A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

Abstract

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

Protocol

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

1. Preparation of Reagents and Starting Material

1. Prepare Madine Darby Canine Kidney-α2,6-sial transferase (MDCK-SIAT1) cells and sterile cell culture medium
   1. Prepare the MDCK-SIAT1 cell culture medium using 500 mL of Dulbecco's Modified Eagle Medium (DMEM) with high glucose, 10% v/v heat-inactivated fetal bovine serum (FBS), 2 mM L- glutamine, 1 mM sodium pyruvate, 1 mg/mL G418 sulfate (<....

Results

Figure 1. MDCK-SIAT1 cell culture. MDCK-SIAT1 cells. (A) MDCK-SIAT1 confluent monolayer (>100% confluency) for virus inoculation; (B) MDCK-SIAT1 cells at log phase with 75 - 95% confluency for TCID₅₀ and MN as.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM) with high Glucose	Life Science	11965	A critical component of Sterile Cell Culture, Virus Propagation and Virus Diluent Media
Fetal bovine serum (FBS)	Hyclone	SH30070.03
Bovine Serum Albumin (BSA) Fraction V, Protase Free	Sigma-Aldrich	3117332001
L-Glutamine	Life Science	25030-081
Sodium pyruvate	Life Science	11360-070
Geneticin G-418 disulfate salt	Sigma-Aldrich	A1720-5G
HEPES	Life Science	15630-080
Penicillin/Streptomycin	Life Science	15140-122
Acetone	VWR	67-64-1	Used at an 80% concentration
Phosphate-Citrate Buffer with Sodium Perborate	Sigma-Aldrich	SLBF2806V
O-Phenylenediamine Dihydrochloride tablet	Sigma-Aldrich	SLBQ1086V	1 tablet per 100ml of cell culture grade water
Sulfuric Acid	Fisher Scientific	A510-P500	Used 0.5M final concentration
Ethanol, Denatured, 4L	VWR	EM-AX0422-3	Used at an 70% concentration
Trypsin-EDTA	Life Science	1748048
RDE II "Seiken"	Denka Seiken	370013
Tween 20	Sigma-Aldrich	P1379-500ml
Anti-NP mouse monoclonal Ab	Millipore pool	MAB 8257 MAB 8258
Anti-mouse IgG HRP	KPL	074-1802
96-well flat-bottom plates	Thermo Scientific	3455
Plate reader	Molecular Device	Spectromax 384 plus
Cell Culture Flask 162 cm2/Vent Cap	Corning/VWR	3151