scholarly journals Multiplex Assays of Luminex for Identification of COVID-19 Antibodies in Patient Serum: Performance Evaluation and Comparison to ELISA

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S92-S92
Author(s):  
M S Shapiro ◽  
X Wang ◽  
D R Mendu ◽  
A Firpo
Keyword(s):  
Dynamic Range ◽  
High Titer ◽  
Mount Sinai Hospital ◽  
Antibody Testing ◽  
Luminex Assay ◽  
Multiplex Assays ◽  
Negative Results ◽  
Broad Dynamic Range ◽  
Circulating Antibodies ◽  
Mount Sinai

Abstract Introduction/Objective Mount Sinai Hospital has received emergency use authorization (EUA) from the FDA for Coronavirus Disease 2019 (COVID-19) antibody testing using ELISA. This serological assay detects and titrates the presence of circulating antibodies to COVID-19. Other platforms have aimed to achieve the credentials of the ELISA instrument, including the multiplex assays of Luminex. The platform is known to have a greater throughput (384 wells vs. 96 wells per microplate) and faster processing speed (8 hours vs. 17 hours). Methods Luminex utilizes beads that couple to the same COVID-19 antigens (mRBD and mSpike) which were utilized for the ELISA assay. The beads are read determining the mean fluorescence intensity (MFI). In order to compare the two methods, our study included 61 patients with COVID-19 at Mount Sinai Hospital, to screen and titrate their sera using Luminex, and to correspond the MFI values with the ELISA titers. Results The Luminex assay has achieved the same level of confidence as ELISA. The 61 patients, representing 30 negatives and 31 positives, are consistently identified as such on both platforms. Our data highlights 32% of patients with a low titer (<1:160), 42% of patients with a high titer (1:160 ~ 1:320), and 26% of patients with a very high titer level (>1:320). These titers correlated well with the MFI values. Based on a cutoff of 80,000 MFI, the sensitivity and specificity of the assay is 98% and 85%, respectively, with no overlapping of MFI between positive and negative results. Conclusion Overall, the study has demonstrated that the Luminex is a strong alternative for the ELISA platform. The Luminex highlights the broad dynamic range with no overlapping between positives and negatives. Migration from ELISA to Luminex, a platform with faster and greater throughput, is therefore, highly desirable.

Informative array testing with multiplex assays

2021 ◽  
Author(s):  
Christopher R. Bilder ◽  
Joshua M. Tebbs ◽  
Christopher S. McMahan
Keyword(s):  
Multiplex Assays ◽  
Array Testing

scholarly journals Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

2014 ◽  
Vol 5 (S1) ◽  
Author(s):  
Suzanne Hagan ◽  
Alan Tomlinson ◽  
Louise Madden ◽  
Anne M Clark ◽  
Katherine Oliver
Keyword(s):  
Dry Eye ◽  
Dry Eye Disease ◽  
Tear Fluid ◽  
Eye Disease ◽  
Multiplex Assays

scholarly journals Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project

2001 ◽  
Author(s):  
R P Koopman ◽  
R G Langlois ◽  
S Nasarabadi ◽  
W J Benett ◽  
J B Richards ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sample Preparation ◽  
Final Report ◽  
Acid System ◽  
Multiplex Assays ◽  
Pcr Multiplex

scholarly journals Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study

2014 ◽  
Vol 18 (53) ◽  
pp. 1-167 ◽  
Author(s):  
Louise Pankhurst ◽  
Louissa Macfarlane-Smith ◽  
James Buchanan ◽  
Luke Anson ◽  
Kerrie Davies ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Infection Control ◽  
Sensitivity And Specificity ◽  
Chain Reaction ◽  
Luminex Assay ◽  
Multiplex Assays ◽  
Infectious Diarrhoea ◽  
Gastrointestinal Pathogens ◽  
Microbiological Testing ◽  
Polymerase Chain

