Development of a Multiplex Bead Assay for the Detection of IgG Antibody Responses to Guinea Worm.
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ABSTRACT:
SUBMITTER: Priest JW
PROVIDER: S-EPMC7695067 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Increased levels of guinea worm (GW) disease transmission among dogs in villages along the Chari River in Chad threaten the gains made by the GW Eradication Program. Infected dogs with preemergent worm blisters are difficult to proactively identify. If these dogs are not contained, blisters can burst upon submersion in water, leading to the contamination of the water supply with L1 larvae. Guinea worm antigens previously identified using sera from human dracunculiasis patients were coupled to polystyrene beads for multiplex bead assay analysis of 41 non-endemic (presumed negative) dog sera and 39 sera from GW-positive dogs from Chad. Because commercially available anti-dog IgG secondary antibodies did not perform well in the multiplex assay, dog IgGs were partially purified, and a new anti-dog IgG monoclonal antibody was developed. Using the new 4E3D9 monoclonal secondary antibody, the thioredoxin-like protein 1-glutathione-S-transferase (GST), heat shock protein (HSP1)-GST, and HSP2-GST antigen multiplex assays had sensitivities of 69-74% and specificities of 73-83%. The domain of unknown function protein 148 (DUF148)-GST antigen multiplex assay had a sensitivity of 89.7% and a specificity of 85.4%. When testing samples collected within 1 year of GW emergence (n = 20), the DUF148-GST assay had a sensitivity of 90.0% and a specificity of 97.6% with a receiver-operating characteristic area under the curve of 0.94. Using sera from two experimentally infected dogs, antibodies to GW antigens were detected within 6 months of exposure. Our results suggest that, when used to analyze paired, longitudinal samples collected 1-2 months apart, the DUF148/GST multiplex assay could identify infected dogs 4-8 months before GW emergence.
| S-EPMC7790091 | biostudies-literature
Project description: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.
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Project description:Serological assays for SARS-CoV-2 antibodies must be validated for performance with a large panel of clinical specimens. Most existing assays utilize a single antigen target and may be subject to reduced diagnostic specificity. This study evaluated a multiplex assay that detects antibodies to three SARS-CoV-2 targets. Human serum specimens (n = 323) with known previous SARS-CoV-2 exposure status were tested on a commercially available multiplex bead assay (MBA) measuring IgG to SARS-CoV-2 spike protein receptor-binding domain (RBD), nucleocapsid protein (NP), and RBD/NP fusion antigens. Assay performance was evaluated against reverse transcriptase PCR (RT-PCR) results and also compared with test results for two single-target commercial assays. The MBA had a diagnostic sensitivity of 89.8% and a specificity of 100%, with serum collection at >28 days following COVID-19 symptom onset showing the highest seropositivity rates (sensitivity: 94.7%). The MBA performed comparably to single-target assays with the ability to detect IgG against specific antigen targets, with 19 (5.9%) discrepant specimens compared to the NP IgG assay and 12 (3.7%) compared to the S1 RBD IgG assay (kappa coefficients 0.92 and 0.88 compared to NP IgG and S1 RBD IgG assays, respectively. These findings highlight inherent advantages of using a SARS-CoV-2 serological test with multiple antigen targets; specifically, the ability to detect IgG against RBD and NP antigens simultaneously. In particular, the 100.0% diagnostic specificity exhibited by the MBA in this study is important for its implementation in populations with low SARS-CoV-2 seroprevalence or where background antibody reactivity to SARS-CoV-2 antigens has been detected. IMPORTANCE Reporting of SARS-CoV-2 infections through nucleic acid or antigen based diagnostic tests severely underestimates the true burden of exposure in a population. Serological data assaying for antibodies against SARS-CoV-2 antigens offers an alternative source of data to estimate population exposure, but most current immunoassays only include a single target for antibody detection. This report outlines a direct comparison of a multiplex bead assay to two other commercial single-target assays in their ability to detect IgG against SARS-CoV-2 antigens. Against a well-defined panel of 323 serum specimens, diagnostic sensitivity and specificity were very high for the multiplex assay, with strong agreement in IgG detection for single targets compared to the single-target assays. Collection of more data for individual- and population-level seroprofiles allows further investigation into more accurate exposure estimates and research into the determinants of infection and convalescent responses.
| S-EPMC9241621 | biostudies-literature
Project description:Influenza hemagglutination inhibition (HI) and virus microneutralization assays (MN) are widely used for seroprevalence studies. However, these assays have limited field portability and are difficult to fully automate for high throughput laboratory testing. To address these issues, three multiplex influenza subtype-specific antibody detection assays were developed using recombinant hemagglutinin antigens in combination with Chembio, Luminex®, and ForteBio® platforms. Assay sensitivity, specificity, and subtype cross-reactivity were evaluated using a panel of well characterized human sera. Compared to the traditional HI, assay sensitivity ranged from 87% to 92% and assay specificity in sera collected from unexposed persons ranged from 65% to 100% across the platforms. High assay specificity (86-100%) for A(H5N1) rHA was achieved for sera from exposed or unexposed to hetorosubtype influenza HAs. In contrast, assay specificity for A(H1N1)pdm09 rHA using sera collected from A/Vietnam/1204/2004 (H5N1) vaccinees in 2008 was low (22-30%) in all platforms. Although cross-reactivity against rHA subtype proteins was observed in each assay platform, the correct subtype specific responses were identified 78%-94% of the time when paired samples were available for analysis. These results show that high throughput and portable multiplex assays that incorporate rHA can be used to identify influenza subtype specific infections.
