Bead-based Immunoassay Research Articles

Electrochemiluminescence Distance and Reactivity of Coreactants Determine the Sensitivity of Bead-Based Immunoassays.

Herein we report the study of electrochemiluminescence (ECL) generation by tris(2,2'-bipyridyl)ruthenium (Ru(bpy)3 2+ ) and five tertiary amine coreactants. The ECL distance and lifetime of coreactant radical cations were measured by ECL self-interference spectroscopy. And the reactivity of coreactants was quantitatively evaluated in terms of integrated ECL intensity. By statistical analysis of ECL images of single Ru(bpy)3 2+ -labeled microbeads, we propose that ECL distance and reactivity of coreactant codetermine the emission intensity and thus the sensitivity of immunoassay. 2,2-bis(hydroxymethyl)-2,2',2''-nitrilotriethanol (BIS-TRIS) can well balance ECL distance-reactivity trade-off and enhance the sensitivity by 236 % compared with tri-n-propylamine (TPrA) in the bead-based immunoassay of carcinoembryonic antigen. The study brings an insightful understanding of ECL generation in bead-based immunoassay and a way of maximizing the analytical sensitivity from the aspect of coreactant.

The Association of Kidney Function with Plasma Amyloid-β Levels and Brain Amyloid Deposition.

Reduced kidney function is related to brain atrophy and higher risk of dementia. It is not known whether kidney impairment is associated with higher levels of circulating amyloid-β and brain amyloid-β deposition, which could contribute to elevated risk of dementia. To investigate whether kidney impairment is associated with higher levels of circulating amyloid-β and brain amyloid-β deposition. This cross-sectional study was performed within the community-based Atherosclerosis Risk in Communities (ARIC) Study cohort. We used estimated glomerular filtration rate (eGFR) based on serum creatinine and cystatin C levels and urine albumin-to-creatinine ratio (ACR) to assess kidney function. Amyloid positivity was defined as a standardized uptake value ratios > 1.2 measured with florbetapir positron emission tomography (PET) (n = 340). Plasma amyloid-β1-40 and amyloid-β1-42 were measured using a fluorimetric bead-based immunoassay (n = 2,569). Independent of demographic and cardiovascular risk factors, a doubling of ACR was associated with 1.10 (95% CI: 1.01,1.20) higher odds of brain amyloid positivity, but not eGFR (odds ratio per 15 ml/min/1.73 m2 lower eGFR: 1.08; 95% CI: 0.95,1.23). A doubling of ACR was associated with a higher level of plasma amyloid-β1-40 (standardized difference: 0.12; 95% CI: 0.09,0.14) and higher plasma amyloid-β1-42 (0.08; 95% CI: 0.05,0.10). Lower eGFR was associated with higher plasma amyloid-β1-40 (0.36; 95% CI: 0.33,0.39) and higher amyloid-β1-42 (0.32; 95% CI: 0.29,0.35). Low clearance of amyloid-β and elevated brain amyloid positivity may link impaired kidney function with elevated risk of dementia. kidney function should be considered in interpreting amyloid biomarker results in clinical and research setting.

Pathogenic antibody response to glucose-6-phosphate isomerase targets a modified epitope uniquely exposed on joint cartilage

ObjectivesTo identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA).MethodsIgG response towards a library of GPI peptides in patients with early RA,...

Acidic pH impact on BSA domains I & II adsorption to a silica dioxide surface.

Bovine Serum Albumin (BSA) is a protein used as a passive blocker, adsorbing to various surfaces to reduce nonspecific interactions. BSA contains three structurally homologous domains but comprised of a different number of basic/acidic amino acids, causing each domain to respond to pH changes differently. At neutral pH, domain I contains many negatively charged residues producing a total net charge of −17. At pH 5.2, these residues become protonated, shifting the net charge to +1. The change in pH alters the charge of BSA and therefore may affect protein-surface interactions. Silica is a common surface used in bead-based immunoassays. Experimental observations suggest that adsorption occurs when both BSA and silica are negatively charged but the mechanism is not fully understood. By using theoretical techniques like molecular dynamics (MD) simulations we can analyze this protein-surface interaction and begin to understand it on a molecular level. In this study, we investigated how the change in pH can impact the adsorption of BSA to silica using MD simulations. BSA binding to silica at pH 5.2 and pH 7.0 were compared by performing simulations using NAMD. We observed that at pH 5.2, domains I and II orient closer to the surface to create many intermolecular interactions, while at pH 7.0 only domain II interacted with the silica surface. This interaction could be due to the change in surface charge of domain I at pH 7.0 and pH 5.2. At pH 7.0 domain I is highly negatively charged impacting the electrostatic interactions that are observed at pH 5.2, where domain I is less negatively charged. With MD simulations, we found pH impacts protein-surface interaction and can affect the mechanism of protein adsorption.

