Conventional ELISA Research Articles

Wavelength-dependent three-dimensional single-molecule superlocalization imaging for yoctomole detection of thyroid-stimulating hormone on a quantum dot nanobiosensor

A wavelength-dependent three-dimensional (3D) superlocalization imaging method on gold nanoislands (GNIs) chip was developed as a supersensitive single-molecule thyroid-stimulating hormone (TSH) nanobiosensor. Scattered and fluorescent signals from gold nanoislands on the substrate and quantum dots (QDs) nanoprobes were simultaneously isolated and acquired within an evanescent field layer generated by total internal reflection (TIR) of incident light using a dual-view device. The 3D TIR fluorescence images of TSH-bound QDs on the GNIs were obtained using z-axis optical sectioning at 10 nm intervals before/after immunoreaction to identify the optimal conditions for detection. The localized centroid position of QD nanoprobes and GNI were distinguished at a subdiffraction limit resolution using 3D Gaussian fitting to the point spread function. The QD TSH nanobiosensor using wavelength-dependent 3D TIR fluorescence-based single-molecule localization microscopy (3D TIRF-SLM) imaging technique showed an excellent detection limit of 90 yoctomoles (∼54 molecules) and a wide linear dynamic range of 1.14 zmol/L − 100 pmol/L for TSH. The detection sensitivity was about 4.4 × 109 times higher than conventional enzyme-linked immunosorbent assay and could successfully quantify TSH in human serum. The wavelength-dependent 3D TIRF-SLM technique may emerge as a reliable platform for ultrahigh-sensitive nanobiosensors at the single-molecule level and early diagnosis with quantification of disease-related ultra-trace biomolecules.

Mix-and-Read Nanobody-Based Sandwich Homogeneous Split-Luciferase Assay for the Rapid Detection of Human Soluble Epoxide Hydrolase.

The soluble epoxide hydrolase (sEH) is possibly both a marker for and target of numerous diseases. Herein, we describe a homogeneous mix-and-read assay for the detection of human sEH based on using split-luciferase detection coupled with anti-sEH nanobodies. Selective anti-sEH nanobodies were individually fused with NanoLuc Binary Technology (NanoBiT), which consists of a large and small portion of NanoLuc (LgBiT and SmBiT, respectively). Different orientations of the LgBiT and SmBiT-nanobody fusions were expressed and investigated for their ability to reform the active NanoLuc in the presence of the sEH. After optimization, the linear range of the assay could reach 3 orders of magnitude with a limit of detection (LOD) of 1.4 ng/mL. The assay has a high sensitivity to human sEH and reached a similar detection limit to our previously reported conventional nanobody-based ELISA. The procedure of the assay was faster (30 min total) and easy to operate, providing a more flexible and simple way to monitor human sEH levels in biological samples. In general, the immunoassay proposed here offers a more efficient detection and quantification approach that can be easily adapted to numerous macromolecules.

Synthesizing Submicron Polyelectrolyte Capsules to Boost Enzyme Immobilization and Enhance Enzyme-Based Immunoassays.

Polyelectrolyte capsules (PCs) exhibit attractive superiorities in enzyme immobilization, including providing a capacious microenvironment for enzyme conformational freedom, highly effective mass transfer, and protecting enzymes from the external environment. Herein, we provide the first systemic evaluation of submicron PCs (SPCs, 500 nm) for enzyme immobilization. The catalytic kinetics results show that SPC encapsulation affected the affinities of enzymes and substrates but significantly enhanced their catalytic activity. The stability test indicates that SPC-encapsulated horseradish peroxidase (HRP) exhibits ultrahigh resistance to external harsh conditions and has a longer storage life than that of soluble HRP. The proposed encapsulation strategy enables 7.73-, 2.22-, and 11.66-fold relative activities when working at a pH as low as 3, at a NaCl concentration as high as 500 mM, and at a trypsin concentration as high as 10 mg/mL. We find that SPC encapsulation accelerates the cascade reaction efficiency of HRP and glucose oxidase. Owing to SPCs enhancing the catalytic activity of the loaded enzymes, we established an amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Escherichia coli O157:H7 using HRP-loaded SPCs. The detection sensitivity of SPC-improved ELISA was found to be 280 times greater than that of conventional HRP-based ELISA. Altogether, we provide an elaborate evaluation of 500 nm SPCs on enzyme immobilization and its application in the ultrasensitive detection of foodborne pathogens. This evaluation provides evidence to reveal the potential advantage of SPCs on enzyme immobilization for enzyme-based immunoassays. It has excellent biological activity and strong stability and broadens the application prospect in urine, soy sauce, sewage, and other special samples.

