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Background Antinuclear antibodies (ANAs) are invaluable biomarkers for the diagnosis of autoimmune diseases (AIDs). This study aims to compare the performances of line immunoassay (LIA), multiplex bead‐based flow fluorescent immunoassay (MBFFI), and magnetic bar code immunofluorescence assay (MBC‐IF) to detect ANA‐Profile‐15S. Methods In total, 184 samples from AID patients and 50 healthy controls (HCs) were collected. Fifteen ANAs (anti‐dsDNA, nucleosome, histone, Sm, PCNA, ribosomal‐P, SS‐A/Ro52, SS‐A/Ro60, SS‐B/La, centromere B [CENP‐B], Scl‐70, U1‐snRNP, AMA‐M2, Jo‐1, and Pm/Scl) were subjected to parallel detection by the LIA, MBFFI, and MBC‐IF. The consistency between assays was analyzed. The discrepant results were further examined by chemiluminescent immunoassay (CLIA). Results Anti‐SS‐A/Ro52 and SS‐A/Ro60 autoantibodies were the most common autoantibodies in ANA positive‐profiles, and were detected with equal efficiency by the LIA, MBFFI, and MBC‐IF (p = 0.101 and p = 0.732, respectively). The three assays showed excellent agreement (consistency range: 66.5%–97.5%), and total consistency was 85.8%. The MBFFI and MBC‐IF assays were in good agreement in terms of ANA‐Profile‐15S determination; the kappa coefficient ranged from 0.59 to 0.95, except for the PCNA and PM‐Scl. Of the 262 re‐assessed divergent results, 124 (47.33%) were positive on CLIA; the various autoantibodies exhibited variable patterns. More importantly, the ANA‐Profile‐15S results of the MBFFI and MBC‐IF accurately identified patients with AID; the area under the curves ranged from 0.642 to 0.919. Conclusions The novel MBFFI and MBC‐IF assay performed well in detecting ANA‐Profile‐15S. The application of MBFFI and MBC‐IF play important roles in laboratory diagnosis of AIDs. Indirect light‐initiated chemiluminescence assay (LICA) was established in this study for the detection of egg white‐sIgE. The application of chemibeads coated with a combined mixture of four main egg white components can avert allergen cross‐reactivity. Performance was evaluated and met clinical requirements.

Background and Aim Primary biliary cholangitis (PBC) is a chronic autoimmune cholangiopathy, characterized by the presence of some autoantibodies in the serum. This study aimed to evaluate the clinical significance of AMA‐M2, anti‐gp210 and anti‐sp100 antibody levels detected by multiplex bead‐based flow fluorescent immunoassay (MBFFI) in PBC. Methods This study cohort included 238 PBC patients, 81 autoimmune hepatitis (AIH) patients, 62 systemic lupus erythematosus (SLE) patients, and 118 healthy controls. Serum AMA‐M2, anti‐gp210 and anti‐sp100 antibody were detected by MBFFI and immunoblotting assay (IBT). The relationship between three antibody levels and cirrhosis, liver function, cholestasis markers and therapeutic effect to ursodesoxycholic acid (UDCA) was evaluated in PBC. Results MBFFI were presented good coincidence rate (87.39%–95.38%) with IBT. The level of AMA‐M2, anti‐gp210 and anti‐sp100 antibodies in PBC patients were higher than other disease group and healthy controls (p ＜ .01). When compared with the healthy controls group, the AUC of AMA‐M2, anti‐gp210 and anti‐sp100 antibodies were 0.9245, 0.7619, and 0.6789, respectively. In addition, gp210 antibody levels have diagnostic value in patients with liver cirrhosis (AUC: 0.7567). We found that when combine detect these three antibodies, the sensitivity was higher than individually detection. High level of serum anti‐gp210 antibody could be related to worse liver function and more severe cholestasis in PBC patients. Moreover, serum antibody levels may decrease or remained flat in patients who responded well to UDCA. Conclusion The detection of AMA‐M2, anti‐gp210 and anti‐sp100 antibody levels by MBFFI showed good performance in the diagnosis of PBC. Serum anti‐gp210 antibody level is related to cirrhosis, poor liver function and severe cholestasis in PBC. The aim of this study was to evaluate the clinical significance of quantitative detection of AMA‐M2, anti‐gp210 and anti‐SP100 antibody levels in PBC.

