An Assay to Measure the Neutralizing Antibody Titer Specific for Respiratory Syncytial Virus

Abstract

Source: Do, L. A. H., et al., An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay. J. Vis. Exp. (2019)

This video demonstrates an assay for measuring neutralizing antibodies against respiratory syncytial virus (RSV). Pre-incubation with serum containing RSV-neutralizing antibodies neutralizes the virus particles, reducing cellular infection and plaque formation. Virus-infected cells are then subjected to fixation, blocking, and fluorophore-conjugated antibody labeling for plaque visualization. This process enables the identification of the highest serum dilution that neutralizes fifty percent of RSV, representing the neutralization titer.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. NOTE: All steps must be performed in a BSL2 hood unless stated differently. Viral titration is required before a plaque-reduction neutralization (PRN) assay to determine the optimal RSV concentration used in the PRN assay. It is recommended to aliquot the virus stocks in a small volume that will be thawed once and used for each NAb assay. …

Representative Results

Figure 1: Viral titration plate layout for quantitation of RSV stocks. v = stock of RSV virus (v1, stock of viral batch 1; v2, stock of viral batch 2; v3, stock of viral batch 3), N = negative control well, X = media added. Color coding is only to aid in the visualization of plate layout. <img alt="Figure 2" src="/files/ftp_upload/…

Materials

Cell line
A549	ATCC	CCL-185	provided by Dr Keith Chappell, University of Queensland
Viral strains
RSV A2	ATCC	VR-1540	lot number 60430286
Reagents
Acetone	Merck	1000142511
Alexa-Fluor donkey anti-goat IgG (stored at 4 °C)	Life Technologies	A11055
CMC sodium salt powder	Sigma-Aldrich	C5678-500G
DMEM (no serum, 3.7 g/L NaHC, P/S) (stored at 4 °C)	Scientific Services – Tissue Culture		MCRI in house supply
Foetal calf Serum (stored in 50ml aliquots at -20 °C)	Interpath	SFBS-F
Goat X RSV antibody	Merck	AB1128
Human polyclonal antiserum to respiratory syncytial virus (RSV) (stored in 45 µL aliquots at -20 °C)	BEI Resources	NR-4022	Free order through BEI Resources upon registration. This serum belong to a panel of human antiserum and immune globulin to RSV (NR-32832)
M199 powder	Life Technologies	31100035
Milk diluent blocking solution (stored at 4 °C)	Australian Biosearch	50-82-01
Penicillin/Streptomycin (stored in 6mL aliquots at -20 °C)	Life Technologies	15140122
s.d.H2O from Milli-Q dispenser	Merck		In-house dispensation
Sterile 1X PBS for culture (stored at 4°C)	Scientific Services – Tissue Culture		MCRI in house supply
Tween 20 polysorbate	Sigma-Aldrich	9005-64-5
General Consumables
Conical Falcon tubes (50 mL)	Invitro Technologies	FAL352070
Filter unit 0.22um (500 mL)	Thermo Fisher	NAL5660020
Sterile Eppendorf tubes (1.5 mL)	Australia PL	AM12400
Sterile flat-bottom plates (96-well with lid)	Interpath	655180
Sterile U-bottom plates (96-well with lid)	Interpath	650180
5ml serological pipette	Sigma-Aldrich	CLS4487-200EA
10 mL serological pipette	Interpath	607180
25 mL serological pipette	Sigma-Aldrich	CLS4251-200EA
Tip Pipette 1-200 µL Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960	Fisher Biotec	TF-200-L-R-S
Tip Pipette 5-20 µL Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960	Fisher Biotec	TF-20-L-R-S
Tip Pipette 100-1000 µLClear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1000	Fisher Biotec	TF-1000-L-R-S
Tip Pipette 1-10 µL Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1001	Fisher Biotec	TXLF-10-L-R-S
Equipments and softwares
ELISpot reader system	AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany
AID ELISpot software version 5.0	AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany
Microsoft Excel 2007