Optimisation and Validation of a conventional ELISA and cut-offs for detecting and quantifying anti-SARS-CoV-2 Spike, RBD, and Nucleoprotein IgG, IgM, and IgA antibodies in Uganda.

There is an urgent need for better immunoassays to measure antibody responses as part of immune-surveillance activities and to profile immunological responses to emerging SARS-CoV-2 variants. We optimised and validated an in-house conventional ELISA to identify and quantify SARS-CoV-2 spike- (S-), receptor binding domain- (RBD-), and nucleoprotein- (N-) directed IgG, IgM, and IgA binding antibodies in the Ugandan population and similar settings. Pre- and post-pandemic specimens were used to compare the utility of mean ± 2SD, mean ± 3SD, 4-fold above blanks, bootstrapping, and receiver operating characteristic (ROC) analyses in determining optimal cut-off optical densities at 450 nm (OD) for discriminating between antibody positives and negatives. "Limits of detection" (LOD) and "limits of quantitation" (LOQ) were validated alongside the assay's uniformity, accuracy, inter-assay and inter-operator precision, and parallelism. With spike-directed sensitivity and specificity of 95.33 and 94.15%, respectively, and nucleoprotein sensitivity and specificity of 82.69 and 79.71%, ROC was chosen as the best method for determining cutoffs. Accuracy measurements were within the expected CV range of 25%. Serum and plasma OD values were highly correlated (r = 0.93, p=0.0001). ROC-derived cut-offs for S-, RBD-, and N-directed IgG, IgM, and IgA were 0.432, 0.356, 0.201 (S), 0.214, 0.350, 0.303 (RBD), and 0.395, 0.229, 0.188 (N). The sensitivity and specificity of the S-IgG cut-off were equivalent to the WHO 20/B770-02 S-IgG reference standard at 100% level. Spike negative IgG, IgM, and IgA ODs corresponded to median antibody concentrations of 1.49, 3.16, and 0 BAU/mL, respectively, consistent with WHO low titre estimates. Anti-spike IgG, IgM, and IgA cut-offs were equivalent to 18.94, 20.06, and 55.08 BAU/mL. For the first time, we provide validated parameters and cut-off criteria for the in-house detection of subclinical SARS-CoV-2 infection and vaccine-elicited binding antibodies in the context of Sub-Saharan Africa and populations with comparable risk factors.

Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay.

The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.

SERS-ELISA using silica-encapsulated Au core-satellite nanotags for sensitive detection of SARS-CoV-2

The sensitive detection of viruses is key to preventing the spread of infectious diseases. In this study, we develop a silica-encapsulated Au core-satellite (CS@SiO2) nanotag, which produces a strong and reproducible surface-enhanced Raman scattering (SERS) signal. The combination of SERS from the CS@SiO2 nanotags with enzyme-linked immunosorbent assay (ELISA) achieves a highly sensitive detection of SARS-CoV-2. The CS@SiO2 nanotag is constructed by assembling 32 nm Au nanoparticles (AuNPs) on a 75 nm AuNP. Then the core-satellite particles are encapsulated with SiO2 for facile surface modification and stability. The SERS-ELISA technique using the CS@SiO2 nanotags provides a great sensitivity, yielding a detection limit of 8.81 PFU mL-1, which is 10 times better than conventional ELISA and 100 times better than lateral flow assay strip method. SERS-ELISA is applied to 30 SARS-CoV-2 clinical samples and achieved 100% and 55% sensitivities for 15 and 9 positive samples with cycle thresholds < 30 and > 30, respectively. This new CS@SiO2-SERS-ELISA method is an innovative technique that can significantly reduce the false-negative diagnostic rate for SARS-CoV-2 and thereby contribute to overcoming the current pandemic crisis.

