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I am trying to perform a modified sandwich ELISA/FluoSpot assay.
Basically what I want to do is coat wells in 96 or 384 well plate with an antibody (a mouse IgE,k) against protein X.
Then I wish to grow cells expressing protein X in the well.
Then after they have expressed some protein X, I wish to add a second antibody (a mouse IgG1,k) against protein X.
And finally a fluorescently conjugated secondary antibody against the second antibody (an anti mouse IgG1).
Then I will look in a microscope and identify cells producing large quantities of protein X and using a special technique I will purify those single cells.
Everything will preferably happen in cell media, preferably with serum, but if that is not feasible PBS can be used for the actual detection part (second antibody, secondary antibody, purification).
I am considering your “Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Cy5 ®), pre-adsorbed (ab6563)”.
Is there any chance this would cross react with the first antibody (mouse IgE)?
Is there any reason to believe this antibody would not work in the experiment above?
Also, I have to use a specific vendor for plates. They only supply “plastic” bottom plates. In your ELISPOT protocol you mention PVDF or nitrocellulose bottom plates. What is the point of the PVDF/nitrocellulose bottoms? I know that protein G can adhere to The “plastic” bottom, so I would expect antibodies would adhere as well or have I misunderstood something?
Asked on Aug 10 2012
Thank you for your inquiry.
I used to perform many ELISPOTs by hand as you with plastic plates. The capture antibody was coated on the plates. The PVDF membrane bottom well plates areused when using a reader to analyze the results. The membranes are pushed through onto sticky tape and read in a reader with special software to identify spots.
I am not aware of a technique where the cells can be identified and sorted according to the production amount of protein X. I am assuming that protein X is secreted and therefore should be captured in the vicinity of the producing cell, but any slight movement of the plate will swirl the cells and make a identification impossible.
ab6563 is in general suitable as secondary to a mouse IgG1. But is is anti heavy and light chain. So it will pick up the light chain too. I suggest to choose an antibody specific to the heavy chain. I can recommend ab136127:
https://www.abcam.com/index.html?datasheet=136127 (or use the following: https://www.abcam.com/index.html?datasheet=136127).
This antibody is not specifically cross absorbed against IgE, but against Mouse IgM, IgG2a, IgG2b, IgG3 and IgA, pooled Human sera and purified Human paraproteins. ab136127 is tested in ELISA and therefore should also work in a ELISPOT setting as secondary.
I hope this information is helpful. Please do not hesitate to contact me again with any further questions in this matter.
Answered on Aug 10 2012