Electrochemiluminescence Amplification in Bead-Based Assays Induced by a Freely Diffusing Iridium(III) Complex.

Heterogeneous electrochemiluminescence (ECL) assays employing tri-n-propylamine as a co-reactant and a tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)3]2+) derivative as an emissive label are integral to the majority of academic and commercial applications of ECL sensing. This model system is an active research area and constitutes the basis of successfully commercialized bead-based ECL immunoassays. Herein, we propose a novel approach to the enhancement of such conventional ECL assays via the incorporation of a second metal coordination complex, [Ir(sppy)3]3- (where sppy = 5'-sulfo-2-phenylpyridinato-C2,N), to the experimental system. By employing ECL microscopy, we are able to map the spatial distribution of ECL emission at the surface of the bead, from [Ru(bpy)3]2+ labels, and solution-phase emission, from [Ir(sppy)3]3-. The developed [Ir(sppy)3]3--mediated enhancement approach elicited a significant improvement (70.9-fold at 0.9 V and 2.9-fold at 1.2 V vs Ag/AgCl) of the ECL signal from [Ru(bpy)3]2+ labels immobilized on the surface of a polystyrene bead. This dramatic enhancement in ECL signal, particularly at low oxidation potentials, has important implications for the improvement of existing heterogeneous ECL assays and ECL-based microscopy, by amplifying the signal, opening new bioanalytical detection schemes, and reducing both electrode surface passivation and deleterious side reactions.

Identification of salivary autoantibodies as biomarkers of oral cancer with immunoglobulin A enrichment combined with affinity mass spectrometry.

Globally, oral cavity squamous cell carcinoma (OSCC) is one of the most common fatal illnesses. Its high mortality is ascribed to the fact that the disease is often diagnosed at a late stage, which indicates an urgent need for approaches for the early detection of OSCC. The use of salivary autoantibodies (autoAbs) as OSCC biomarkers has numerous advantages such as easy access to saliva samples and efficient detection of autoAbs using well-established secondary reagents. To improve OSCC screening, we identified OSCC-associated autoAbs with the enrichment of salivary autoAbs combined with affinity mass spectrometry (MS). The salivary IgA of healthy individuals and OSCC patients was purified with peptide M-conjugated beads and then applied to immunoprecipitated antigens (Ags) in OSCC cells. Using tandem MS analysis and spectral counting-based quantitation, the level of 10 Ags increased in the OSCC group compared with the control group. Moreover, salivary levels of autoAbs to the 10 Ags were determined by a multiplexed bead-based immunoassay. Among them, seven were significantly higher in early-stage OSCC patients than in healthy individuals. A marker panel consisting of autoAbs to LMAN2, PTGR1, RAB13, and UQCRC2 was further developed to improve the early diagnosis of OSCC.

Plasma metabolomics, lipidomics and cytokinomics profiling predict disease recurrence in metastatic colorectal cancer patients undergoing liver resection.

In metastatic colorectal cancer (mCRC) patients (pts), treatment strategies integrating liver resection with induction chemotherapy offer better 5-year survival rates than chemotherapy alone. However, liver resection is a complex and costly procedure, and recurrence occurs in almost 2/3rds of pts, suggesting the need to identify those at higher risk. The aim of this work was to evaluate whether the integration of plasma metabolomics and lipidomics combined with the multiplex analysis of a large panel of plasma cytokines can be used to predict the risk of relapse and other patient outcomes after liver surgery, beyond or in combination with clinical morphovolumetric criteria. Peripheral blood metabolomics and lipidomics were performed by 600 MHz NMR spectroscopy on plasma from 30 unresectable mCRC pts treated with bevacizumab plus oxaliplatin-based regimens within the Obelics trial (NCT01718873) and subdivided into responder (R) and non-R (NR) according to 1-year disease-free survival (DFS): ≥ 1-year (R, n = 12) and < 1-year (NR, n = 18). A large panel of cytokines, chemokines, and growth factors was evaluated on the same plasma using Luminex xMAP-based multiplex bead-based immunoassay technology. A multiple biomarkers model was built using a support vector machine (SVM) classifier. Sparse partial least squares discriminant analysis (sPLS-DA) and loading plots obtained by analyzing metabolomics profiles of samples collected at the time of response evaluation when resectability was established showed significantly different levels of metabolites between the two groups. Two metabolites, 3-hydroxybutyrate and histidine, significantly predicted DFS and overall survival. Lipidomics analysis confirmed clear differences between the R and NR pts, indicating a statistically significant increase in lipids (cholesterol, triglycerides and phospholipids) in NR pts, reflecting a nonspecific inflammatory response. Indeed, a significant increase in proinflammatory cytokines was demonstrated in NR pts plasma. Finally, a multiple biomarkers model based on the combination of presurgery plasma levels of 3-hydroxybutyrate, cholesterol, phospholipids, triglycerides and IL-6 was able to correctly classify patients by their DFS with good accuracy. Overall, this exploratory study suggests the potential of these combined biomarker approaches to predict outcomes in mCRC patients who are candidates for liver metastasis resection after induction treatment for defining personalized management and treatment strategies.