Wound Healing after Acellular Dermal Substitute Positioning in Dermato-Oncological Surgery: A Prospective Comparative Study

MatriDerm and Integra are both widely used collagenic acellular dermal matrices (ADMs) in the surgical setting, with similar characteristics in terms of healing time and clinical indication. The aim of the present study is to compare the two ADMs in terms of clinical and histological results in the setting of dermato-oncological surgery. Ten consecutive patients with medical indications to undergo surgical excision of skin cancers were treated with a 2-step procedure at our Dermatologic Surgery Unit. Immediately after tumor removal, both ADMs were positioned on the wound bed, one adjacent to the other. Closure through split-thickness skin grafting was performed after approximately 3 weeks. Conventional histology, immunostaining and ELISA assay were performed on cutaneous samples at different timepoints. No significant differences were detected in terms of either final clinical outcomes or in extracellular matrix content of the neoformed dermis. However, Matriderm was observed to induce scar retraction more frequently. In contrast, Integra was shown to carry higher infectious risk and to be more slowly reabsorbed into the wound bed. Sometimes foreign body-like granulomatous reactions were also observed, especially in Integra samples. Even in the presence of subtle differences between the ADMs, comparable global outcomes were demonstrated after dermato-oncological surgery.

Droplet-free digital immunoassay based on electrochemiluminescence

Here, we report a droplet-free digital immunoassay for goat immunoglobulin G (IgG) based on electrochemiluminescence (ECL) for simplified digital immunoassay. Digital enzyme-linked immunosorbent assay (ELISA) enabled ultrasensitive detection using digital counting immuno-reaction-induced femtoliter-sized droplets. However, the complicated preparation process of femtoliter-sized droplet assay limits the simplicity of digital ELISA. In this study, droplet-free digital ELISA using ECL was developed with digital counting ECL-induced beads instead of droplets along with a labeling reaction of immuno-reacted beads with ECL luminophore of [Ru(bpy)3]2+ in bulk solution. After antigen–antibody reaction on the beads using poly horseradish peroxidase (HRP) as a label, HRP reaction with [Ru(bpy)3]2+-conjugated tyramide was induced to modify the bead with [Ru(bpy)3]2+. The number of beads emitting ECL was counted on an indium tin oxide (ITO) electrode using tri-n-propylamine as a co-reactant. From result of poly HRP assay, our digital assay system required two poly HRP molecules on the bead to confirm ECL. Finally, a digital immunoassay was performed. The limit of detection for goat IgG was 0.7 pg/mL (4.7 fmol/L). Our simplified digital ELISA was less sensitive than conventional digital ELISA by 10-100-fold, however, we successfully performed digital ELISA based on ECL without complicated procedures for droplet array.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

Development of small-sized fluorescence detector for pipette tip-based biosensor for on-site diagnosis

A small-sized fluorescence detector (referred to as a pipette tip [PT]-reader) was developed for a pipette tip-based biosensor. The PT-reader allows us to measure the fluorescence intensity of a solution in a truncated cone-shaped pipette tip with only the tip inserted into the PT-reader. A pipette holder made from a mixture of polydimethylsiloxane (PDMS) and carbon black was capable of the rigorous position arrangement of a truncated cone shaped-pipette tip and the prevention of stray light. The detection performance of the PT-reader was evaluated by measurement of resorufin. The limit of detection (LOD; 3σ) and the relative standard deviation (RSD, n = 4) were estimated to be 0.46 μM and 0.47–4.1%, respectively. This performance was comparable to that of a desktop-type fluorescence microplate reader. In addition, the PT-reader was applied to the quantification of immunoglobulin A (IgA), and the LOD (3σ) of IgA was estimated to be 1.0 ng/mL. The quantitation values of IgA in human saliva obtained by the PT-based enzyme-linked immunosorbent assay (PT-ELISA) were in agreement with those obtained by conventional ELISA. The PT-reader is expected to be useful for low-cost and user-friendly measurements, and the technique of device development proposed in this study will contribute to the progress of on-site medical diagnosis.

Gold nanoclusters-manganese dioxide composite-based fluorescence immunoassay for sensitive monitoring of fenitrothion degradation in Chinese cabbage

Understanding the residues and degradation of organophosphorus pesticides (OPs) in crops has attracted increasing attention. Herein, we designed a sensitive fluorescence immunoassay (FIA) by employing nanobody-linked alkaline phosphatase (Nb-ALP) and gold nanoclusters anchored manganese dioxide (AuNCs-MnO2) composite. In immunoassay protocol, Nb-ALP is used to competitively recognize the coating antigen and pesticide. After competitive immunoreaction, alkaline phosphatase catalyzes l-ascorbic acid-2-phosphate to produce ascorbic acid that can trigger the decomposition of the AuNCs-MnO2 composite, regulating the fluorescence response. As a proof-of-concept, fenitrothion (FNT) is chosen as the target analyte. As a result, the developed FIA exhibits high detection sensitivity (IC10 = 5.78 pg/mL), which is about 56-times higher than that of the conventional enzyme-linked immunosorbent assay. The developed FIA has been successfully applied for precisely monitoring the degradation of FNT in Chinese cabbage with excellent anti-interference ability and reproducibility, paving the way for the determination of pesticide residues in real food samples.

