

Herein, several components including mesoporous silica nanoparticles (MSNs) as a reservoir, iron (III) ion to trigger color change, gold nanoparticle (AuNP) as an imaging agent and tannic acid (TA) to...

Lateral Flow Immunoassay Coupled with Copper Enhancement for Rapid and Sensitive SARS-CoV-2 Nucleocapsid Protein Detection

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) is still raging all over the world. Hence, the rapid and sensitive screening of the suspected population is in high demand. The nucleocapsid protein (NP) of SARS-CoV-2 has been selected as an ideal marker for viral antigen detection. This study describes a lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles for rapid NP antigen detection, in which sensitivity was improved through copper deposition-induced signal amplification. The detection sensitivity of the developed LFIA for NP antigen detection (using certified reference materials) under the optimized parameters was 0.01 μg/mL and was promoted by three orders of magnitude to 10 pg/mL after copper deposition signal amplification. The LFIA coupled with the copper enhancement technique has many merits such as low cost, high efficiency, and high sensitivity. It provides an effective approach to the rapid screening, diagnosis, and monitoring of the suspected population in the COVID-19 outbreak.

Allergen Shedding in Human Milk from Mothers of Preterm Infants: Proteomic and Peptidomic Feasibility and Pilot Analysis

Rationale: There is little information regarding the allergen content of milk feeds in the preterm population. Previous studies have evaluated specific proteins/peptides via ELISA, but no studies have performed a broad analysis of the allergenic peptide content and protease activity of milk feeds in this population. Preterm infants spend a critical window of time for immune development in the Newborn Intensive Care Unit (NICU), and may receive fortified donor milk, maternal milk or formula feeds via nasogastric tube or bottle instead of fresh breastmilk via breastfeeding. Methods: To evaluate feasibility, we initially performed mass spectrometry on four human milk samples (two term and two preterm) from the Mommy’s Milk Human Milk Biorepository (HMB) which included maternal surveys of diet and environmental exposures. We analyzed the results against the University of Nebraska FASTA database and UniProt for a total of 2211 protein sequences. We then further analyzed 5 samples from the Microbiome, Atopy and Prematurity (MAP) pilot study along with formula and human milk fortifier controls and performed not only mass spectrometry, but also peptidomic and protease activity analysis. Results: Each HMB sample had between 806 and 1007 proteins, with 37 to 44 non-human proteins/sample encompassing 26 plant and animal species. Bovine proteins were the most numerous; seven unique Bos taurus proteins were found in all four samples, and three contained Bos d 5. Cat, dog, mosquito, salmon, and crab were detected in all four samples. All donors ingested fish, shellfish and tree nuts, and all had salmon and crab proteins in their milk samples; two almond proteins were detected in three samples. Aeroallergens, including dust mite (Der f 28, Der f 25) and mold (Cla h 4) were identified in all samples. Two samples contained allergens to latex (Hev b 9) and chicken (Gal d 10). One sample contained several unique proteins, including carrot, two molds (including Pen c 19) and Der f 33-like protein. In the preterm MAP samples, 784 digested non-human proteins were identified, 30 were non-bovine in origin. Proteins from 23 different species including aeroallergens, food, and contact allergens were identified. Protease activity was highest in human milk samples without human milk fortifier and lowest in preterm formula. Conclusions: These findings represent the first preterm milk feed mass spectrometry and protease analysis with identification of known allergenic proteins to food, contact and aeroallergens. The varying degree of protein detection may reflect variable individual secretion and augmentation of feeds. This raises questions of whether the composition of milk feeds in the NICU impact the development of atopic disease in the preterm population and whether the complex interaction between allergens, proteases, and other human milk components can serve to induce sensitization or tolerance to allergens in infants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody lateral flow assay for antibody prevalence studies following vaccination: a diagnostic accuracy study

Background: Lateral flow immunoassays (LFIAs) are able to achieve affordable, large scale antibody testing and provide rapid results without the support of central laboratories. As part of the development of the REACT programme extensive evaluation of LFIA performance was undertaken with individuals following natural infection. Here we assess the performance of the selected LFIA to detect antibody responses in individuals who have received at least one dose of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Methods: This was a prospective diagnostic accuracy study. Sampling was carried out at renal outpatient clinic and healthcare worker testing sites at Imperial College London NHS Trust. Two cohorts of patients were recruited; the first was a cohort of 108 renal transplant patients attending clinic following two doses of SARS-CoV-2 vaccine, the second cohort comprised 40 healthcare workers attending for first SARS-CoV-2 vaccination and subsequent follow up. During the participants visit, finger-prick blood samples were analysed on LFIA device, while paired venous sampling was sent for serological assessment of antibodies to the spike protein (anti-S) antibodies. Anti-S IgG was detected using the Abbott Architect SARS-CoV-2 IgG Quant II CMIA. A total of 186 paired samples were collected. The accuracy of Fortress LFIA in detecting IgG antibodies to SARS-CoV-2 compared to anti-spike protein detection on Abbott Assay Results: The LFIA had an estimated sensitivity of 92.0% (114/124; 95% confidence interval [CI] 85.7% to 96.1%) and specificity of 93.6% (58/62; 95% CI 84.3% to 98.2%) using the Abbott assay as reference standard (using the threshold for positivity of 7.10 BAU/ml) Conclusions: Fortress LFIA performs well in the detection of antibody responses for intended purpose of population level surveillance but does not meet criteria for individual testing.