A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum
Published: January 31, 2024
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 Prepare Madine Darby Canine Kidney-α2,6-sial transferase (MDCK-SIAT1) cells and sterile cell culture medium Prepare the MDCK-SIAT1 cell culture medium using 500 mL of Dulb…
Representative 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 assays. <img alt="Figure 2" src="/files/ftp_upload/21908/21908_Figure2.j…
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
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A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum. J. Vis. Exp. (Pending Publication), e21908, doi: (2024).