One-step impedimetric NT-proBNP aptasensor targeting cardiac insufficiency in artificial saliva

Currently, sensitive and accurate approaches for diagnosis, rapid assessment, and cardiac biomarker monitoring in patients with heart failure are needed. In this context, the advantages of aptamers over traditional antibodies have been employed to fabricate a single-step impedimetric N-terminal pro b-type natriuretic peptide (NT-proBNP)-modified gold microelectrode array. The development of an electrochemical aptasensing platform was based on the coimmobilization of alkanethiol self-assembled monolayers and amine-terminated aptamer that specifically recognized cardiac NT-proBNP protein resulting in charge electron transfer. Electroimpedimetric signals of the sensor were observed to be linear to the NT-proBNP concentrations in the range of 5.0 × 10−3 to 1.0 pg mL−1 (R2 = 0.9624), while achieving a low detection limit of 5.0 × 10−3 pg mL−1. Clinically relevant detection levels for NT-proBNP were achieved in a simple, rapid, and label-free measurement using artificial saliva, which was highlighted to be specific, regenerative, and selective over potential interferers occurring during the processes of cardiac insufficiency, Therefore, the novel NT-proBNP aptasensor is a promising point-of-care tool exhibiting safe, non-invasive, affordable, and non-prescription home use accessible to overcome the limitations associated with conventional ELISA and previous aptasensing.

Detection of Periodontal Disease Marker with Geometrical Transformation of Ag Nanoplates.

Alkaline phosphatase (ALP) and interleukin-1beta (IL-1β) are crucial salivary biomarkers for the diagnosis of periodontal disease that harms the periodontal tissue along with tooth loss. However, there has been no way of sensitive and portable detection of both biomarkers in saliva with multivariate signal readout. In this work, we design the multicolorimetric ALP and IL-1β sensing platform based on geometrical transformation of silver nanoplate transducer. By utilizing enzymatic activity of ALP that dephosphorylates p-aminophenol phosphate (p-APP) to p-aminophenol (p-AP), localized surface plasmon resonance properties of silver nanoplate vary with ALP and show a distinct color change from blue to yellow based on a controlled seed transformation from triangular to hexagonal, rounded pentagonal, and spherical shape. The multicolor sensor shows an ALP detection range of 0-25 U/L with a limit of detection (LOD) of 0.0011 U/L, which is the lowest range of LOD demonstrated to date for state-of-the-art ALP sensor. Furthermore, we integrate the sensor with the conventional ELISA to detect IL-1β for multicolor signaling and it exhibits a linear detection range of 0-250 pg/mL and an LOD of 0.066 pg/mL, which is 2 orders of magnitude lower than the monochromic conventional ELISA (LOD of 3.8 pg/mL). The ALP multicolor sensor shows high selectivity with a recovery of 100.9% in real human saliva proving its reliability and suitability for the readily accessible periodontal diagnosis with multivariate signal readout.

DropLab: an automated magnetic digital microfluidic platform for sample-to-answer point-of-care testing—development and application to quantitative immunodiagnostics

In point-of-care testing (POCT), tests are performed near patients and results are given rapidly for timely clinical decisions. Immunodiagnostic assays are one of the most important analyses for detecting and quantifying protein-based biomarkers. However, existing POCT immunodiagnostics mainly rely on the lateral flow assay (LFA), which has limited sensitivity or quantification capability. Although other immunodiagnostic assays, such as enzyme-linked immunosorbent assays (ELISAs), offer more sensitive and quantitative results, they require complex liquid manipulations that are difficult to implement in POCT settings by conventional means. Here, we show the development of DropLab, an automated sample-in-answer-out POCT immunodiagnostic platform based on magnetic digital microfluidic (MDM) technology. DropLab performs microbead-based ELISA in droplets to offer more sensitive and quantitative testing results. The intricate liquid manipulations required for ELISA are accomplished by controlling droplets with magnetic microbeads using MDM technology, which enables us to achieve full automation and easy operations with DropLab. Four ELISAs (the sample in triplicates and a negative control) can be run in parallel on the thermoformed disposable chip, which greatly improves the throughput and accuracy compared to those of other POCT immunodiagnostic devices. DropLab was validated by measuring two protein targets and one antibody target. The testing results showed that the limit of detection (LOD) of DropLab matched that of the conventional ELISA in a microwell plate. DropLab brings MDM one step closer to being a viable medical technology that is ready for real-world POCT applications.

