[lab2015]

I recently performed a competitive ELISA for the first time to detect a protein in urine samples. The final colour has a reverse relationship to the concentration of the protein. In the protocol, they didn't say what blank should be used for the plate reading. There is 6 standards [standard 0 (concentration 0 ng/ml), standards 1-5] and 2 controls. I used standard 0 as a blank. All the absorbance readings were negative. I would appreciate any advice about how can I know what blank should I use? I used a 4 parametric logistic curve with the readings I got, as requested in the kit, and the results for my samples were in the expected range but can I use these results with the fact that all absorbance readings were negative?
Please help.
Regards,

Carol

[Yggdrasil]

The appropriate blank would probably contain all of the materials except for one of the components responsible for generating the color.  Without knowing more of the details of the method, it's hard to suggest the appropriate blank.  One solution would just be to blank the spectrophotometer with water or buffer, take the measurements, and figure out the correct background subtraction afterwards.

[Arkcon]

It might help if we saw your data. If I understand correctly, the inverse relationship means that the blank gives the most "signal" in whatever plate reader.  And then less and less through higher standards.  It seems like you can still use the blank, but even with the inverse response, you still get a line, you still determine a function, and you can still determine an unknown.

[lab2015]

The appropriate blank would probably contain all of the materials except for one of the components responsible for generating the color.  Without knowing more of the details of the method, it's hard to suggest the appropriate blank.  One solution would just be to blank the spectrophotometer with water or buffer, take the measurements, and figure out the correct background subtraction afterwards.

Thank you so much for your reply. how can I blank the spectrophotometer with water or buffer? should I leave the blank wells empty and add water or buffer just before reading the plate?

C

[Yggdrasil]

The appropriate blank would probably contain all of the materials except for one of the components responsible for generating the color.  Without knowing more of the details of the method, it's hard to suggest the appropriate blank.  One solution would just be to blank the spectrophotometer with water or buffer, take the measurements, and figure out the correct background subtraction afterwards.

Thank you so much for your reply. how can I blank the spectrophotometer with water or buffer? should I leave the blank wells empty and add water or buffer just before reading the plate?

C

It's hard to say without knowing more details about the experimental procedure.  Adding water or buffer to the wells just before performing the experiment could work.

[Babcock_Hall]

I find it helpful to distinguish setting the spectrophotometer reference from making a blank, although I am not sure how universal the terminology is.  I use the word blanks to describe a type of control experiment in which something is left out (often the macromolecule in a biochemical experiment), but they need not have zero absorbance values.  The substance used to set the reference of a spectrometer is usually defined to have an absorbance of zero.

[Yggdrasil]

I've generally heard the term blank used to describe the spetrophotometric reference and never an experimental control.  I'd generally refer to the control experiments as "negative controls," "no enzyme controls," or "no substrate controls."
 

[lab2015]

I am really confused about the competitive ELISA assay because I need a zero blank to read the plate against, which should have no colour, but how can I do this? can I skip adding the conjugate? would any one who did competitive ELISA advice me about this please? 

*MOD Edit: remove own quote* 

[Babcock_Hall]

Application of experimental design techniques to optimize a competitive ELISA
Journal of Immunological Methods
Volume 190, Issue 2, 19 April 1996, Pages 151–161
G. Sitta Sittampalam, Wendell C. Smith, Thomas W. Miyakawa, David R. Smithc Clyde McMorris,
doi:10.1016/0022-1759(95)00262-6

I have not yet read this article, but this one or one like it may be of help.

[lab2015]

Thank you all for your replys.
It appeared that I shouldn't use any blank!

[Babcock_Hall]

I have not done this assay myself, but hat sounds very odd.  What makes you say that?