It looks like you still don't have a good assay to test your protein. Currently you're putting your hopes on the mouse polyclonal antibody made by your colleague. Jumping from a not-very-good-assay into another one, won't give you much information. If I were you, I would try to look for expression of the 37 KDa protein in a Western Blot using the mouse polyclonal antibody. If this works, then your ELISA would probably work. But it's best to use a standard structural technique like immunoblot to gain information instead of a newly developed technique like ELISA and doesn't give you structural information. Use a gradient gel, say 4-20% acrylamide.

To improve the probability of detection, I would use Golgi Plug or Brefeldin A to block the secretion of the protein and make it concentrate inside the secretory pathway (ER, Golgi, endosomes, etc). A few hours is enough (3-4 hrs), take a whole lysate (easily done with cell culture, apply 40 uL of 2xLaemmli buffer to a monolayer (2cm2; one well of a 24 well plate) and shear DNA with an insulin syringe). Load into SDS-PAGE and detect with mouse polycloncal antibody.

Once you detect your protein with Western blot, then you're in a position to study possible stimulation of expression with ligands or drugs. Good luck.

You could also try Dot Blot with your supernatant. We did that successfully with a 30kDa secreted protein. Just spot your supernatant on a Nitrocellulose or PVDF Membrane and let it dry, block, incubate with your antibody, secondary antibody and detection via HRP. The method is supereasy and straight forward

I agree with Benjamin Jurek, dot blots are more sensitive than ELISAs (an ELISA plate binds only a few % of the sample protein, a PVDF membrane 100%), linear over 5 orders of magnitude (with fluorescence or chemiluminescence detection), cheaper (membrane instead of plates) and quicker too.

However, I always use PVDF, which in experiments with 125I-labelled Hsc70 bound reproducibly nearly all protein, while NC bound only about 60% with considerable variability. Also, NC has the bad habit of fragmenting during development.

You can simply do a spot blot (pippet 1 µL sample onto the membrane), but quantitation is more precise if you use dot-blots with a 96-well vacuum manifold, but the good ones with a gasket of silicone rubber, not the cheap ones sealed with filter paper. I use https://www.bio-rad.com/de-de/sku/1706545-bio-dot-apparatus?ID=1706545

Sorry, if I was not clear. I have used the polyclonal mouse antibody and conducted a WB analysis and done immunostaining and it seems to be very specific. There is just not alot of endogenously expressed protein, I have to use 30ug of protein to detect protein expression on the membrane. Thank you everyone I will do more reading and see if the Dot Blot is applicable!

I AGREE WITH BENJAMIN JUREK

(I would like to know if there is a technique called ELISA cell. ELISPOT is similar?)

Mª Angeles Zorrilla Lopez-Perea : yes, there is, see https://en.wikipedia.org/wiki/ELISpot

It is a technique desighned for detecting and quantifying soluble substances such as peptide, protein, antibodies and hormones.