Hi everyone,
I am currently trying to work out inactivation procedures for influenza virus. I am interested in using heat treatment for cell culture supernatants, but I'm concerned that heating a sample used for ELISA may negatively impact how well the antibodies will be able to recognize potentially denatured proteins. It's been shown previously that heating samples containing influenza at 70C for 30 minutes will completely inactivate the virus, but do you all think that heating at the temperature for such a lengthy period of time might potentially make the samples useless for ELISA, etc?
I recognize that this is far from an ideal situation, but have any of you ever heated supernatants before using them for ELISA, etc?
Thanks for any input!!
heat inactivation could denature your proteins and affect binding by ELISA. However, it depends on what you are testing for in your ELISA. As for antibodies, we inactivate our serum samples for 30min @56C which works well and does not affect antibody binding in our ELISAs. Several groups have looked at the effect of heat inactivation on influenza viruses. Zou et al. Virol. J. 10:289 (2013), have shown that heat treating avian influenza viruses (H7N9) @56C for 30min or UV irradiation for 30min resulted in complete loss of infectivity. You can treat your supernatant with heat or UV and then look for infectious virus to make sure that you have inactivated the virus prior to running in your ELISA. As a control, you can spike a positive sample in culture supernatant, treat with various heat or UV conditions, and compare to untreated to see if this effects your ELISA.
Hi Alex,
I agree that heat inactivation would denature your proteins (the point of heat inactivation), which may then make them difficult to test by ELISA. The ELISA antibody pair you use would be the finally judge of that, but Abs that bind to denatured proteins are typically different than ones that bind well to the native proteins.
Have you tried inactivating your influenza virus using UV? That works wonderfully, and leaves the proteins in your solution intact for ELISA. This is often done in virus studies. UV cross-links the virus genomic RNA and renders it useless, but doesn't affect proteins. I'm happy to send a protocol about how to do this.
heat inactivation could denature your proteins and affect binding by ELISA. However, it depends on what you are testing for in your ELISA. As for antibodies, we inactivate our serum samples for 30min @56C which works well and does not affect antibody binding in our ELISAs. Several groups have looked at the effect of heat inactivation on influenza viruses. Zou et al. Virol. J. 10:289 (2013), have shown that heat treating avian influenza viruses (H7N9) @56C for 30min or UV irradiation for 30min resulted in complete loss of infectivity. You can treat your supernatant with heat or UV and then look for infectious virus to make sure that you have inactivated the virus prior to running in your ELISA. As a control, you can spike a positive sample in culture supernatant, treat with various heat or UV conditions, and compare to untreated to see if this effects your ELISA.
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