Trace detection of bromadiolone and brodifacoum in biological and food samples: A label-free fluorescence immunoassay based on an in situ formation strategy

The widespread use of bromadiolone and brodifacoum poses a threat to public health and initiates poisoning incidents. Conventional colorimetric enzyme-linked immunosorbent assays (ELISAs) suffer from poor sensitivity, making it difficult to meet the trace detection of analytes. In this work, we constructed a sensitive and label-free fluorescence immunoassay based on the in situ formation strategy, for the quantification of bromadiolone and brodifacoum in biological and food samples. Alkaline phosphatase, an essential enzyme in ELISA, catalyzed the substrate sodium ascorbyl phosphate to L-ascorbic acid, which reacted with ο-phenylenediamine and triggered an in situ fluorescence signal with excitation/emission wavelengths of 350/430 nm. To reduce the reaction time, Fe3+ was utilized for the in situ immunoassay system. The fluorescence signal system was introduced to conventional ELISA without any label procedures, thus resulting in higher sensitivity with a 3.8-fold improvement in the half-maximal inhibitory concentration for bromadiolone, compared with colorimetric ELISA. The calculated limits of detection for bromadiolone and brodifacoum were 0.45–0.76 and 0.34–0.58 ng/mL (ng/g) in the human serum and corn samples, respectively, with recovery values ranging from 82.0 % to 93.1 %, and a coefficient of variation lower than 13.4 %. Due to its advantages of high sensitivity, cost-effectiveness, and easy substrate fabrication, this immunoassay may offer promise for applications in poisoning diagnosis and food analysis of bromadiolone and brodifacoum.

Single-molecule fluorescence methods for protein biomarker analysis.

Proteins have been considered key building blocks of life. In particular, the protein content of an organism and a cell offers significant information for the in-depth understanding of the disease and biological processes. Single-molecule protein detection/sequencing tools will revolutionize clinical (proteomics) research, offering ultrasensitivity for low-abundance biomarker (protein) detection, which is important for the realization of early-stage disease diagnosis and single-cell proteomics. This improved detection/measurement capability delivers new sets of techniques to explore new frontiers and address important challenges in various interdisciplinary areas including nanostructured materials, molecular medicine, molecular biology, and chemistry. Importantly, fluorescence-based methods have emerged as indispensable tools for single protein detection/sequencing studies, providing a higher signal-to-noise ratio (SNR). Improvements in fluorescent dyes/probes and detector capabilities coupled with advanced (image) analysis strategies have fueled current developments for single protein biomarker detections. For example, in comparison to conventional ELISA (i.e., based on ensembled measurements), single-molecule fluorescence detection is more sensitive, faster, and more accurate with reduced background, high-throughput, and so on. In comparison to MS sequencing, fluorescence-based single-molecule protein sequencing can achieve the sequencing of peptides themselves with higher sensitivity. This review summarizes various typical single-molecule detection technologies including their methodology (modes of operation), detection limits, advantages and drawbacks, and current challenges with recent examples. We describe the fluorescence-based single-molecule protein sequencing/detection based on five kinds of technologies such as fluorosequencing, N-terminal amino acid binder, nanopore light sensing, and DNA nanotechnology. Finally, we present our perspective for developing high-performance fluorescence-based sequencing/detection techniques.

Inflammasome Activation in Human Macrophages: IL-1β Cleavage Detection by Fully Automated Capillary-Based Immunoassay.

