Use of spectrophotometer for ELISA - no access to microplate reader - will this work? (Nov/21/2009 )
Thanks so much, anything you can help with would be great.
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.
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.
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).