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

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 as...

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Main Authors: Gao, Jin, Couzens, Laura, Eichelberger, Maryna C.
Format: Online
Language:English
Published: MyJove Corporation 2016
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091984/
id pubmed-5091984
recordtype oai_dc
spelling pubmed-50919842016-11-15 Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay Gao, Jin Couzens, Laura Eichelberger, Maryna C. Immunology 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 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥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. MyJove Corporation 2016-09-06 /pmc/articles/PMC5091984/ /pubmed/27684188 http://dx.doi.org/10.3791/54573 Text en Copyright © 2016, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Gao, Jin
Couzens, Laura
Eichelberger, Maryna C.
spellingShingle Gao, Jin
Couzens, Laura
Eichelberger, Maryna C.
Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay
author_facet Gao, Jin
Couzens, Laura
Eichelberger, Maryna C.
author_sort Gao, Jin
title Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay
title_short Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay
title_full Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay
title_fullStr Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay
title_full_unstemmed Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay
title_sort measuring influenza neuraminidase inhibition antibody titers by enzyme-linked lectin assay
description 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 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥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.
publisher MyJove Corporation
publishDate 2016
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091984/
_version_ 1613709325712228352

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