Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens

Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna F. O.; Krieger, Marco Aurélio; Foti, Leonardo

doi:10.1016/j.jim.2016.05.002

Cited by 8 publications

(8 citation statements)

References 26 publications

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“…It is thought that during this incubation, anti-E.coli antibodies present in the serum bound to their target proteins, thus removing them from the pool of available antibodies that could bind to the bacterial proteins coupled to the microspheres during the assay. This has previously been shown to be an effective technique to reduce or remove background signals from MMIAs [65]. In addition, one microsphere region was covalently coupled to E.coli cell lysate and acted as a control during the assays-any serum samples that had MFI readings above background levels to this microsphere region would be discounted from further analysis.…”

Section: Discussionmentioning

confidence: 99%

Development of a multiplex microsphere immunoassay for the detection of antibodies against highly pathogenic viruses in human and animal serum samples

et al. 2020

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Surveillance of highly pathogenic viruses circulating in both human and animal populations is crucial to unveil endemic infections and potential zoonotic reservoirs. Monitoring the burden of disease by serological assay could be used as an early warning system for imminent outbreaks as an increased seroprevalance often precedes larger outbreaks. However, the multitude of highly pathogenic viruses necessitates the need to identify specific antibodies against several targets from both humans as well as from potential reservoir animals such as bats. In order to address this, we have developed a broadly reactive multiplex microsphere immunoassay (MMIA) for the detection of antibodies against several highly pathogenic viruses from both humans and animals. To this aim, nucleoproteins (NP) of Ebola virus (EBOV), Marburg virus (MARV) and nucleocapsid proteins (NP) of Crimean-Congo haemorrhagic fever virus, Rift Valley fever virus and Dobrava-Belgrade hantavirus were employed in a 5-plex assay for IgG detection. After optimisation, specific binding to each respective NP was shown by testing sera from humans and non-human primates with known infection status. The usefulness of our assay for serosurveillance was shown by determining the immune response against the NP antigens in a panel of 129 human serum samples collected in Guinea between 2011 and 2012 in comparison to a panel of 88 sera from the German blood bank. We found good agreement between our MMIA and commercial or in-house reference methods by ELISA or IIFT with statistically significant higher PLOS NEGLECTED TROPICAL DISEASES

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Section: Discussionmentioning

confidence: 99%

Development of a multiplex microsphere immunoassay for the detection of antibodies against highly pathogenic viruses in human and animal serum samples

et al. 2020

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“…Immunoreactions were performed in low binding 96-well plates (SARSTEDT) and all incubations were performed at 37 °C on a microplate shaker (set at 300 RPM) and protected from light. A blocking step using E. coli lysate was performed as previously described [17]. For the wash steps, a Hydroflex plate washer with a magnetic support (TECAN, Durban, NC, USA) was used.…”

Section: Methodsmentioning

confidence: 99%

Detection of IgG3 antibodies specific to the human immunodeficiency virus type 1 (HIV-1) p24 protein as marker for recently acquired infection

et al. 2018

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Reducing the risk of human immunodeficiency virus type 1 (HIV-1) transmission is still a public health priority. The development of effective control strategies relies on the quantification of the effects of prophylactic and therapeutic measures in disease incidence. Although several assays can be used to estimate HIV incidence, these estimates are limited by the poor performance of these assays in distinguishing recent from long-standing infections. To address such limitation, we have developed an assay to titrate p24-specific IgG3 antibodies as a marker of recent infection. The assay is based on a recombinant p24 protein capable to detect total IgG antibodies in sera using a liquid micro array and enzyme-linked immunosorbent assay. Subsequently, the assay was optimised to detect and titrate anti-p24 IgG3 responses in a panel of sequential specimens from seroconverters over 24 months. The kinetics of p24-specific IgG3 titres revealed a transient peak in the 4 to 5-month period after seroconversion. It was followed by a sharp decline, allowing infections with less than 6 months to be distinguished from older ones. The developed assay exhibited a mean duration of recent infection of 144 days and a false-recent rate of ca. 14%. Our findings show that HIV-1 p24-specific IgG3 titres can be used as a tool to evaluate HIV incidence in serosurveys and to monitor the efficacy of vaccines and other transmission control strategies.