Interleukin (IL)-1β is a key mediator of inflammation and activates via pattern recognition receptors (PRR) of the inflammasome family by proteolytic maturation. Proteolysis is driven by proteases such as caspase-1 (also known as IL-1 converting enzyme, ICE) and converts the intact pro-IL-1β ~31kDa pro-peptide into a mature, ~17kDa form that can exit cells through nanomolecular pores or via microvesicles. Whereas pro-IL-1β fails to trigger IL-1 receptor (IL-1R) activation, mature IL-1β, upon release from the cell, triggers pleiotropic downstream effects, establishing an inflammatory state. Hence, being able to detect IL-1β conversion is physiologically relevant for measuring inflammation, but it cannot be easily accomplished by conventional ELISA or flow cytometry as most commercially available antibodies do not discriminate mature and pro-form. Furthermore, unlike for other cytokines, the mere induction and translation of IL1B mRNA cannot serve as a proxy of inflammasome PRR activation. Rather the cleavage of IL-1β needs to be verified. Hence, conventional immunoblotting has emerged as the gold standard for demonstrating inflammasome activation as the difference in molecular weight between pro- and mature form can easily be detected. However, conventional immunoblotting suffers from poor standardization, quantification, and reproducibility, may require sample concentration, and is also not suitable for medium to high throughput. Some of these shortcomings are prohibitive for analysis of human primary samples but can be overcome by fully automated capillary-based immunoassay as we outline here. We here provide a practical guide to quantify pro- vs mature IL-1β directly from unconcentrated supernatants of human monocyte-derived macrophages. The assay may be useful for more standardized and medium-throughput analysis in these cells or other biospecimen.

Supersensitive detection of single-histamine molecule on nanoplates by turn-on small molecule fluorescence sandwich immunoassay.

We develop a supersensitive "turn-on format" fluorescence sandwich immunoassay for detecting small single molecules. Gold nanoplate-based biotin antibodies and streptavidin-fluorophores were used instead of streptavidin-horseradish peroxidase reacting with a biotin tracer in a microplate-based competitive enzyme-linked immunosorbent assay (ELISA). Our platform showed a low detection limit of 5 zeptomolar (5 × 10-21 M), 5.4 × 1010 times higher detection sensitivity than the conventional tune-off format ELISA.

A bovine serum albumin and squaraine dye assembly fluorescent probe for pepsin detection

Gastroesophageal reflux disease (GERD) is a highly prevalent condition. Screening for GERD remains a great challenge due to the non-specific nature of the symptoms. Now, salivary pepsin detection has become an effective tool for diagnosing GERD due to its non-invasive and highly specific advantages. However, the diagnosis of GERD by detecting pepsin in saliva still has factors such as long reaction time, poor selectivity, and low detection sensitivity, which need to be improved. In this work, bovine serum albumin (BSA) and a squaraine dye (SQ) were employed to build a BSA-SQ assembly with a fluorescent “on-off” change for rapid and sensitive detection of pepsin. In a Gly-HCl (pH = 2.6) buffer system, SQ had a significant aggregation caused-quenching (ACQ) effect. As SQ was embedded in the cavity of BSA, this improved its dispersion and inhibited the ACQ effect of SQ, resulting in enhanced fluorescence of the system. At this stage, pepsin catalyzed the hydrolysis of BSA, leading to the aggregation of SQ and the quenching of the fluorescence of BSA-SQ. The high catalytic activity of pepsin makes BSA-SQ suitable for the qualitative and quantitative detection of pepsin, showing a good linear relationship in the range of 0.5–40 μg/mL with a detection limit of 97 ng/mL. Finally, the use of this fluorescent probe method to detect pepsin in human saliva showed results consistent with conventional ELISA methods with relative errors between 0.4 and 5.5 %, confirming the great potential of BSA-SQ for the rapid diagnosis of GERD.

Development of a bead-based multiplex immunoassay for simultaneous quantitative detection of IgG serum antibodies against seven vaccine-preventable diseases

Serosurveillance and seroprevalence studies should be carried out to monitor vaccine-preventable diseases. Multiplex immunoassay (MIA) systems are useful tools for this purpose, allowing the simultaneous quantitative detection of antibodies in one small serum sample, which presents an advantage over conventional methods, such as enzyme-linked immunosorbent assays (ELISAs). Therefore, we developed a multiplex immunoassay for the measurement of antibodies against seven vaccine-preventable infections (measles, rubella, mumps, tetanus, diphtheria, pertussis and Haemophilus influenza type b (Hib) infection). In our multiplex system, heterologous inhibition generally did not exceed 10%, while homologous inhibition varied between 90 and 98%. The intra- and inter-assay variability was ≤11%. The results of in-house MIA showed satisfactory correlation with commercial ELISAs, with Spearman correlation coefficients from 0.90 to 0.98. At the cut-off values defined for our MIA the serostatus can be determined with high sensitivity (89–100%) and specificity (92–98%). Thus, the developed in-house MIA represents a feasible alternative to conventional ELISAs and could be used for large-scale serosurveillance/seroprevalence studies of vaccine-preventable diseases.