BackgroundEvery year approximately 5000–9000 patients are admitted to a hospital with diarrhoea, which in up to 90% of cases has a non-infectious cause. As a result, single rooms are ‘blocked’ by patients with non-infectious diarrhoea, while patients with infectious diarrhoea are still in open bays because of a lack of free side rooms. A rapid test for differentiating infectious from non-infectious diarrhoea could be very beneficial for patients.ObjectiveTo evaluate MassCode multiplex polymerase chain reaction (PCR) for the simultaneous diagnosis of multiple enteropathogens directly from stool, in terms of sensitivity/specificity to detect four common important enteropathogens:Clostridium difficile,Campylobacterspp.,Salmonellaspp. and norovirus.DesignA retrospective study of fixed numbers of samples positive forC. difficile(n = 200),Campylobacterspp. (n = 200),Salmonellaspp. (n = 100) and norovirus (n = 200) plus samples negative for all these pathogens (n = 300). Samples were sourced from NHS microbiology laboratories in Oxford and Leeds where initial diagnostic testing was performed according to Public Health England methodology. Researchers carrying out MassCode assays were blind to this information. A questionnaire survey, examining current practice for infection control teams and microbiology laboratories managing infectious diarrhoea, was also carried out.SettingMassCode assays were carried out at Oxford University Hospitals NHS Trust. Further multiplex assays, carried out using Luminex, were run on the same set of samples at Leeds Teaching Hospitals NHS Trust. The questionnaire was completed by various NHS trusts.Main outcome measuresSensitivity and specificity to detectC. difficile,Campylobacterspp.,Salmonellaspp., and norovirus.ResultsNucleic acids were extracted from 948 clinical samples using an optimised protocol (200Campylobacterspp., 199C. difficile, 60S. enterica, 199 norovirus and 295 negative samples; some samples contained more than one pathogen). Using the MassCode assay, sensitivities for each organism compared with standard microbiological testing ranged from 43% to 94% and specificities from 95% to 98%, with particularly poor performance forS. enterica. Relatively large numbers of unexpected positives not confirmed with quantitative PCR were also observed, particularly forS. enterica,Giardia lambliaandCryptosporidiumspp. As the results indicated thatS. entericadetection might provide generic challenges to other multiplex assays for gastrointestinal pathogens, the Luminex xTag®gastrointestinal assay was also run blinded on the same extracts (937/948 remaining) and on re-extracted samples (839/948 with sufficient material). ForCampylobacterspp.,C. difficileand norovirus, high sensitivities (> 92%) and specificities (> 96%) were observed. ForS. enterica, on the original MassCode/Oxford extracts, Luminex sensitivity compared with standard microbiological testing was 84% [95% confidence interval (CI) 73% to 93%], but this dropped to 46% on a fresh extract, very similar to MassCode, with a corresponding increase in specificity from 92% to 99%. Overall agreement on the per-sample diagnosis compared with combined microbiology plus PCR for the main four/all pathogens was 85.6%/64.7%, 87.0%/82.9% and 89.8%/86.8% for the MassCode assay, Luminex assay/MassCode extract and Luminex assay/fresh extract, respectively. Luminex assay results from fresh extracts implied that 5% of samples did not represent infectious diarrhoea, even though enteropathogens were genuinely present. Managing infectious diarrhoea was a significant burden for infection control teams (taking 21% of their time) and better diagnostics were identified as having major potential benefits for patients.ConclusionsOverall, the Luminex xTag gastrointestinal panel showed similar or superior sensitivity and specificity to the MassCode assay. However, on fresh extracts, this test had low sensitivity to detect a key enteric pathogen,S. enterica; making it an unrealistic option for most microbiology laboratories. Extraction efficiency appears to be a major obstacle for nucleic acid-based tests for this organism, and possibly the whole Enterobacteriaceae family. To improve workflows in service microbiology laboratories, to reduce workload for infection control practitioners, and to improve outcomes for NHS patients, further research on deoxyribonucleic acid-based multiplex gastrointestinal diagnostics is urgently needed.FundingThe Health Technology Assessment programme of the National Institute for Health Research.

