Optimization of a Quantitative Micro-neutralization Assay

The overall goal of this micro-neutralization assay is to characterize all types of current circulating influenza viruses and the activity of antibodies and antiviral measurements in a quantitative, high-throughput, and highly sensitive manner. This method can help answer key questions in the virology field, such as antigenic characterization of influenza viruses, quantitative neutralization of antibodies, and antiviral activities. The main advantage of this technique is that it characterized the entire infected cell population, including visible and invisible plaques, and single cell infections.

The micro-neutralization assay will be demonstrated by Dr.Yipu Lin, assistant director of the WHO Collaborating Center for Influenza in London. The biosafety level, or BSL, for the following protocol is BSL 2 for seasonal influenza viruses and BSL 3+for potential pandemic influenza viruses. To begin, aliquot sufficient cells into 96-well plates.

Incubate the cells at 37 degrees Celsius and 5%CO2 for two to three days to reach confluence. Using 70%ethanol, sterilize a multi-well plate washer, and use PBS or VGM to rinse it. Then, use 200 microliters of virus growth medium, or VGM, to wash the confluent cells.

Aspirate the VGM from the cells, and immediately add 50 microliters of VGM to each well. Next, add 900 microliters of VGM to each of six sterile tubes. Then, pipette 100 microliters of virus into the first tube and mix well.

Prepare serial dilutions of the virus starting with the first tube, changing the tips between each dilution. Add 50 microliters of each virus dilution starting with the highest dilution to duplicate wells of a 96-well plate. Then, place the plate at 37 degrees Celsius for two to three hours to allow the virus to infect the cells.

After preparing the overlay according to the text protocol, remove the inoculum from the wells. Then, add 200 microliters of overlay to each well, and incubate at 37 degrees Celsius overnight undisturbed. Add 200 microliters of ice cold 4%PFA in PBS A to the wells, and incubate at four degrees Celsius for 30 minutes or room temperature for 20 minutes.

Following the incubation, aspirate the PFA, and use 200 microliters per well of PBS A to wash the plate twice. Add 100 microliters of permeabilization buffer to the plate, and incubate at room temperature for 30 minutes. Then, use PBS A to wash the plate twice.

Next, add 50 microliters per well of a mouse monoclonal antibody against influenza type A to the wells, and incubate at room temperature with shaking for one hour. Following the incubation, use 300 microliters of wash buffer to wash the well three times. Then, add 50 microliters of an HRP conjugated goat secondary antibody to the samples, and incubate at room temperature for one hour.

After washing the wells three times as before, add 50 microliters of substrate per well, and incubate at room temperature for 30 minutes or until the development of a blue color is clearly visible. To stop the reaction, use 200 microliters of distilled water to wash the wells twice. Then, after air drying the plate, store it in a dark place.

Place a well plate in the scanning area of a flatbed scanner, using the L-shaped position limit to ensure an optimum and repeatable imaging location can be obtained. Scan the plate using the settings shown here. Then, run the well plate reader software to calculate the required virus concentration by clicking the Load Image button to load an image.

Next, in the global threshold tab, slide the red bar in the histogram to adjust the sampling threshold. Then, click the update button to examine the effect on an image. Now, tick the Calculate Neutralization/Titration box.

Click the Sampling button to quantify the infected cell population, or ICP. Then when prompted, click the Save button to save the sampling results. To obtain the titration results, load or input the well plate definition map.

In Titration:Optimal Virus Population indicate the threshold. Select the Titration Process tab to calculate the titration results. Then, check the titration results before clicking the Save and Close button to save the results.

Refer to the supplement S1 for more detailed instruction of the software. To carry out virus neutralization, prepare the cell monolayer two or three days in advance. Then, add 50 microliters of VGM to each well of the plate.

Use columns 11 and 12 for the virus control and cell control respectively. Add 50-microliter aliquots of a one in 20 dilution of receptor destroying enzyme, or RDE, treated serum to the first row of columns one to 10. Perform two-fold serial dilutions by transferring 50 microliters from row A to row H and discarding 50 microliters from row H.Then, add 50 microliters of diluent to each well of the cell control column.

Add 50 microliters of the virus to each well of the plate, except for the cell control column. Incubate the plate at 37 degrees Celsius for two to three hours. Then, remove the inoculum, and add the overlay to each well.

Incubate the plate overnight at 37 degrees Celsius undisturbed. Then, fix and stain the plates as demonstrated earlier in this video. To quantify neutralization, place a well plate in the scanning area of a flatbed scanner as demonstrated earlier in this video.

Scan the plate and run the well plate reader software to calculate the required viral titres as before. After loading or inputting the well plate map, in Neutralization:Infection Deduction indicate the threshold. Then, select Neutralization Process to calculate the titres.

Finally, check the titres, and click the Save and Close button to save the neutralization results. Shown here are titration results from recent antigenic characterization experiments of eight input viruses with dilutions between 1.0 times 10 to the negative one and 1.0 times 10 to the negative six. The graph illustrates that ICPs decrease with an increase in virus dilution.

The curves were normalized against the ICPs of the same viruses that yielded infections in all cells within a well. Listed in this table are the virus dilutions that produced 30%of ICP saturation that were chosen as the input virus dilution for neutralization. This figure shows the results from a neutralization experiment using one input virus against five antisera.

The graph illustrates the infection process with the increase in serum dilution. The normalized positive population on the vertical axis represents the ratio of ICPs from the corresponding antisera response against the average ICP of the reference virus. Finally, the neutralization titres were determined as the reciprocals of the antiserum dilutions corresponding to 50%ICP reduction.

This assay focuses on consistency, speed, and detection sensitivity, including quantitated titration of input viruses, high-throughput imaging, and data processing. It is cost-effective, user friendly, and has been routinely used in viral antigenic studies.