There's a need for an accurate and high throughput method for measuring RSV neutralizing antibodies, as an important marker of protection against respiratory syncytial virus. This method is highly reproducible with inter-assay variations for the reference antiserum being less than 10%We believe this assay can be readily established in many laboratories worldwide at relatively low cost. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A, and can also be adapted for RSV subgroup B, and different sample types.
Demonstrating the procedure will be Reuben Tse, a member of the research team at MCRI. To seed plates on day one, first resuspend A549 cells in DMEM media, with 10%FCS and 1, 000 units Penicillin-Streptomycin mixture at a concentration of 400, 000 cells per milliliter. Then seed 96-well flat-bottom sterile plates with 40, 000 A459 cells at a density of 100 microliters per well.
Incubate the plate at 37 degrees Celsius in 5%carbon dioxide environment overnight. To prepare the serum dilution on day two, first thaw the human RSV reference serum and serum test samples at room temperature. Then place them in a water bath at 56 degrees Celsius for 30 minutes to heat and activate them.
Next, prepare more than 110 microliters of a one to 100 dilution of the reference serum by mixing 1.5 microliters of the reference serum with FCS-free DMEM media with Penicillin-Streptomycin mixture to a final volume of 150 microliters. Pipette 110 microliters of this dilution into well A12 of a 96-well U-bottom sterile plate. To make serial one to two dilutions down the plate, first add 55 microliters of FCS-free DMEM media with Penicillin-Streptomycin mixture to wells B12 to H12.
Then with a new tip transfer 55 microliters from well A12 to well B12. Pipette up and down five times to mix the solution, and continue until you reach well H12. Pipette off the excess 55 microliters of the solution from well H12.
Next, prepare a one to 100 dilution of serum test samples, and add 110 microliters of each dilution to corresponding wells A1 to A9 and E1 to E9 of the 96-well sterile plate. Then add 55 microliters of FCS-free DMEM media with Penicillin-Streptomycin mixture to wells in columns one through nine. Make serial one to two dilutions of row A to row D and from row E to row H as previously described.
Add 55 microliters of FCS-free DMEM media with Penicillin-Streptomycin mixture to wells in columns 10 and 11. To prepare the virus stock, first put a frozen vial of RSV in a 37 degree Celsius water bath to thaw it rapidly, then place the vial on ice. Next, add the virus to the DMEM media containing Penicillin-Streptomycin mixture at approximately 200 PFU per well to make a total volume of 55 milliliters for 100 wells.
To prepare the virus serum mixture, first add 55 microliters of the diluted virus to all wells of columns one through nine and wells of rows E through H of columns 10 and 11. Then add 55 microliters of FCS-free DMEM media with Penicillin-Streptomycin mixture to all wells of rows A through D of columns 10 and 11, and incubate the plate at 37 degrees Celsius in 5%carbon dioxide environment for one hour. Prior to the inoculation of A549 cells with RSV, examine the cell plate under a light microscope to confirm that A549 cell monolayers are 80%confluent.
Then invert the plate gently to discard the media, and lightly blot the plate on a sterile absorbent paper towel. After each, wash with filtered sterile PBS. Next add 100 microliters per well of the virus serum mixture to the corresponding wells on the A549 cell plate according to the plate template provided in the manuscript, and incubate the plate at 37 degrees Celsius in 5%carbon dioxide for one hour.
At the end of the incubation, use a pipette to remove 90 microliters per well, and add 100 microliters of the media containing 1XM199, 1.5%CMC, and 2%FCS in Penicillin-Streptomycin mixture. Incubate the plate at 37 degrees Celsius in 5%carbon dioxide for three days. To fix and develop an assay plate, first invert the RSV-infected A549 cell plate gently to discard the media containing M199, CMC, 2%FCS, and Penicillin-Streptomycin mixture.
Then slowly add 200 microliters of Fixation Buffer to each well. Incubate the plate at minus 20 degrees Celsius for 20 minutes. Then discard the fixation buffer and gently blot on an absorbent paper towel.
Leave the plate face down to dry for 10 minutes. Then with filtered PBS buffer containing 05 polysorbate 20, make 5%milk blocking solution. Add 200 microliters of the blocking solution to each well and incubate the plate at room temperature for 30 minutes.
At the end of incubation invert the plate gently to discard the solution and blot on an absorbent paper towel. To make the primary antibody, dilute one to 500 goat XRSV antibody in the blocking solution. Add 50 microliters to each well and incubate the plate at 37 degrees Celsius for one hour.
Wash the plate three times with filtered PBS-polysorbate. Next, make the secondary antibody by diluting one to 5, 000 Alexa Fluor donkey anti-goat immunoglobulin G in the blocking solution. Add 50 microliters to each well and incubate at 37 degrees Celsius for one hour.
Wash the plate five times with filtered PBS-polysorbate. To count plaques with an automated spots reader, first use an unused 96-well culture plate to collaborate the instrument by entering the count settings in the FITC channel. Then read the cell plate, wrap it in foil, and store it at four degrees Celsius.
Next, check the images of wells for artifactual plaques or disrupted cell monolayers, and exclude wells with disrupted cell monolayers. The mean number of plaques of the no-serum control wells was calculated and used to determine the 50%neutralization cut-off that would result in 50%inhibition of virus activity. The reciprocal serum dilution was plotted on the x-axis and the number of plaques on the y-axis of a semi-log graph from a line drawn between the two points.
The 50%neutralization titres of the test serum samples were red and serum dilutions with counts immediately above or below the 50%value of the no-serum control wells were identified. RSVAPRN assays in two different adult cohorts from Gambia and healthy volunteers in Melbourne resulted in the Gambian adults having a lower mean NAB titre compared to the Melbourne adults. The shown human RSV-reference serum demonstrates very low variability with the coefficient of variation of 6.82%This improved high throughput RSV plaque reduction neutralization assay can be easily implemented in many laboratories, and will support the international harmonization effort of WHO for RSV neutralizing antibody assays.
This will be critical for the evaluation of novel RSV vaccine candidates in the future.
