An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

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 …

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