Abstract 454: Impact of Uremia on Human Adipose Phenotype

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Karen J Ho ◽  
Hui Xue ◽  
Christine R Mauro ◽  
Binh T Nguyen ◽  
Peng Yu ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Multiple Regression Analysis ◽  
Coronary Disease ◽  
Independent Predictor ◽  
Elective Surgery ◽  
Health Impact ◽  
Independent Variables ◽  
Multiplex Assays ◽  
Uremic Patients ◽  
Subcutaneous Adipose

Background: Recognition of adipose-related signaling participation in vascular processes is increasing, though direct interrogation of clinically relevant human adipose has been sparse. Few scenarios rival uremia for health impact. To advance understanding of the spectrum/determinants of human adipose biology, we compared fresh extremity adipose from patients with/without uremia. Methods: Subcutaneous adipose and clinical data was collected from patients undergoing open elective surgery. Protein was isolated, and mediator levels determined via multiplex assays. Results: In the 71 patient cohort, mean age=63.4y; 63.3% had diabetes, 49.2% had hyperlipidemia and 53.5% had coronary disease. Compared to non-uremic patients, uremic patients had 1/10th the levels of leptin, 1/3rd the levels of adiponectin, and 3-fold higher levels of resistin (Table 1). Females had 6-fold higher leptin, 1.5-fold higher adiponectin and 2-fold higher TNF but equivalent resistin. There were no differences in mediator levels when stratified by age. In both the obese/non-obese strata, we observed a concordant pattern of association (magnitude/significance) of uremia and leptin/adiponectin/resistin. No differentials in other mediators emerged upon BMI stratification. Multiple regression analysis for leptin/adiponectin/resistin (with age/gender/uremia as independent variables) showed uremia as the highest independent predictor of all three mediators, followed by gender, whereas age had no effect. Conclusions: Based on direct biopsy of living human adipose, uremia is associated with adiposopathy, including perturbations in the adipose derived hormones leptin, adiponectin, and resistin. Compared with other parameters including BMI, uremia overall dominated in determining adipose phenotype.

PSX-A-8 Late-Breaking: Development of multiplex assays for equine cytokines IL-1β, IL-4, IL-6, IL-8, IL-10, and TNFα

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 371-371
Author(s):  
Sarah H White-Springer
Keyword(s):  
Polyclonal Antibodies ◽  
Positive Control ◽  
Viable Option ◽  
Meso Scale Discovery ◽  
Multiplex Assays ◽  
Tnf Α ◽  
Before And After ◽  
Bronchoalveolar Fluid ◽  
Cytokine Assay ◽  
Necrosis Factor

Abstract Equine-specific assays to quantify cytokine concentrations are limited and often have a restricted range such that physiological concentrations of many cytokines are below detectable limits of the assay. We aimed to develop custom multiplex assays for equine interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) α using the Meso Scale Discovery U-PLEX platform. Equine-specific ELISA kits containing unlabeled and biotinylated polyclonal antibodies and the specific recombinant equine cytokine were purchased for each cytokine. Each biotinylated antibody was coupled to a linker specific for a unique spot within each well of the U-PLEX plates. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to optimization in multiplex. Two preliminary experiments were performed: 1) multiplexed conjugation of equine IL-10 and TNFα to the U-PLEX plates; and 2) multiplexed conjugation of equine IL-1β, IL-4, IL-6, and IL-8 to the U-PLEX plates. Standard curves were run at concentrations ranging from 0 to 5,000 pg/mL for TNFα and IL-8, to 12,500 pg/mL for IL-10, to 25,000 pg/mL for IL-1β and IL-6, and to 50,000 pg/mL for IL-4. The minimum average concentrations measured by the standard curves were 0.065, 0.006, 0.017, 0.00013, 0.196, and 0.050 pg/mL for IL-1β, IL-4, IL-6, IL-8, IL-10, and TNFα, respectively. Test samples of equine serum (n = 5) and bronchoalveolar fluid (n = 3) before and after exercise and of ConA-stimulated equine peripheral mononuclear cell supernatants (positive control) were analyzed for each multiplexed assay. With the exception of serum from one horse, all samples ran within detectable limits of each assay. This preliminary work indicates the U-PLEX platform is a viable option to simultaneously quantify concentrations of multiple equine cytokines, allowing for expansion of research efforts focused on understanding immune responses in the horse.

scholarly journals Updates in immune-based multiplex assays

2019 ◽  
Vol 40 (1) ◽  
pp. 1-2 ◽  
Author(s):  
Cristiana Tanase ◽  
Radu Albulescu ◽  
Monica Neagu
Keyword(s):  
Multiplex Assays
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