Multiplexed Bead-Based Peptide Immunoassays for the Detection of Antibody Reactivities.

Antigenic peptides are commonly used in serological test settings such as enzyme-linked immunosorbent assays (ELISA) to determine reactive antibodies (ABs) from serum or plasma samples. The use of synthetic peptides provides advantages like lower production effort and easier incorporation of specific chemical modifications compared to full-length antigenic proteins. Multiplexed antibody (AB) profiling methods such as microarray technologies enable the simultaneous identification of multiple novel biomarkers for the use in early disease diagnostics, vaccine development, or monitoring of immune responses. Despite various benefits they still show major limitations which can be overcome with bead-based assay technologies like the multi-analyte profiling (xMAP) technology developed by Luminex. In this chapter we introduce our established workflow for AB profiling with a multiplexed bead-based peptide immunoassay. The workflow is based on copper-catalyzed click chemistry to immobilize designed synthetic peptides onto uniquely color-coded paramagnetic beads in an orientation-specific manner. The individual peptide-coupled beads can be distinguished by their unique emission spectra during readout in the xMAP instrument and therefore allow testing of up to 500 different antigenic peptides in one multiplexed reaction. The multistep process described in this chapter is divided into separate sections for peptide design, coupling of functionalized peptides to MagPlex beads via click chemistry, confirmation of successful peptide immobilization, processing of serum or plasma samples, or preferably purified IgG thereof, with the multiplexed bead-based peptide immunoassay and subsequent data export and analysis.

Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoassays.

Antibody (AB) testing or serotesting for reactive ABs against antigenic proteins is broadly used. Parallel examination of many antigens is of high interest to identify autoantibodies (AAB) or differential antigenic reactivities in many biological settings like allergy and infectious autoimmune, cancerous, or systemic disease. The resulting AAB profiles can be used for diagnosis, prognosis, and monitoring of such conditions. Protein microarrays have been used for AB profiling over the past decade but show some significant limitations which make them unsuitable for clinical applications. Alternative multiplexing platforms such as bead arrays were shown to provide a versatile tool for the confirmation and efficient analysis of high numbers of biological samples. Luminex' bead-based xMAP technology combines advantages such as multiplexing and lower demand for sample volume and at the same time overcomes the challenges of microarrays. It works faster, shows better antigen stability, is more reproducible, and allows the analysis of up to 500 analytes in one sample well. In this chapter we introduce our established workflow for the use of the xMAP technology for AB profiling including an overview of the method principle and protocols for the covalent immobilization of proteins to the MagPlex beads, confirmation of protein coupling, the execution of a multiplexed bead-based protein immunoassay, and subsequent data handling.

Circulating cytokine and chemokine patterns associated with cytomegalovirus reactivation after stem cell transplantation.