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“…Serum samples were diluted 1:100 in 0.3% Tween 20-PBS containing 5% skimmed milk and a 5% E. coli lysate (from BL21 CodonPlus RIL cells) that were then incubated at 37˚C for 60 minutes with stirring. The E. coli lysate was obtained as per Crestani et al [30]. Samples were tested in duplicate as follows: 50 μl of diluted sera were added to each well together with 50 μl of 0.3% Tween 20-PBS containing 5% skimmed milk, and 2500 r2B2t-and 2500 rT24H-coupled microspheres and left to incubate on a plate shaker for 30 minutes at room temperature.…”

Section: Multiplex Bead-based Fluorescent Assaymentioning

confidence: 99%

Multiple-bead assay for the differential serodiagnosis of neglected human cestodiases: Neurocysticercosis and cystic echinococcosis

et al. 2022

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Background Neurocysticercosis (NCC), and cystic echinococcosis (CE) are two neglected diseases caused by cestodes, co-endemic in many areas of the world. Imaging studies and serological tests are used in the diagnosis of both parasitic diseases, but cross-reactions may confound the results of the latter. The novel multiplex bead-based assay with recombinant antigens has been reported to increases the diagnostic accuracy of serological techniques. Methodology We set-up an immunoassay based on the multiplex bead-based platform (MBA), using the rT24H (against Cysticercus cellulosae, causing cysticercosis) and r2B2t (against Echinococcus granulosus sensu lato, causing CE) recombinant antigens, for simultaneous and differential diagnosis of these infections. The antigens were tested on 356 sera from 151 patients with CE, 126 patients with NCC, and 79 individuals negative for both diseases. Specificity was calculated including sera from healthy donors, other neurological diseases and the respective NCC or CE sera counterpart. The diagnostic accuracy of this assay was compared with two commercial ELISA tests, Novalisa and Ridascreen, widely used in the routine diagnosis of cysticercosis and CE, respectively. Main findings For the diagnosis of NCC, sensitivity ranged from 57.94–63.49% for the rT24H-MBA, and 40.48–46.03% for Novalisa ELISA depending on exclusion or inclusion of sera having equivocal results on ELISA from the analysis; specificities ranged from 90.87–91.30% and 70.43–76.96%, respectively. AUC values of the ROC curve were 0.783 (rT24H) and 0.619 (Novalisa) (p-value < 0.001). For the diagnosis of CE, the sensitivity of the r2B2t-MBA ranged from 68.87–69.77% and of Ridascreen ELISA from 50.00–57.62%; specificities from 92.47–92.68% and from 74.15–80.98%, respectively. AUC values were 0.717 and 0.760, respectively. Conclusions/Significance Overall, the recombinant antigens tested with the bead-based technology showed better diagnostic accuracy than the commercial assays, particularly for the diagnosis of NCC. The possibility of testing the same serum sample simultaneously for the presence of antibodies against both antigens is an added value particularly in seroprevalence studies for cysticercosis linked to control programs in endemic areas where these two parasites coexist.

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Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens

Cited by 8 publications

References 26 publications

Development of a multiplex microsphere immunoassay for the detection of antibodies against highly pathogenic viruses in human and animal serum samples

Development of a multiplex microsphere immunoassay for the detection of antibodies against highly pathogenic viruses in human and animal serum samples

Detection of IgG3 antibodies specific to the human immunodeficiency virus type 1 (HIV-1) p24 protein as marker for recently acquired infection

Multiple-bead assay for the differential serodiagnosis of neglected human cestodiases: Neurocysticercosis and cystic echinococcosis

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