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ELISA of Sindbis virus from media


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#1 Andras

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Posted 16 March 2010 - 08:00 AM

Dear all,
I have been looking for a quick way to check for sindbis in my cell cultures (either for contamination, or for presence -obviously I want it where it should be, and do not want it in my samples).
RT-PCR works just fine, but the whole procedure with isolation, purification, RT, and PCR takes about two days -hardly a quick, routine test. (It'd be great if it worked with RNA directly...)
There's ways to titrate the virus, but then again, I need to grow cells on a plate first, and add the media to those -a two day protocol, if the incubation is included. Three, if the seeding of the plate is included.

I was thinking maybe the media simply could be used in ELISA to test for Sindbis antibody.

media added to the ELISA plate
media removed, plate washed
primary ab
secondary ab
colorimetric reaction of some sort
and I could even quantify the virus (well, sort of)

Does anyone know if it's possible like that? Since the sindbis ab is precious, I'd rather ask first, and try later...

Thank you very much for your help

#2 sgt4boston

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Posted 17 March 2010 - 02:32 AM

Unclear as to what you wish to detect. You described detecting the virus...then you mention detecting the antibody to the virus.

If you wish to detect the presence of the ab to the virus you would have to coat the plate with protein specfic to the surface (?) marker of the virus that the ab would recognize. then you could add the media (may or maynot have ab) allow binding, wash, and conjugated ab specific to the virus incubate wash, substrate. Or if you do not have conj specific ab add another ab to virus incubate , wash, 2ndary ab, incubate, wash, substrate.

Your procedure could work if there are no other proteins in the media which could block the plate and you have adsorption based on the media/buffer/ph. Have not heard of the procedure you are trying.

If you wish to detect the virus then it would be a different approach.

#3 Andras

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Posted 22 March 2010 - 03:05 AM

Unclear as to what you wish to detect. You described detecting the virus...then you mention detecting the antibody to the virus.

If you wish to detect the presence of the ab to the virus you would have to coat the plate with protein specfic to the surface (?) marker of the virus that the ab would recognize. then you could add the media (may or maynot have ab) allow binding, wash, and conjugated ab specific to the virus incubate wash, substrate. Or if you do not have conj specific ab add another ab to virus incubate , wash, 2ndary ab, incubate, wash, substrate.

Your procedure could work if there are no other proteins in the media which could block the plate and you have adsorption based on the media/buffer/ph. Have not heard of the procedure you are trying.

If you wish to detect the virus then it would be a different approach.


Yeah. Apologies; the "ELISA test for sindbis antibody" was somewhat misleading.
I'm interested in the presence of virus in the media using ELISA and sindbis antibody - these are BHK and MCF7 cultures, so no antibodies are expected to turn up by themselves.




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