| S-EPMC8958803 | biostudies-literature
Project description:Numerous infectious diseases impacting livestock impose an important economic burden and in some cases also represent a threat to humans and are classified as zoonoses. Some zoonotic diseases are transmitted by vectors and, due to complex environmental and socio-economic factors, the distribution of many of these pathogens is changing, with increasing numbers being found in previously unaffected countries. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies to five important pathogens of livestock (three of them zoonotic) that are currently emerging in new geographical locations: Rift Valley fever virus (RVFV), Crimean-Congo haemorrhagic fever virus (CCHFV), Schmallenberg virus (SBV), Bluetongue virus (BTV) and the bacteria complex Mycobacterium tuberculosis. Using the Luminex platform, polystyrene microspheres were coated with recombinant proteins from each of the five pathogens. The mix of microspheres was used for the simultaneous detection of antibodies against the five corresponding diseases affecting ruminants. The following panel of sera was included in the study: 50 sera from sheep experimentally infected with RVFV, 74 sera from calves and lambs vaccinated with SBV, 26 sera from cattle vaccinated with Mycobacterium bovis, 30 field sera from different species of ruminants infected with CCHFV and 88 calf sera infected with BTV. Finally, to determine its diagnostic specificity 220 field sera from Spanish farms free of the five diseases were assessed. All the sera were classified using commercial ELISAs specific for each disease, used in this study as the reference technique. The results showed the multiplex assay exhibited good performance characteristics with values of sensitivity ranging from 93% to 100% and of specificity ranging from 96% to 99% depending on the pathogen. This new tool allows the simultaneous detection of antibodies against five important pathogens, reducing the volume of sample needed and the time of analysis where these pathogens are usually tested individually.
| S-EPMC8246919 | biostudies-literature
Project description:The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.
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Project description:Guinea worm Dracunculus medinensis causes debilitating disease in people and is subject to an ongoing global eradication programme. Research and controls are constrained by a lack of diagnostic tools. We developed a specific and sensitive LAMP method for detecting D. medinensis larval DNA in copepod vectors. We were able to detect a single larva in a background of field-collected copepods. This method could form the basis of a "pond-side test" for detecting potential sources of Guinea worm infection in the environment, in copepods, including in the guts of fish as potential transport hosts, enabling research, surveillance and targeting of control measures. The key constraint on the utility of this assay as a field diagnostic, is a lack of knowledge of variation in the temporal and spatial distribution of D. medinensis larvae in copepods in water bodies in the affected areas and how best to sample copepods to obtain a reliable diagnostic sample. These fundamental knowledge gaps could readily be addressed with field collections of samples across areas experiencing a range of worm infection frequencies, coupled with field and laboratory analyses using LAMP and PCR.
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Project description:Isocitrate dehydrogenase 1 and 2 (IDH) mutations are frequently found in various cancer types such as gliomas, chondrosarcomas and myeloid malignancies. Their molecular detection has recently gained wide recognition in the diagnosis and prognosis of these neoplasms. For that purpose various molecular approaches have been used but a universally accepted method is still lacking. In this study we aimed to develop a novel bead-based liquid assay using Locked nucleic acids (LNA)-modified oligonucleotide probes for multiplexed detection of the most frequent IDH1 (p.R132C, p.R132G, p.R132H, p.R132L, p.R132S) and IDH2 (p.R140Q, p.R172K) mutations. The method includes four steps: 1) PCR amplification of the targeted fragments with biotinylated primers; 2) Direct hybridization to barcoded microbeads with specific LNA-modified oligonucleotide probes; 3) Incubation with phycoerythrin coupled streptavidin; 4) Acquisition of fluorescent intensities of each set of beads on a flow platform (LuminexCorp., USA). We tested the performance of the assay on both artificial plasmid constructs and on clinical samples from 114 patients with known or suspected myeloid malignancies. The method appeared to be superior to direct sequencing having a much higher sensitivity of 2.5% mutant alleles. Applying this method to patients' samples we identified a total of 9 mutations (one IDH1 p.R132C, seven IDH2 p.R140Q and one IDH2 p.R172K). In conclusion, this method could be successfully implemented in the diagnostic work-up for various tumors known to harbor IDH1/2 mutations (e.g. myeloid malignancies, gliomas, etc.). International initiatives are needed to validate the different existing methods for detection of IDH1/2 mutations in clinical settings.