Objectives Detection of antinuclear antibodies (ANA) by immunofluorescence assay (IFA) is increasingly substituted by multiplex bead-based immunoassay (MBA) and line-blot immunoassay (LIA). This study is to compare the diagnostic performance of MBA and LIA ANA assays on clinically characterized patient samples. Methods A total of 728 serum samples from 385 patients with systemic autoimmune rheumatic diseases (SARD), 204 patients with non-SARD diseases, and 139 apparently healthy subjects were tested with the BioPlex 2200 ANA Screen and EuroLine ANA Profile 3 as the representative MBA and LIA technologies and HEp-2 ANA IFA. Clinical data were collected independent of laboratory analysis and later related to the ANA test results. The clinical diagnostic performances were analyzed using Analyse-it software. Results The MBA demonstrated higher area under curve (AUC) compared to LIA (0.814 vs 0.761, p  = 0.002) and HEp-2 IFA (0.814 vs 0.771, p  = 0.008). The MBA and LIA ANA methods showed higher specificity (83.8% and 77.0% vs 67.6%, p  < 0.001 and p  = 0.005) but lower sensitivity (79.0% and 75.3% vs 86.5%, p  < 0.001) compared to HEp-2 IFA. The MBA and LIA ANA revealed substantial to excellent agreements on specific antinuclear antibodies except anti-dsDNA, with the total agreement from 92.3 to 99.9% and Cohen’s kappa from 0.71 to 0.98. The MBA demonstrated significantly higher sensitivity (58.1% vs 19.8%, p  < 0.001) and comparable specificity (95.9% vs 97.5%, p  = 0.221) on anti-dsDNA assay for the diagnosis of SLE compared to LIA. Conclusions The MBA and LIA ANA assays have higher specificity but lower sensitivity compared to HEp-2 IFA. There are good agreements between MBA and LIA ANA for the specific antinuclear antibodies except for anti-dsDNA. The MBA ANA demonstrated better assay performance compared to LIA as the MBA possesses higher sensitivity and specificity in the diagnosis of SARD. Key Points • The multiplex bead-based immunoassay (MBA) ANA outperformed line-blot immunoassay (LIA) and traditional HEp-2 IFA. • There are good agreements between the MBA BioPlex 2200 ANA Screen and LIA EuroLine ANA Profile 3 for the most of specific antinuclear antibodies except anti-dsDNA. • Additional anti-dsDNA testing is suggested when EuroLine ANA Profile 3 is used for the aid of SLE diagnosis and management. • The positive predictive value of both multiplex ANA assays can be substantially increased without significantly affecting negative predictive value by using at least two specific antinuclear antibodies for reporting a positive ANA  result.

Type 2 diabetes mellitus (T2DM) is a major public health concern that significantly increases the risk of diabetic retinopathy (DR), a leading cause of visual impairment worldwide. This study aimed to identify molecular markers of inflammation (INF) and angiogenesis (ANG) in the aqueous humor (AH) of patients with non-proliferative diabetic retinopathy (NPDR). We conducted an observational, multicenter, case–control study including 116 participants classified into T2DM with NPDR, T2DM without DR, and non-diabetic controls (SCG) undergoing cataract surgery. AH samples were collected intraoperatively and analyzed for 27 cytokines using multiplex immunoassay. Eighteen immune mediators were detected in AH samples, and several were significantly elevated in the NPDR group, including the interleukins (IL) -1β, -6, -8, -15, -17, as well as the granulocyte–macrophage colony stimulating factor (GM-CSF), basic fibroblast growth factor (bFGF), interferon gamma-induced protein (IP-10), macrophage inflammatory protein 1 beta (MIP-1b), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T cell-expressed and -secreted protein (RANTES), and the vascular endothelial growth factor (VEGF). These molecules are involved in retinal INF, blood–retinal barrier breakdown, and pathological neovascularization. Our findings reveal a distinct pro-INF and pro-ANG profile in the AH of NPDR patients, suggesting that these cytokines may serve as early diagnostic/prognostic biomarkers for DR. Targeting these molecules could provide novel therapeutic strategies to mitigate retinal damage and vision loss in diabetic patients.

