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Use of spectrophotometer for ELISA


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3 replies to this topic

#1 gnarl

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Posted 21 November 2009 - 10:15 PM

So, I am doing an experiment that involves ELISA. (This is a science fair project, by the way) I am using a facility without a microplate reader, but they do have a uv-vis spectrophotometer. Thus, I plan to do the actual assay in the 96 well plate (because the high binding plate is necessary). Once I have added the stop solution and the wavelength is ready to be read, I will dilute the samples to 2ml, then transfer to the spectrophotometer cuvette to be assayed. My mentor has told me that they have done this before so it is fine, but I was wondering - have any of you seen this before? I have seen some very old journal articles (like 1980s) that have used spectrophotometers for ELISAs, but nothing recent. Also, I was wondering, should the samples be diluted with water or do I need some kind of buffer?
Thanks so much, anything you can help with would be great.


#2 prof. moriarty

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Posted 21 November 2009 - 11:22 PM

I have not actually seen this before. You are right, before the invention of the microplate reader scientists did use cuvettes for ELISAs.
But then the microplate reader was born, and things changed a bit - made it quite a bit faster as well.
The only thing I'd be concerned about if I were you is the transfer process, and how that might make your results inaccurate. Obviously you're familiar with the ELISA principle - since the antibodies that are at the core of the ELISA reaction are bound to the microwell plate, I'm not sure how everything would respond if you transferred the solution and just left the antibodies there.
Sorry I'm not much help, I've just very little experience with this. But, you say that your mentor has done this before, so you should be fine as long as your mentor has done it with quantitative ELISA. I assume that you are looking for quantitative results?
Best of luck with science fair, by the way. ELISA is quite advanced for a student, so I congratulate you. I hope others can help you.

Edited by prof. moriarty, 21 November 2009 - 11:24 PM.


#3 Gerard

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Posted 22 November 2009 - 03:50 AM

You can do the reading from the extinctions with a spectrophotometer and it will be okay, the only drawback is that the measured OD will be lower than with a microplate reader.
you will dilute ca 250 ul to 2000 ul, a 8 times dilution but the ligtpath will increase from ca 3mm to 10 mm thus the overall dilution will be about a factor 2.5.
This will influens your sensivity of the assay a little.

I would make the dilutions with the substate buffer but water also will work.

Edited by Gerard, 22 November 2009 - 03:53 AM.

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Pluralitas non est ponenda sine necessitate
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#4 mdfenko

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Posted 23 November 2009 - 11:52 AM

personally, if i had the option, i would dilute to 1 ml and use a semi-micro cuvette. this will keep the readings higher (if they are too high then you can dilute further).
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