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Streptavidin binding directly to antigen, what is going on


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#1 bdg25

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Posted 30 July 2014 - 09:44 AM

Hi All,

I am very confused by my results but have repeated them several times with very consistent results.  Essentially, I have a sandwich ELISA using biotinylated detection antibody. I coat the plate with capture antibody overnight (anti-HPV 18 E7). The next day I block with 3% BSA, then apply HeLa cell Lysate, then apply biotinylated detection antibody. Finally I apply streptavidin-HRP. As a negative control I have a set of wells with capture antibody, cell lysate, no detection antibody, and streptavidin-HRP. Without the detection antibody, I still get the same signal! It's specific too -- if I don't use cell lysate or use another type of cell lysate, there is no signal with just streptavidin-HRP. It is only present when there is antigen, but doesn't depend on the presence of detection antibody. How can this be??



#2 tkf

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Posted 30 July 2014 - 11:07 AM

May be there are naturally-biotinylated proteins in your cell lysate.



#3 bdg25

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Posted 30 July 2014 - 11:15 AM

Maybe, but it would have to be specifically my protein of interest, not any protein in the lysate because it is a sandwich ELISA, the lysate is not being directly adsorbed to the plate. I wouldn't be surprised to see some signal due to nonspecific capture, but there's an absorbance measurement of about 2 with or without detection antibody. It's not like a high background, it's a strong signal only present when antigen is present


Edited by bdg25, 30 July 2014 - 11:20 AM.


#4 tkf

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Posted 30 July 2014 - 11:23 AM

Repeat your ELISA experiment with the detection antibody followed by an HRP-conjugated antibody to your detection antibody. That should tell you if your protein of interest is biotinylated or not in the lysate.

 

Also, non specific proteins will stick to your plate (how much depends on the total protein concentration of your lysate) and will generate a strong signal strep-HRP if they are biotinylated.

 

Good Luck.


Edited by tkf, 30 July 2014 - 11:27 AM.


#5 bdg25

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Posted 30 July 2014 - 11:30 AM

Repeat your ELISA experiment with the detection antibody followed by an HRP-conjugated antibody to your detection antibody. That should tell you if your protein of interest is biotinylated or not in the lysate.

 

Also, non specific proteins will stick to your plate (how much depends on the total protein concentration of your lysate) and will generate a strong signal strep-HRP if they are biotinylated.

 

Good Luck.

 

Thanks, I appreciate it. Unfortunately my capture and detection antibody are from the same species.  Anti-mouse HRP will just bind the capture antibody.  I realize you'll get nonspecific proteins stuck to the plate, but using a control cell line of the same number of cells/mL I get very little signal, and I get the identical signal between having my lysate of interest + detection antibody and having lysate of interest without detection antibody. I agree it's possible this is 100% due to background activity, but based on my controls I find it really unlikely.  

 

I'll try with a direct format, no capture antibody, and use anti-mouse HRP to see what happens. thanks



#6 tkf

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Posted 30 July 2014 - 11:53 AM

Ah I see..






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