For virus extraction and inactivation validation purposes I need to find a method that will reduce cytoxicity observed on TCID50 assays while using AVL buffer. Anybody have any helpful suggestions? I thought perhaps of dilution, but im not sure if this will work?Unfortunately we cannot concentrate our virus stock enough to circumvent the 2 Log kill factor associated with the AVL buffer.
Dilution would be the simplest to try. You may have to dilute the AVL containing the possible still-active virus enough such that it is spread across multiple wells or even 10 cm dish(es) of host cells. You will need controls with a known low TCID50 inoculum spread across the same number of wells to make sure you see the expected CPE (if your virus produces CPE). Also, I would include a similar control in which equivalently diluted AVL is added to the cells after adsorption/penetration of control low TCID50's, with the AVL left on the cells for the same length of time as in your infectivity assay, in order to control for buffer effects that inhibit virus replication after infection (induced stress pathways, etc.) Regarding the latter, I've had conditions in my experiments where the virus was spiked in and the cell monolayers looked good over the 5 day course of the assay (no overt cytotoxicity) but inhibitors in the viral inoculum during the adsorption period precluded future CPE formation. Further dilution of the inoculum and/or limiting the exposure time of the AVL containing inoculum to the cells -/+ washing away the AVL after adsorption may help.
Or, you could dialyze or microconcentrate/buffer substitute your AVL flow-thru such that the virus is maintained but the AVL is replaced with tissue culture media. Not knowing which virus you are working on, the virion may or may not be amenable to exposure to dialysis membranes or microconcentrator filters without inactivation. Controls will be key for all of this. If detergent in the AVL produces micelles, they may be retained and not pass through the dialysis or filtration membrane.
Good luck.
Dilution would be the simplest to try. You may have to dilute the AVL containing the possible still-active virus enough such that it is spread across multiple wells or even 10 cm dish(es) of host cells. You will need controls with a known low TCID50 inoculum spread across the same number of wells to make sure you see the expected CPE (if your virus produces CPE). Also, I would include a similar control in which equivalently diluted AVL is added to the cells after adsorption/penetration of control low TCID50's, with the AVL left on the cells for the same length of time as in your infectivity assay, in order to control for buffer effects that inhibit virus replication after infection (induced stress pathways, etc.) Regarding the latter, I've had conditions in my experiments where the virus was spiked in and the cell monolayers looked good over the 5 day course of the assay (no overt cytotoxicity) but inhibitors in the viral inoculum during the adsorption period precluded future CPE formation. Further dilution of the inoculum and/or limiting the exposure time of the AVL containing inoculum to the cells -/+ washing away the AVL after adsorption may help.
Or, you could dialyze or microconcentrate/buffer substitute your AVL flow-thru such that the virus is maintained but the AVL is replaced with tissue culture media. Not knowing which virus you are working on, the virion may or may not be amenable to exposure to dialysis membranes or microconcentrator filters without inactivation. Controls will be key for all of this. If detergent in the AVL produces micelles, they may be retained and not pass through the dialysis or filtration membrane.
Good luck.
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