Human cytomegalovirus (HCMV) reactivation is the leading viral complication after allogeneic haematopoietic stem cell transplantation (allo-HSCT). Understanding of circulating cytokine/chemokine patterns which accompany HCMV reactivation and correlate with HCMV DNAemia magnitude is limited. We aimed to characterise plasma cytokine/chemokine profiles in 36 allo-HSCT patients (21 with HCMV reactivation and 15 without HCMV reactivation) at four time-points in the first 100-day post-transplant. The concentrations of 31 cytokines/chemokines in plasma samples were analysed using a multiplex bead-based immunoassay. Cytokine/chemokine concentrations were compared in patients with high-level HCMV DNAemia, low-level HCMV DNAemia or no HCMV reactivation, and correlated with immune cell frequencies measured using mass cytometry. Increased plasma levels of T helper 1-type cytokines/chemokines (TNF, IL-18, IP-10, MIG) were detected in patients with HCMV reactivation at the peak of HCMV DNAemia, relative to non-reactivators. Stem cell factor (SCF) levels were significantly higher before the detection of HCMV reactivation in patients who went on to develop high-level HCMV DNAemia (810-52 740 copies/mL) vs. low-level HCMV DNAemia (< 250 copies/mL). High-level HCMV reactivators, but not low-level reactivators, developed an elevated inflammatory cytokine/chemokine profile (MIP-1α, MIP-1β, TNF, LT-α, IL-13, IL-9, SCF, HGF) at the peak of reactivation. Plasma cytokine concentrations displayed unique correlations with circulating immune cell frequencies in patients with HCMV reactivation. This study identifies distinct circulating cytokine/chemokine signatures associated with the magnitude of HCMV DNAemia and the progression of HCMV reactivation after allo-HSCT, providing important insight into immune recovery patterns associated with HCMV reactivation and viral control.

Relationships of body composition and adipocytokines with outcomes in metastatic castration-resistant prostate cancer patients receiving docetaxel chemotherapy.

We evaluated the relationships of body composition and serum adipocytokine levels with progression-free survival (PFS) and overall survival (OS) in metastatic castration-resistant prostate cancer (mCRPC) patients receiving docetaxel. The medical records of mCRPC patients who received docetaxel between January 2011 and December 2015 at Fudan University Shanghai Cancer Center (Shanghai, China) were reviewed. The following body composition parameters were calculated using computed tomography: skeletal muscle index (SMI), visceral adipose tissue index (VATI), and subcutaneous adipose tissue index (SATI). Pretreatment serum adipocytokine levels, including interleukin 6, insulin, leptin, monocyte chemoattractant protein-1, adiponectin, and resistin, were measured using the multiplex bead-based immunoassays. Cox regression and Kaplan-Meier methods were used for survival analyses. Of the 453 mCRPC patients initially identified, 105 were included in the analysis. High VATI group patients had longer PFS (median, 10 months vs 7 months, P = 0.008) and OS (median, 24 months vs 15 months, P = 0.017), compared with low VATI group patients. SMI and SATI were not significantly associated with PFS or OS. Of the six detected adipocytokines, only leptin was associated with mCRPC prognosis. High leptin group patients had shorter PFS (median, 7 months vs 12 months, P = 0.0018) and OS (median, 17 months vs 22 months, P = 0.042), compared with low leptin group patients. Multivariate analysis showed that a high VATI was an independent protective factor for PFS and OS, while a high leptin level was an independent risk factor for PFS and OS. Therefore, VATI and serum leptin levels could provide important information concerning mCRPC prognosis.

Dicer Suppresses Hepatocellular Carcinoma via Interleukin-8 Pathway.

Elevated level of interleukin-8 (IL-8) promotes hepatocellular carcinoma (HCC) development and contributes to poor prognosis. Previously, we have proved that Dicer inhibits HCC progression. In this study, we evaluated the potential interaction between IL-8 and Dicer as well as their influence on HCC. Hepatocellular carcinoma cells of SMMC-7721 were divided into 2 groups for subsequent analysis: pCMV-Dicer group for Dicer-overexpressing lentivirus transfected cells (pCMV-Dicer cells) and pCMV-NC group for empty lentivirus transfected cells (pCMV-NC cells). Cell Counting kit-8 (CCK8), wound healing, and transwell were used to evaluate the inhibitory effect of Dicer overexpression on proliferation, migration, and invasion of HCC cells. The level of IL-8 was measured by flow cytometry bead-based immunoassays. Male nude BALB/c mice injected with pCMV-Dicer or pCMV-NC cell suspensions was used for transplant of HCC tumor. We found that the secretion of IL-8 was reduced in the medium of pCMV-Dicer cells (P = .027). Recombinant human IL-8 (rhIL-8) reversed the inhibitory effect of Dicer on proliferation (P < .01), migration (P = .003), and invasion (P = .001), whereas IL-8 inhibitor of reparixin enhanced inhibitory effect of Dicer on proliferation (P < .05), migration (P = .008), and invasion (P = .000). Lenvatinib downregulated the IL-8 level of HCC cells (P = .000) as well as promote Dicer-induced inhibition for HCC cells referring to proliferation (P < .05), migration (P = .000), and invasion (P = .000). Animal experiments also demonstrated that Dicer cooperated with lenvatinib to inhibit the growth of HCC tumors (P < .05). Dicer cooperated with lenvatinib to inhibit HCC growth via downregulating IL-8, and Dicer displayed its potential capability to enhance the anti-tumor effect of lenvatinib.