Serological assays for SARS-CoV-2 play a pivotal role in the definition of whether patients are infected, the understanding of viral epidemiology, the screening of convalescent sera for therapeutic and prophylactic purposes, and in obtaining a better understanding of the immune response towards the virus. The aim of this study was to investigate the performance of a bead-based multiplex assay. This assay allowed for the simultaneous testing of IgG antibodies against SARS-CoV-2 spike, S1, S2, RBD, and nucleocapsid moieties and S1 of seasonal coronaviruses hCoV-22E, hCoV-HKU1, hCoV-NL63, and hCoV-OC43, as well as MERS and SARS-CoV. We compared the bead-based multiplex assay with commercial ELISA tests. We tested the sera of 27 SARS-CoV-2 PCR-positive individuals who were previously tested with different ELISA assays. Additionally, we investigated the reproducibility of the results by means of multiple testing of the same sera. Finally, the results were correlated with neutralising assays. In summary, the concordance of the qualitative results ranged between 78% and 96% depending on the ELISA assay and the specific antigen. Repeated freezing–thawing cycles resulted in reduced mean fluorescence intensity, while the storage period had no influence in this respect. In our test cohort, we detected up to 36% of sera positive for the development of neutralising antibodies, which is in concordance with the bead-based multiplex and IgG ELISA.

Although radiotherapy is a common form of treatment for head and neck cancer, it may lead to tissue damage in the salivary and lacrimal glands, possibly affecting cytokine expression in the gland fluid of treated individuals. Cytokine profiles in saliva and tear fluid of 29 radiated head and neck cancer patients and 20 controls were screened using a multiplex assay. Correlations between cytokine expression and clinical oral and ocular manifestations were examined, and cellular pathways influenced by these cytokines were assessed using the Functional Enrichment Analysis Tool. Significantly elevated cytokines identified in patient saliva were CCL21, IL-4, CX3CL1, CCL2, CXCL1 and CCL15. Many of these cytokines correlated positively with objective signs of oral dryness, and reduced saliva production in the patients. Although CCL21 and IL-4 levels were significantly lower in patient tear fluid, they correlated with subjective ocular symptoms. These increased salivary cytokines affected pro-inflammatory and apoptotic cellular pathways, including T cell signalling, several interleukin signalling pathways, TNF and TGF-β receptor signalling, and the apoptotic p53 pathway. In conclusion, the upregulated salivary cytokines identified suggest an interplay between innate and adaptive immunity, affecting immunoregulatory cellular pathways. Whether this is due to late effects of radiotherapy or tissue repair remains to be investigated.

Background High throughput protein expression studies can be performed using bead-based protein immunoassays, such as the Luminex® xMAP® technology. Technical variability is inherent to these experiments and may lead to systematic bias and reduced power. To reduce technical variability, data pre-processing is performed. However, no recommendations exist for the pre-processing of Luminex® xMAP® data. Results We compared 37 different data pre-processing combinations of transformation and normalization methods in 42 samples on 384 analytes obtained from a multiplex immunoassay based on the Luminex® xMAP® technology. We evaluated the performance of each pre-processing approach with 6 different performance criteria. Three performance criteria were plots. All plots were evaluated by 15 independent and blinded readers. Four different combinations of transformation and normalization methods performed well as pre-processing procedure for this bead-based protein immunoassay. Conclusions The following combinations of transformation and normalization were suitable for pre-processing Luminex® xMAP® data in this study: weighted Box-Cox followed by quantile or robust spline normalization (rsn), asinh transformation followed by loess normalization and Box-Cox followed by rsn.

