HI Zhi-Wei Lin -

Having not seen your data have you looked at other drugs or specifically only those two drugs are giving saturated abs values? It could be due to the chemistry of those two particular drugs i.e they are reacting with your ELISA components in the absence of biological test sample. What did the control give for just the compounds without test sample in the presence of ELISA components one by one? Perhaps give that a try. If the control compounds react with your ELISA components/secondary substrate you could then subtract the basal absorbance value from those two controls compared to the wells that have the test sample containing your primary target.

Blake Balcomb

Thanks for your reply!

The drugs are in human plasma. Just human plasma alone gave results close to background noise, so its not the content of plasma that's causing this.

When the drugs are not in plasma, instead in assay buffer provided by the kit give saturated abs values.

PBS itself should not cause a problem.

So the ELISA kit detected the drugs? Did the drugs still saturate when you only add one of them?

Did it ONLY saturate when it is in the assay buffer? And not when it's in plasma?

Is the drug actual pharamceutical or just the active component? And are the drugs a small molecule or protein?

If is some weird interaction with your ELISA kit components, you will need to just test them one at a time. I really can't figure what it could be...

Is one of the antibodies in your ELISA kit a polyclonal ab?

Shen-An Hwang

Thank you for reply!

The drugs aren't combined. Both are giving us troubles.

The drug stocks we have saturate in plasma and assay buffer. But when using the standard provided by the kit, both plasma and assay buffer work.

That's what we are looking into now! The drug stocks we got contain the main active component only. We just ordered the clinical formulation, will see how that goes. The drug is a protein.

Not sure if the anitbodies (receptors) in kit are polyclonal. Both drugs are monclonal and inhibitors to the antibody.

Is the drug(s) a recombinant protein? Do you know what cell line (human, bacteria, yeast...etc) it is produced in? The only thing I can think of that may be different between the drug and the standard is glycoslyation, becuase usually standards are not glycoslyated and drugs (at least more now than before) are since I think most uses human cell lines? If one of your antibodies in your ELISA kit is a polyclonal (monoclonal may also crossreact...but polyclonal is more likely), maybe it's picking up the glycoslyation patterns?

Is this a regular ELISA? or competition ELISA? Is the drug a biologic (since you said that they are monoclonal)? If the drug is an antibody or has antibody structures, such as the FC or the constant region...you might be looking at false positives caused by antibody-antibody binding (kind of like heterophilic antibody issues that may arise when we immunoassay serum/plasma). There are commerically available reagents that can block these... might give them a try...Or you can try increasing you BSA concentration and adding tween in your diluent.

Hi Zhi-Wei Lin -

Okay, good that your plasma control is negative. Your assay kit and standards provided in the kit are working fine which is great that you know its not the kit or plasma. Since you do say the absorbance is ONLY saturated when you add these two drugs to the assay buffer provided by the kit then the drug compounds themselves are most likely the cause of the high absorbance. The high absorbance is either that the drugs have a high intrinsic absorbance due to aromatics/ring structures of the compounds since they would be in the range of wavelength for ELISA readings. The other alternative for the compounds giving high absorbance in the assay buffer is that they are precipitating out in that buffer. Thus I would suggest to test the absorbance of the drugs alone in the buffer and no antibodies. i.e if your compounds alone in buffer solution give an absorbance reading you can still subtract that background intrinsic absorbance of those to compounds to the reactions wells. Essentially you need to test the control of checking the absorbance of those two drugs in buffer alone. We often synthesize small molecule inhibitors that give their own florescence or absorbance alone in reaction buffer. So I believe this is where your problem lies.

Shen-An Hwang Blake Balcomb

Thank you both so very much for the help. Sorry for a late reply, I was working on other projects while waiting for new drugs to come in.

New drug came in today in its clinical formulation. Ran ELISA kit, still failed the spike-recovery test.

The drug is a humanized IgG4 monoclonal antibody that has fc region. The ELISA kits we get (tried 3 companies so far) specify that they only recognize Nivolumab, the drug, and nothing else within human plasma. I contacted their R&D team and am waiting on their response in terms of the composition of their standards.

Shen-An Hwang What kind of reagents should I look into for blocking?

We will run some controls tomorrow, specifically blank plasma and drug-only/no-antibody.

Do you know if commercial elisa kits contain tween in blocking and washing?

Again thank you both so much.

Zhi-Wei Lin

Excellent let me know how it goes!

Just an FYI, our testing lab has tried many of these ELISA kits...and I'm not sure that they were able to get it working to what the manufacturer's claim. I'm not sure if they ever found a kit that worked well... s I can't even give you an answer as to which ones to try.o

As to what type of blockers... you can try increasing BSA and adding Tween20, if you haven't. Or their are commerical reagents...I've located a handy guide to explain in the following link...

https://meridianlifescience.com/documents/Blocker%20insert/MLS%20Blocker%20Practical%20Guide%20English_2018_WEB.PDF

Blake Balcomb Shen-An Hwang

We tried re-blocking the plates with 0.05% Tween20 for 1 hour as well as adding that to washing buffers. Didn't see any signs of improvement. We know that plasma was giving nonspecific abs, but simply subtracting plasma control doesn't always work... Additionally, those two drugs (monoclonal anitbodies) still lead to saturated abs values when prepared in assay buffer instead of plasma.

Do you have protocols for re-blocking the commercially available plates? Should we try longer period of incubation?