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.

Toward mechanistic understanding of electrochemiluminescence generation by tris(2,2′-bipyridyl)ruthenium(II) and peroxydisulfate

Electrochemiluminescence (ECL) generation by tris(2,2’-bipyridyl)ruthenium (Ru(bpy)32+) and S2O82− is one of most frequently studied cathodic systems. However, the reaction mechanism is yet to be fully resolved, since the reaction involving highly reactive intermediates occurs at the negative potential limit of aqueous solutions. In this work we used bismuth microwire (BiMW) as the working electrode and ECL microscopy (ECLM) as the imaging tool to study the mechanism of cathodic ECL generation by Ru(bpy)32+/S2O82−. Thanks to its high hydrogen evolution overpotential, BiMW electrode could provide us with a wide negative potential window in aqueous solutions. While ECLM allowed us to determine the thickness of ECL layer (TEL), which was found to vary from ca. 3.4 μm to 3.0 μm with increasing the concentration of S2O82−. Given the chemical reduction of S2O82− by Ru(bpy)3+ is essential for extending the concentration profile of SO4•−, the slightly changed TEL is likely related to the instability of Ru(bpy)3+ in aqueous solutions. Finally, no ECL was detected for Ru(bpy)32+-labelled microbeads, implying that S2O82− could not work as the cathodic coreactant like tri-n-propylamine (TPrA) as the anodic one in bead-based immunoassays.

Ergometrine sensing in rye flour by a magnetic bead-based immunoassay followed by flow injection analysis with amperometric detection

A certain group of mycotoxins, the ergot alkaloids, has caused countless deaths throughout human history. They are found in rye and other cereals and ingesting contaminated foods can cause serious health problems. To identify contaminated food exceeding the legal limits for ergot alkaloids, a portable and cost-effective test system is of great interest to the food industry. Rapid analysis can be achieved by screening for a marker compound, for which we chose ergometrine. We developed a magnetic bead-based immunoassay for ergometrine with amperometric detection in a flow injection system using a handheld potentiostat and a smartphone. With this assay a limit of detection of 3 nM (1 μg L−1) was achieved. In spiked rye flour, ergometrine levels from 25 to 250 μg kg−1 could be quantified. All results could be verified by optical detection. The developed assay offers great promise to meet the demand for on-site ergometrine detection in the food industry.

The Relationship between Extracellular Volume Compartments and Matrix Metalloproteinases-2 in Left Ventricular Remodeling after Myocardial Infarction.

Matrix metalloproteinases (MMPs) can affect myocardial extracellular volume (ECV) and its compartments, and this can provide more detailed information about the mechanism of adverse left ventricular (LV) remodeling (AR) after acute myocardial infarction (MI). To investigate the role of changes (Δ) in ECV compartments (matrix volume (MVi) and cell volume (CVi)) in the development of AR after MI, and their relationship with MMP-2 expressions. Ninety-two first MI patients who underwent 3 Tesla cardiovascular magnetic resonance imaging performed 2 weeks (baseline) and 6 months post-MI. We measured T1 mapping with MOLLI sequences. ECV was performed post-gadolinium enhancement. ECV and LV mass were used to calculate MVi and CVi. AR was defined as an increase of ≥ 12% in LV end-diastolic volume in 6 months. MMPs were measured using a bead-based multiplex immunoassay system at first day (baseline) and 2 weeks post-MI. P <0.05 was accepted as statistically significant. Mean ECV and mean MVi baseline levels were higher in AR group compared to without AR group (42.9±6.4 vs 39.3±8.2%, p= 0.037; 65.2±13.7 vs 56.7±14.7 mL/m2, p=0.010; respectively). CVi levels was similar between groups. A positive correlation was found between baseline levels of MMP-2 and baseline levels of ECV (r=0.535, p<0.001) and MVi (r=0.549, p<0.001). Increased ΔMVi levels was independently predictor of AR (OR=1.03, p=0.010). ΔMVi had superior diagnostic performance compared to ΔECV in predicting AR (ΔAUC: 0.215±0.07, p<0.001). High MVi levels are associated with AR, and ΔMVi was independently predictor of AR. This may be associated with MMP-2 release due to increased inflammatory response.

