Coating with monoclonal/polyclonal - (Aug/28/2008 )

Have run out of affinity purified polyclonal and the same protein G purified-HRP conjugated polyclonal.

And am struggling to get my ELISA working with non-affinity purified polyclonal and a new monoclonal, would like to get OD of at least 1.0 for the highest standard, (previously got 1.6).

Using the protein G purified polyclonal as coat and the remaining polyclonal HRP conjugate as detect gives an OD of 0.5, (so difference between non-affinity purified and affinity purified polyclonal coat is 1.6-0.5 = 1.1)

Had hoped that I wouldn't need to affinity purify the polyclonal but try to use the monoclonal to increase OD instead.

Am doing sandwich ELISA and when polyclonal is the coat and monoclonal detect I get a decent standard curve, (but only followed by alkaline phosphatase after overnight incubation - yes overnight - gives OD 1.0), but when monoclonal is the coat and polyclonal detect, (using HRP), I either get no OD or some high readings but nothing that you can call a standard curve.

Is there a problem with the monoclonal not coating in all the wells? Am currently using sodium carbonate pH9.6.

Am I wasting my time expecting the monoclonal to increase sensitivity when all it can do is increase specificity, therefore I will have to affinity purify the polyclonal after all?

Thanks in advance for your kind suggestions.

Hi Vectra,
I suggest you try different buffers for coating your monoclonal and try the ELISA without blocking.
Sorry you didn't get any other replies.

regards,

Vectra

Don't try to change so many parameters at the same time. Keep your one working conjugate and purchase several other abs for capture. Screen them with a negative, midpoint and high standard. Leave your coating condition constant. Leave your blocking condition constant.

I have never heard of not blocking a plate...you would just be setting yourself up for high non specific binding.

Vectra: how's your ELISA going now?

I'm having similar difficulty coating with monoclonal. You said you incubate overnight for colour development? Is this in the cold room?

I've tried PBS and carbonate buffer to coat. Starting to run out of ideas 9and antibody).

Here's one that might help coating, warm the plates and Mab to 37C first and incubate for an hour at 37C before overnight in the cold room.

Also, we have a team in my lab who don't block. It is done. I'm blocking at the moent but I'll run a plate to see if blocking really improves ODs or not.

Anyone else got any ideas to improve coating?

first of all I suggest to always use monoclonal (if possible) as coating ab, since it should increase the sensitivity. I've always obtained better results with monoclonal as capturing ab rather than polyclonal. The coating with carbonate usually works, but not always. Once I had some monoclonals, which did not remain onto the plate at all with carbonate, but only with PBS. Try also citrate at pH 5.
Have you checked the presence of your monoclonal simply using anti-ms in some well?

I don't think you can increase the sensitivity of your assay by using monoclonals. I would still use the MAb as coating though. But your problem doesn't seem to be the monoclonals as much as the polyclonal sera.
Does lessening the dilution of the polyclonals improve things? The problem might also be the amount of HRP conjugated onto the polyclonals. Have you tried increasing the amount of HRP?

well, if you use polyclonals both as capturing and as primary abs, the sensitivity may be the same or just a shade more, but, generally, you lose in specificity. Since specificity is often a priority, you should use monoclonals...some good ones. Now: if you use polyclonal as coating abs and monoclonal as primary abs, the sensitivity usually diminishes. However I did not understand which way VECTRA obtained 1.6 OD. Furthermore, if you purify a serum with proteinG you have to keep in mind that you will find in the "purified" solution also a lot of other antibodies, since proteinG capture all the IgG in the serum. Thus the best way to purify your abs and obtain the best results should be affinity purify by using the antigen itself, best if after a previous proteinG pre-purification. However, serum can be used as primary abs with good results, hoping not to get too a high background. Finally, I can tell you that TMB solution is very very delicate: time ago ELISAs done by mister A in our lab developed very slowly and with very low final OD, while ELISAs done by others developed normally. Mister A made something wrong with TMB solutions (we still don't know what)
SATANCLAUS, have you thought about my problem (in the other topic?)