Name: A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum
Uploaded: 2024-01-31T13:36:00
Description: All influenza viruses should be handled according to appropriate biosafety level requirements (BSL-2 or higher) as defined in the Biosafety on ...

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a microneutralization assay to measure neutralizing antibody titers in human serum

Source: Gross, F. L. et al., <a href="https://app.jove.com/v/56448">Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates Microneutralization assays to measure neutralizing antibodies using Madin-Darby Canine Kidney cells overexpressing &#945;2,6-linked sialic acid in human sera. This assay is often used for influenza human serology

<img alt="Figure 1" src="/files/ftp_upload/21908/21908_Figure1.jpg" /> 
Figure 1. MDCK-SIAT1 cell culture.&#160;MDCK-SIAT1 cells. (A) MDCK-SIAT1 confluent monolayer (&#62;100% confluency) for virus inoculation; (B) MDCK-SIAT1 cells at log phase with 75 - 95% confluency for TCID50&#160;and MN assays.
<img alt="Figure 2" src="/files/ftp_upload/21908/21908_Figure2.j...

A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

All influenza viruses should be handled according to appropriate biosafety level requirements (BSL-2 or higher) as defined in the Biosafety on ...

For a microneutralization assay, take a multi-well plate containing heat-inactivated, serially diluted human serum with decreasing concentrations of influenza-specific neutralizing antibodies.
Introduce pathogenic influenza viruses. Serum antibodies interact with the viruses and neutralize them; however, higher serum dilutions exhibit limited virus neutralization.
Seed with Madine-Darby Canine Kidney cells, or MDCK cells, overexpressing human sialic acid residues on their surface.
In the higher dilution, non-neutralized influenza viruses fuse with the cells&#39; sialic acid, releasing the viral genome into the cell cytoplasm.
The viral genes are transcribed and translated to viral proteins and nucleoproteins within the host cell.
Treat with a high concentration of acetone for fixation and permeabilization.
Introduce primary antibodies that interact with viral nucleoproteins.
Overlay with enzyme-conjugated secondary antibodies that interact with primary antibodies. Wash and treat with the enzyme&#39;s substrate, producing a color product.
Quantify the color intensities and plot them against the serum dilution; the highest serum dilution, achieving fifty percent virus neutralization represents the neutralization titer.

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163 Views. Source: Gross, F. L. et al., Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells. J. Vis. Exp. (2017) This video demonstrates Microneutralization assays to measure neutralizing antibodies using Madin-Darby Canine Kidney cells overexpressing α2,6-linked sialic acid in human sera. This assay is often used for influenza human serology 

Video: A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum - Concept

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

JoVE Journal

Immunology and Infection

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. This method is highly reproducible, with inter-assay variations for the reference antiserum being less than 10%. We believe this assay can be readily established in many laboratories worldwide at relatively low cost. Development of an improved, high-throughput assay that measures RSV NAbs represents a significant step forward for the standardization of this method internationally as well as being critical for the evaluation of novel RSV vaccine candidates in the future.

An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay ...

Medicine

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

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