Calculating infectious viral titers is a basic experimental approach. However, the classical method of using plaque assays is not the most reliable for viruses that did not have cytopathic effects, such as for human coronaviruses. In this video, we demonstrate an alternative method for detection and titering a virus using an enzymatic antigen detection technique known as an immunoperoxidase assay.
Here we will show you how to collect your viral samples, prepare the cells for testing, and finally, the immuno peroxidase assay using serial dilution to determine the viral titer. Hi, my name is Dr.Pierre Albo, director of Laboratory of Neuroimmunology at the AM RIP Institute in Laval, Quebec, Canada. Today we will demonstrate how to quantify human color viruses strains OT 43 and two nine E, which are prototype strains of these viruses which don't produce plaques in culture.
So we have developed an assay to go around this problem. This called Immunoperoxidase assay. To demonstrate this protocol, I will introduce you, my student, Gabrielle Marol, my technician Francis Nader, and my research associate Ellan Jacob.
So let's get started. First, we want to prepare our tissue samples. We start by pre weighing the sterile tubes we will use to collect each organ.
Once we've determined the weight of each empty tube, we'll quickly dissect the organs of interest in sterile conditions, then keep them on ice. It's important to remember to rinse the instruments with 70%ethanol between each dissection. After we've collected the organs, we determine the weight of each biological sample.
Next, we will homogenize the tissue samples with 10%sterile PBS with a poly tron homogenizer in a laminar flow hood. Turn the homogenizer on and then raise and lower the tube to disrupt the tissue. After homogenizing each sample, wash the apparatus once with sterile water, once with 70%ethanol, and finally with PBS before processing the next tissue sample.
After the samples have been homogenized, centrifuge the tubes at four degrees Celsius for 20 minutes. At 1000 Gs collect the supernatants in new sterile tubes. At this point, viral samples can be processed for immunoperoxidase detection.
Otherwise, you can immediately freeze them at minus 80 degrees Celsius and store them until you're ready for assays. Next, we wanna prepare the cell samples. There are different methods for preparing adherent and non-adherent cells, but both involve collecting intracellular and extracellular viral titers.
In order to determine extracellular viral production in adherent cells, we collect supernatants and sterile tubes from the infected cells, which are to be tested at appropriate times following infection. For estimation of intracellular viral titers, we want to first remove the medium, wash the cells in warm and sterile PBS, and then add the same volume or a known volume of medium, and perform three cycles of free thaw at minus 80 degrees Celsius. This will lice the cells and release viral particles into the medium.
For non-adherence cells, we first centrifuge the cells at a thousand Gs for five minutes. After pelleting the cells, we collect the supernatants in sterile tubes. This represents the extracellular portion of infectious virus.
Next, we suspend the cell pellet in the same volume as the collected supernatant or in a known volume of culture medium. Now we perform three cycles of free thaw at minus 80 degrees Celsius to ly cells and release viral particles in the medium. This represents the intracellular portion of infectious virus.
After lysis of either adherent or non-adherent cells, centrifuge them for five minutes at a thousand gs. Now the supernatant can be collected in new sterile tubes and titers can be analyzed with the immunoperoxidase assay. Again, the sample can also be immediately frozen and minus 80 degrees C and stored until assay.
In order to test the productivity of our human coronavirus infection in our biological samples, we want to use an immuno peroxidase assay on coronavirus susceptible cells First trips and treatment is used to create a suspension of susceptible cells from a confluent monolayer. When working with human coronavirus, 2 29 EL 1 32 cells are used as susceptible cells and for human coronavirus OC 43, the HRT 18 cell line is used after generating a suspension of susceptible cells. They're plated in a 96 well plate for tissue culture.
If using a multi-channel pipette dispense a hundred microliters per well of cell suspension using the appropriate concentration of cells. The number of susceptible cells per well will depend on the type of cells used and when the inoculation of cell will take place. After plating, please see our Springer protocol for more detailed information.
Before beginning the immuno peroxidase assay susceptible cells need to be infected with the samples that we've prepared earlier. When the susceptible cells have reached 70 to 80%con fluency, remove the medium from each 96 well plate by wrapping the plate in sterile tissue paper and gently flicking it face down on another sheet of sterile paper. Next, add into each well a hundred microliters of alpha MEM supplemented with 1%FBS.
Each sample should be tested in four adjacent columns. In the same plate, put a hundred microliters per well of aliquots that are to be tested in the first row. Typically susceptible cells are inoculated with serial logarithmic dilution of the infected samples.
To do this, add 11 microliters per well of the same sample again in four wells. Now in the second row of the 96 well plate then mix with the multi-channel pipette by pipetting up and down three times and transfer 11 microliters of each well in the third row and so forth until the last row of the plate. Once you've inoculated the cells, incubate the plates in a humidified chamber at 33 degrees Celsius with 5%CO2 for human coronavirus.
2290 infected cells are incubated for five days and for human coronavirus. OC 43 infected cells are incubated for four. After which time we can proceed to virus detection.
After incubation, we are ready to detect the virus. We first start by removing the medium completely. We do this by flicking plates onto non-sterile paper towels in the laminar flow hood.
We then delicately rinse the cells by gently filling wells with PBS. Using a multi-channel pipette. Flick the PBS out of the wells and remove any residual PBS by wrapping each plate in paper tissue.
After the cells have been washed, you will want to fix them with a hundred percent methanol containing 0.3%hydrogen peroxide. Add the methanol mix to the cells and let the plate sit for 15 to 30 minutes At room temperature following the 30 minute incubation, the fixative is removed by flicking plates over the sink. Residual fixative is removed by flicking it face down onto several paper towels that have been laid on the benchtop.
Then let it totally air dry, face up for approximately 15 to 30 minutes while the cells are drying. You can prepare an antibody solution specific to the virus at the appropriate dilution in PBS. Next, you will add a hundred microliters of the specific viral antibody to each well in incubate plates for two hours at 37 degrees Celsius.
After incubation, completely remove the medium by flicking it over the sink. Rinse the plate by filling the wells with PBS. Then flick the plate into the sink and rinse with PBS.
Two more times again. Remove all residual PBS by flicking it face down onto non-sterile paper towels laying on the benchtop. Next, add a hundred microliters to each well of secondary antibody at one to 1000.
Dilution in PBS, incubate the plate for two hours at 37 degrees without CO2 30 minutes before the end of the incubation. Prepare the detection reagent, which is a mixture of DAB with PBS and 0.01%hydrogen peroxide. Rinse the plate three times in PBS as done previously.
After the plate has been washed, add a hundred microliters of developing reagent to each. Well incubate for 10 to 20 minutes at room temperature or until the positive control is stained. In order to stop the reaction, wash the plate once with water as previously done with PBS and fill each well with a hundred microliters deionized water.
Finally read the plates with the light microscope to quantify all wells presenting stained cells. Now at long last, we can determine the titer of our virus. This could be calculated using the car method, the equation for which you can find in the Springer protocol that accompanies this video.
We've just shown you how to titrate human coronaviruses when doing this procedure. It's important to remember that you are working with samples containing both viruses and that you need to work in sterile conditions. So that's it.
Thanks for watching and good luck with your experiments.