Altered PTPN22 and IL10 mRNA Expression Is Associated with Disease Activity and Renal Involvement in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with very heterogeneous clinical behavior between affected individuals. Therefore, the search for biomarkers clinically useful for the diagnosis, prognosis, and monitoring of the disease is necessary. Here, we determined the association between PTPN22, IL10, OAS2, and CD70 mRNA expression with the clinical characteristics and with the serum levels of IL-10, IFN-γ, and IL-17 in SLE patients. Forty patients with SLE and 34 control subjects (CS) were included, mRNA expression was determined by real-time qPCR and cytokine levels were quantified by a multiplex bead-based immunoassay. Compared to CS, SLE patients showed increased IL10 mRNA and high IL-10 and IL-17 serum levels; in contrast, PTPN22 mRNA and IFN-γ were decreased. PTPN22 and IL10 gene expression was negatively correlated with Mex-SLEDAI score and were notably downregulated in SLE patients with lupus nephritis. Interestingly, SLE patients with renal damage were the ones with the lowest levels of PTPN22 and IL10 mRNA and the highest SLEDAI scores. No associations were observed for OAS2 and CD70 mRNA and IL-10, IL-17, and IFN-γ. In conclusion, we suggest that the assessment of IL10 and PTPN22 mRNA could be useful for monitoring disease activity in SLE patients showing renal involvement.

Borrelia multiplex: a bead-based multiplex assay for the simultaneous detection of Borrelia specific IgG/IgM class antibodies

BackgroundLyme borreliosis (LB) is the most common tick-borne infectious disease in the northern hemisphere. The diagnosis of LB is usually made by clinical symptoms and subsequently supported by serology. In Europe, a two-step testing consisting of an enzyme-linked immunosorbent assay (ELISA) and an immunoblot is recommended. However, due to the low sensitivity of the currently available tests, antibody detection is sometimes inaccurate, especially in the early phase of infection, leading to underdiagnoses.MethodsTo improve upon Borrelia diagnostics, we developed a multiplex Borrelia immunoassay (Borrelia multiplex), which utilizes the new INTELLIFLEX platform, enabling the simultaneous dual detection of IgG and IgM antibodies, saving further time and reducing the biosample material requirement. In order to enable correct classification, the Borrelia multiplex contains eight antigens from the five human pathogenic Borrelia species known in Europe. Six antigens are known to mainly induce an IgG response and two antigens are predominant for an IgM response.ResultsTo validate the assay, we compared the Borrelia multiplex to a commercial bead-based immunoassay resulting in an overall assay sensitivity of 93.7% (95% CI 84.8–97.5%) and a specificity of 96.5% (95%CI 93.5–98.1%). To confirm the calculated sensitivity and specificity, a comparison with a conventional 2-step diagnostics was performed. With this comparison, we obtained a sensitivity of 95.2% (95% CI 84.2–99.2%) and a specificity of 93.0% (95% CI 90.6–94.7%).ConclusionBorrelia multiplex is a highly reproducible cost- and time-effective assay that enables the profiling of antibodies against several individual antigens simultaneously.

Cytokine/Chemokine assessment as a complementary diagnostic tool for inflammatory skin diseases.

Inflammatory skin conditions are the 4th leading cause of non-fatal health burden in the general population worldwide. The diagnosis of skin lesions due to systemic drug reactions, viral or bacterial exanthems, or in patients with psoriasis, atopic dermatitis or contact dermatitis is often difficult and relies heavily upon conventional histopathologic examination. Conversely, it is widely accepted that the cutaneous profile of inflammatory markers, or 'inflammatory signature', is differentially expressed in various skin conditions. In this pilot study, we investigated the possibility of inflammatory skin disease diagnosis from an immunological perspective in small punch biopsies. We collected lesional and perilesional punch biopsies from 139 patients suffering from a variety of inflammatory skin conditions and attending the Dermatology Department at the Princess Alexandra Hospital in Brisbane, Australia. Using bead-based immunoassays we were able to measure 13 out of 17 inflammatory markers from a pre-selected multi-analyte panel and to detect significant differences between lesional and perilesional biopsies from each individual patient. Hierarchical and unbiased clustering methods based on inflammatory signatures grouped psoriasis and atopic dermatitis lesions into individual clusters in contrast to other skin conditions, highlighting the potential of inflammatory signatures to be used as diagnostic differentiators and to inform alternative targets in anti-inflammatory treatment strategies.

A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.