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sandwich ELISA without standard curve


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4 replies to this topic

#1 lucky2610

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Posted 15 March 2011 - 03:44 AM

HI,

I'm just a beginner in ELISA. I would like to question if it is possible to carry out classic sandwich ELISA assay without standard? I mean, I have antibody (capture and detection), but I don't have pure antigen for standard curve.

Concretely, I'm interested in the detection of staphylococcal enterotoxin G (extracellular protein, 28 kDa) with help of commercial mouse antibody.

Thanks for every suggestion!!!

#2 HBImolbiol

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Posted 15 March 2011 - 01:48 PM

HI,

I'm just a beginner in ELISA. I would like to question if it is possible to carry out classic sandwich ELISA assay without standard? I mean, I have antibody (capture and detection), but I don't have pure antigen for standard curve.

Concretely, I'm interested in the detection of staphylococcal enterotoxin G (extracellular protein, 28 kDa) with help of commercial mouse antibody.

Thanks for every suggestion!!!



It depends on the purpose of the ELISA. If the purpose in quantitation then yes you need a standard curve. If the purpose is ID no you do not need a standard curve per say but you DO need positive and negative controls of known quantity before you can conclude that you signals you are seeing are indeed due to specific recognition of the antigen. If you cannot purchase purified enterotoxin G then you will need to find your own source of standard. It doesn't necessarily need to be purified but you need another method to quantify how much is there (i.e. HPLC).

Edited by HBImolbiol, 15 March 2011 - 01:52 PM.


#3 lucky2610

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Posted 16 March 2011 - 01:40 AM

Hi HBImolbiol,

Thank you for your suggestion! It is really helpful.

Yes, I need quantification, therefore I also need a standard. However, I have never realized that I wouldn't need pure enterotoxin!

Do I understand it correct that I could for example only pre-isolate the standard (28 kDa extracellular protein) with help of easy gel permeation chromatography to get the fraction with my protein and then quantify the standard concentration on HPLC? (It is slightly out of topic, but it is possible to quantify concentration on HPLC without standard of known concentration...;o)

And then could I use this fraction as a standard for ELISA?

Or do you think that I only could prepare this way positive control?

Thank you again!

#4 HBImolbiol

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Posted 16 March 2011 - 08:33 AM

Hi HBImolbiol,

Thank you for your suggestion! It is really helpful.

Yes, I need quantification, therefore I also need a standard. However, I have never realized that I wouldn't need pure enterotoxin!

Do I understand it correct that I could for example only pre-isolate the standard (28 kDa extracellular protein) with help of easy gel permeation chromatography to get the fraction with my protein and then quantify the standard concentration on HPLC? (It is slightly out of topic, but it is possible to quantify concentration on HPLC without standard of known concentration...;o)

And then could I use this fraction as a standard for ELISA?

Or do you think that I only could prepare this way positive control?

Thank you again!


Analytical HPLC would use a conventional protein standard (i.e. BSA) and base the protein concentration on peptide bond absorbance (A214). You would just need to convince yourself that the peaks used for quantification on the HPLC trace of your sample actually correspond to enterotoxin and not other proteins, which will depend on the quality of the separation and availability of appropriate negative controls (ie same protein milieu without enterotoxin).

Edited by HBImolbiol, 16 March 2011 - 08:34 AM.


#5 lucky2610

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Posted 21 March 2011 - 12:53 PM

Hi,

thank you again:)

I have now one question slightly out of topic... Do you think, that there is a difference between using mouse and sheep antibody for the detection of staphylococcal extracelullar protein - in cultivating media? Concretely, I have protocol which is optimized for sheep antibody...will it work with mouse antibody?




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