| S-EPMC3786925 | biostudies-literature
Project description:BackgroundOral fluid (hereafter, saliva) is a non-invasive and attractive alternative to blood for SARS-CoV-2 IgG testing; however, the heterogeneity of saliva as a matrix poses challenges for immunoassay performance.ObjectivesTo optimize performance of a magnetic microparticle-based multiplex immunoassay (MIA) for SARS-CoV-2 IgG measurement in saliva, with consideration of: i) threshold setting and validation across different MIA bead batches; ii) sample qualification based on salivary total IgG concentration; iii) calibration to U.S. SARS-CoV-2 serological standard binding antibody units (BAU); and iv) correlations with blood-based SARS-CoV-2 serological and neutralizing antibody (nAb) assays.MethodsThe salivary SARS-CoV-2 IgG MIA included 2 nucleocapsid (N), 3 receptor-binding domain (RBD), and 2 spike protein (S) antigens. Gingival crevicular fluid (GCF) swab saliva samples were collected before December 2019 (n = 555) and after molecular test-confirmed SARS-CoV-2 infection from 113 individuals (providing up to 5 repeated-measures; n = 398) and used to optimize and validate MIA performance (total n = 953). Combinations of IgG responses to N, RBD and S and total salivary IgG concentration (μg/mL) as a qualifier of nonreactive samples were optimized and validated, calibrated to the U.S. SARS-CoV-2 serological standard, and correlated with blood-based SARS-CoV-2 IgG ELISA and nAb assays.ResultsThe sum of signal to cutoff (S/Co) to all seven MIA SARS-CoV-2 antigens and disqualification of nonreactive saliva samples with ≤15 μg/mL total IgG led to correct classification of 62/62 positives (sensitivity [Se] = 100.0%; 95% confidence interval [CI] = 94.8%, 100.0%) and 108/109 negatives (specificity [Sp] = 99.1%; 95% CI = 97.3%, 100.0%) at 8-million beads coupling scale and 80/81 positives (Se = 98.8%; 95% CI = 93.3%, 100.0%] and 127/127 negatives (Sp = 100%; 95% CI = 97.1%, 100.0%) at 20-million beads coupling scale. Salivary SARS-CoV-2 IgG crossed the MIA cutoff of 0.1 BAU/mL on average 9 days post-COVID-19 symptom onset and peaked around day 30. Among n = 30 matched saliva and plasma samples, salivary SARS-CoV-2 MIA IgG levels correlated with corresponding-antigen plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.83, S: ρ = 0.82; all p < 0.001). Correlations of plasma SARS-CoV-2 nAb assay area under the curve (AUC) with salivary MIA IgG (N: ρ = 0.68, RBD: ρ = 0.78, S: ρ = 0.79; all p < 0.001) and with plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.79, S: ρ = 0.76; p < 0.001) were similar.ConclusionsA salivary SARS-CoV-2 IgG MIA produced consistently high Se (> 98.8%) and Sp (> 99.1%) across two bead coupling scales and correlations with nAb responses that were similar to blood-based SARS-CoV-2 IgG ELISA data. This non-invasive salivary SARS-CoV-2 IgG MIA could increase engagement of vulnerable populations and improve broad understanding of humoral immunity (kinetics and gaps) within the evolving context of booster vaccination, viral variants and waning immunity.
| S-EPMC9911157 | biostudies-literature
Project description:The family of polyomaviruses, which cause severe disease in immunocompromised hosts, has expanded substantially in recent years. To accommodate measurement of IgG seroresponses against all currently known human polyomaviruses (HPyVs), including the Lyon IARC polyomavirus (LIPyV), we extended our custom multiplex bead-based HPyV immunoassay and evaluated the performance of this pan-HPyV immunoassay. The VP1 proteins of 15 HPyVs belonging to 13 Polyomavirus species were expressed as recombinant glutathione S-transferase (GST) fusion proteins and coupled to fluorescent Luminex beads. Sera from healthy blood donors and immunocompromised kidney transplant recipients were used to analyze seroreactivity against the different HPyVs. For BK polyomavirus (BKPyV), the GST-VP1 fusion protein-directed seroresponses were compared to those obtained against BKPyV VP1 virus-like particles (VLP). Seroreactivity against most HPyVs was common and generally high in both test populations. Low seroreactivity against HPyV9, HPyV12, New Jersey PyV, and LIPyV was observed. The assay was reproducible (Pearson's r2 > 0.84, P < 0.001) and specific. Weak but consistent cross-reactivity between the related viruses HPyV6 and HPyV7 was observed. The seroresponses measured by the GST-VP1-based immunoassay and a VP1 VLP-based enzyme-linked immunosorbent assay were highly correlated (Spearman's ρ = 0.823, P < 0.001). The bead-based pan-HPyV multiplex immunoassay is a reliable tool to determine HPyV-specific seroresponses with high reproducibility and specificity and is suitable for use in seroepidemiological studies.
| S-EPMC5869810 | biostudies-literature