Development of an ELISA-array for simultaneous detection of five encephalitis viruses

Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fu‐Cheng; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingqing; Liu, Hong; Yang, Yinhui

doi:10.1186/1743-422x-9-56

Cited by 18 publications

(13 citation statements)

References 19 publications

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“…Indirect immunofluorescence assay (IFA) was performed to characterize the expression of JEV E protein in BHK-21 cells transfected with equal amounts of RNA transcripts. Briefly, the transfected BHK-21 cells were fixed with cold acetone and incubated with the mouse monoclonal antibody (MAb) 4D5, specific for JEV E protein (14), followed by goat anti-mouse IgG-fluorescein isothiocyanate (FITC) conjugate as previously described (12). Mutant viruses recovered from viral RNA-transfected cells (passage 0), as well as the viruses after passage on Vero cells for 5 (passage 5) and 10 (passage 10) rounds, were analyzed by plaque assays as previously described (12).…”

Section: Cells and Virusesmentioning

confidence: 99%

Rational Design of a Flavivirus Vaccine by Abolishing Viral RNA 2′- O Methylation

Dong

et al. 2013

J Virol

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Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2=-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5= end of viral RNA. The function of 2=-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2=-O methylation is replicative, but its viral RNA lacks 2=-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2=-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2=-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2=-O methylationdefective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2=-O methyltransferases. Live attenuated vaccine represents the best medical intervention to prevent many viral diseases, such as those caused by vaccinia virus, poliovirus (Sabin), yellow fever virus (YFV; YF-17D), Japanese encephalitis virus (JEV SA14-14-2), and MMR (measles, mumps, and rubella viruses). The attenuated vaccine replicates to a low level but induces immune response and memory that are sufficient to prevent virulent virus infection. The traditional method of developing an attenuated vaccine is by passaging the virus through a foreign host (e.g., tissue culture or live animals). The attenuation of a vaccine strain is empirically achieved through accumulation of random mutations during passaging while maintaining immunogenicity. The function of each accumulated mutation in the vaccine strain needs to be analyzed to understand the mechanism of attenuation. As an alternative approach for vaccine development, viral attenuation could be rationally designed by altering the ability of virus to antagonize innate immunity (1). Such rationally designed virus is replicative and induces protective immunity; however, the virus is quickly eliminated due to its enhanced sensitivity to the antiviral effect of the host innate immune response.RNA and DNA viruses that replicate in the cytoplasm cannot use the cellular nuclear capping machinery and thus have evolved viral methyltransferas...

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Section: Cells and Virusesmentioning

confidence: 99%

Rational Design of a Flavivirus Vaccine by Abolishing Viral RNA 2′- O Methylation

Dong

et al. 2013

J Virol

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“…Relative to a single antigen serological test, a multiplex (i.e., a multi-antigen) serological test can provide the following advantages: a) simultaneous interpretation of the magnitude of response to multiple viral antigens and vaccine components (8- 11), b) differential diagnosis of infection (12,13), c) robust and quantitative measurement of antibody response, and d) increase in sensitivity due to greater coverage of immunogenic epitopes (13)(14)(15)(16)(17)(18)(19). The increased specificity and sensitivity of printed multiplex ELISA are particularly valuable when the assay is used for patient populations and cohorts with varying severity of COVID-19 or time since infection, as mild and asymptomatic infections are associated with lower antibody titers compared to severe cases (20,21) and antibody response may differentially wane depending on the cognate antigen (22).…”

Section: Introductionmentioning

confidence: 99%

“…Multiplexed ELISA platforms employ a printed array of antigens (ELISA-array) (8,9,13,14,(24)(25)(26), microspheres coated with antigens or antibodies (bead-based ELISA) (27)(28)(29)(30), or a cocktail of antigens (cocktail ELISA) (19,(31)(32)(33). ELISA-array and bead-based ELISA platforms spatially separate the antigens on a surface, whereas conventional and cocktail ELISA coat the whole surface with the same antigen(s).…”

Section: Introductionmentioning

confidence: 99%

multiSero: open multiplex-ELISA platform for analyzing antibody responses to SARS-CoV-2 infection

Byrum

Waltari

Janson

et al. 2021

Preprint

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Serology has provided valuable diagnostic and epidemiological data on antibody responses to SARS-CoV-2 in diverse patient cohorts. Deployment of high content, multiplex serology platforms across the world, including in low and medium income countries, can accelerate longitudinal epidemiological surveys. Here we report multiSero, an open platform to enable multiplex serology with up to 48 antigens in a 96-well format. The platform consists of three components: ELISA-array of printed proteins, a commercial or home-built plate reader, and modular python software for automated analysis (pysero). We validate the platform by comparing antibody titers against the SARS-CoV-2 Spike, receptor binding domain (RBD), and nucleocapsid (N) in 114 sera from COVID-19 positive individuals and 87 pre-pandemic COVID-19 negative sera. We report data with both a commercial plate reader and an inexpensive, open plate reader (nautilus). Receiver operating characteristic (ROC) analysis of classification with single antigens shows that Spike and RBD classify positive and negative sera with the highest sensitivity at a given specificity. The platform distinguished positive sera from negative sera when the reactivity of the sera was equivalent to the binding of 1 ng mL-1 RBD-specific monoclonal antibody. We developed normalization and classification methods to pool antibody responses from multiple antigens and multiple experiments. Our results demonstrate a performant and accessible pipeline for multiplexed ELISA ready for multiple applications, including serosurveillance, identification of viral proteins that elicit antibody responses, differential diagnosis of circulating pathogens, and immune responses to vaccines.

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“…Various high throughput methods such as inkjet printing (Delaney et al, 2009) and roll-to-roll printing methods such as flexographic printing (Phillips et al, 2012), microcontact printing (Austin and Holway, 2011;Bernard et al, 1998Bernard et al, , 2000, capillary drop patterning (Delehanty, 2004) and spot and line deposition (Kang et al, 2012) have been utilized for biomolecule deposition on a range of surfaces, such as glass slides, nitrocellulose coated slides, plastics, paper and polymeric membranes. Different printing techniques that are of potential interest in biomolecule printing are illustrated in the Fig.…”

Section: Introductionmentioning

confidence: 99%

Printing technologies for biomolecule and cell-based applications

Ihalainen

Määttänen

Sandler

2015

International Journal of Pharmaceutics

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Development of an ELISA-array for simultaneous detection of five encephalitis viruses

Cited by 18 publications

References 19 publications

Rational Design of a Flavivirus Vaccine by Abolishing Viral RNA 2′- O Methylation

Rational Design of a Flavivirus Vaccine by Abolishing Viral RNA 2′- O Methylation

multiSero: open multiplex-ELISA platform for analyzing antibody responses to SARS-CoV-2 infection

Printing technologies for biomolecule and cell-based applications

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