Serological assays that simultaneously detect antibodies to multiple targets of SARS-CoV-2 and to other structurally related coronaviruses provide a holistic picture of antibody response patterns. Well-validated multiplex immunoassays are scarce. Here, we evaluated the performance of an 11-plex serological assay capable of detecting antibodies directed to four antigenic targets of SARS-CoV-2 and to S1 proteins of other human pathogenic coronaviruses. We used 620 well-characterized sera (n = 458 seropositive and n = 110 seronegative for SARS-CoV-2 in the pre-SARS-CoV-2 era and n = 52 seronegative for SARS-CoV-2 in the era of SARS-CoV-2) as positive and negative standards. We calculated the sensitivity, specificity, as well as positive and negative predictive values, including a 95% confidence interval. The difference in mean fluorescence intensity (95% CI) was used to assess a potential cross-reaction between antibodies to SARS-CoV-2 and the other coronaviruses. The sensitivity (95% CI) of detecting anti-SARS-CoV-2 antibodies to four antigenic targets ranged from 83.4% (76.7–86.7) to 93.7% (91.0–95.7) and the specificity from 98.2% (93.6–99.8) to 100% (96.7–100). We observed no obvious cross-reaction between anti-SARS-CoV-2 antibodies and antibodies to the other coronaviruses except for SARS-CoV-1. The high sensitivity and specificity warrant a reliable utilization of the assay in population-based seroprevalence surveys or vaccine efficacy studies.

Recent interests in comprehensive protein surveys and protein biomarker studies have led to an increased demand for simultaneous measurement of multiple proteins in a single sample. Among various multiplex techniques, bead-based immunoassays, which use encoded particles attached with capture probes, have demonstrated distinct advantages of fluid-phase kinetics, high precision, and flexible target selection. In particular, encoded hydrogel particles composed of porous, hydrophilic, three-dimensional polymers have received positive attention because they enhance the binding kinetics of proteins, reduce protein denaturation, and increase the loading density of capture probes. Microfluidic techniques have been extensively used to fabricate the encoded hydrogel particles for multiplex immunoassays, enabling mass-production of highly monodisperse particles with complex morphologies in mild synthesis conditions. In this paper, we review microfluidic techniques available for the synthesis of encoded hydrogel particles and the important design parameters that determine the particles’ immunoassay performance. We also discuss currently reported multiplex immunoassay platforms that are based on encoded hydrogel particles.

Background Currently, the reference standard assay for the serodiagnosis of neurocysticercosis (NCC) is the lentil lectin-bound glycoproteins/enzyme-linked immunoelectrotransfer blot (LLGP-EITB). The main disadvantage of this technique is the complexity of obtaining and purifying the LLGP extract. This could be solved by replacement with highly specific recombinant antigens from Taenia solium . Based on previous studies, we selected and produced the recombinant Ts8B2 and T24H proteins and applied them to three diagnostic techniques: western blot (WB), enzyme-linked immunosorbent assay (ELISA) and the multiplex bead-based assay (MBA). Methods The Ts8B2 and T24H cDNA sequences were expressed in a prokaryotic system and the corresponding expression products purified; three recombinant proteins were further characterized: T24H-his, GST-T24H and GST-Ts8B2. The proteins on WB, ELISA and MBA were tested against 149 sera from patients with NCC confirmed by brain imaging, 40 sera from patients with other parasitic diseases, and 131 sera from US. individuals without evidence of neurocysticercosis (clinical/serological/brain imaging). The sensitivity and specificity of each antigen by WB were calculated by counting the number of true positive, false positive, true negative and false negative results. Using the receiver operating characteristic (ROC) curves, the cut-off values for the ELISA and MBA were established as well as the sensitivity and specificity of each assay. Results All three antigens showed a high sensitivity on WB in active NCC cases with two or more viable cysts and low sensitivity for cases with single viable cyst or calcified lesions and inactive NCC. WB showed the highest specificity and sensitivity out of the three diagnostic techniques. The recombinant T24H-his was the best diagnostic reagent in WB (100% sensitivity, 99.4% specificity), exhibiting similar results to the LLGP-EITB, against the same panel of NCC sera. The GST-T24H antigen worked better than the others in ELISA and MBA protocols (88.3 and 96.1% sensitivity, respectively and 96.5% specificity). Conclusions The sensitivity and specificity that we obtained were similar to results from a previous study using a similar recombinant antigen (rT24H), suggesting that recombinant antigens may be good alternatives to crude extracts in a variety of diagnostic techniques. Furthermore, these antigens can be applied in the development of point-of-care tests which would be useful in NCC field studies.