An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Published: April 30, 2024

Abstract

Source: Gao, J., et al. Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay. J. Vis. Exp. (2016).

This video demonstrates an assay measuring influenza neuraminidase (NA) inhibiting antibody titers in a serum sample. A serum sample containing antibodies against NA is added to a fetuin-coated microwell plate. Upon adding the virus, the antibodies prevent viral NA from cleaving fetuin and exposing its galactose moiety. A peroxidase-conjugated lectin is then added, which binds to the exposed galactose. The NA-inhibiting antibody titer is determined using a colorimetric test, where a peroxidase substrate gets cleaved by the immobilized lectin-conjugated peroxidase to produce color.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Preparation of Reagents and Starting Material Note: Refer to the Materials Table to obtain the source of all reagents. Fetuin-coated Plates Prepare the coating buffer by mixing 10 ml of the 10x coating buffer with 90 ml of deionized H2O. Prepare a stoc…

Representative Results

Table 1. Root cause analysis of assays that do not meet performance criteria. This table provides possible causes and solutions for problems that may occur when performing the ELLA. Problem Possible cause(s) Solution Weak signal or no plateau reached in virus titration i) low …

Declarações

The authors have nothing to disclose.

Materials

Coating buffer KPL 50-84-01
Fetuin Sigma F3385
5X MES, pH 6.5 KD-Medical PBS-0134
CaCl2 Sigma C7902
30% BSA Sigma A8327
Tween 20 Sigma P1379
Lectin PNA-HRPO Sigma L7759
PBS-T Sigma P3563-10PAK
o-Phenylenediamine dihydrochloride Sigma P8287
Phosphate-citrate buffer Sigma P4922
Sulfuric acid Sigma 258105
96-well Maxisorp plates NUNC 439454
96-well round bottom well plates NUNC 267245
Plate sealers Thermo Scientific 14-245-192B
Chicken eggs Charles River Laboratories 9 day old embryonated specific pathogen free (SPF) chicken eggs
Multichannel pipette Variety of suppliers e.g., Rainin, Eppendorf 8 or 12 channel manual pipettem 50-250 µl volume
Pipette tips Depends on pipettor brand Depends on pipettor brand
Plate reader, with 490 nm filter Perkin Elmer Victor V
Water bath set to 37 °C Variety of suppliers Variety of models
Water bath set to 56 °C Variety of suppliers Variety of models
Refrigerator set to 4 °C Variety of suppliers Variety of models
Incubator set to 37 °C Variety of suppliers Variety of models

Tags

Play Video

Citar este artigo
An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers. J. Vis. Exp. (Pending Publication), e22105, doi: (2024).

View Video