Dear All,
Can we use two poly clonal antibodies generated against specific antigen in rabbit and mice simultaneously in the ELISA?
I think it would help if you could describe the assay in more detail.
The key question to ask, however, is whether any of the antibodies or labelled detection reagents that you may use can crossover onto the 'wrong' reagent. For example if you had an anti-rabbit reagent in your assay, it needs to be specific for rabbit IgG and not recognise mouse at all.
If one of your polyclonal antibodies is the detection reagent and the other one will be a labelled detector, I see no major issues. It is unusual to have polyconal mouse antibody (at least in any quantity) so I am not sure if my guesswork on the assay format is correct. Please provide a few more details if you can.
Hi Anil, I agree with Nick.
More details are needed, but If your detection Ab is labeled with HRP, AP, biotin, etc. then there should not be a problem.
If your detection Ab in not labeled, then you have a problem.
I think it was probably obvious, I meant..
If one of your polyclonal antibodies is the CAPTURE reagent and the other one will be a labelled detector, I see no major issues.
I suspect you will have far more of the rabbit polyclonal than mouse polyclonal, in which case using the rabbit Ab for plate coating and labelled mouse Ab for detection might work best. A mouse monoclonal would be preferred if you have one, either as capture reagent or labelled detector; you would have to try both ways to see what works best.
Thank You all for your nice suggestions.
Actually, I have generated polyclonal antibodies (pAbs) against recombinant protein of M.tuberculosis in both Rabbit and Mice. Now I want to use these two pAbs (one antibody as detection antibody and other one as capture antibody) in the designing of sandwich ELISA for the detection of antigen in TB patient serum. ...
Thus, I want to known can I proceed this assay or not??
Once again Thank You Every One..
Sir, I am new in this field. I am planning to use mouse mAbs (that will be HRP conjugate using commercial Ab labeling kit) as detection Abs and Rabbit pAbs as capture antibody via sandwich ELISA.....Kindly suggest ..
Okay, this is good, a polyclonal/monoclonal combination is better than poly-poly in terms of likely selectivity. You could do the sandwich assay either way round (mAb capture or poly capture). I think mAb capture is more common, but I have not done a detailed survey on this. It is not possible to predict whether any particular mAb will be better as a capture reagent or as a detector. Ideally you will have a lot of mAbs from which to choose a suitable one. I have to declare a commercial interest before recommending labeling kits, but my company does provide easy-to-use kits. There are several suppliers of HRP kits however. Finally, you may want to consider mAb-mAb if you have enough antibodies to screen out a good pair because a polyclonal reagent will always run out at some point.
Dear All,
I am facing problem in the leveling of mAbs with HRP enzyme. I have tried leveling with Abcam-HRP leveling kit.But i could not found level mAbs. After literature survey, I found glycine (mAbs eluted via Tris glycine buffer) may interfere in the mAbs leveling as it have free NH2 group. So, I have tried to remove glycine from the mAbs via buffer exchange (HEPES). I want to known, how can i detect free glycine in the mAbs solutions.
Thank you sir for your suggestion. I am using amicon centrifugation technique for removal of glycine. But i am not sure,how much amount of glycine remained after 5 buffer exchange.
Thanking you
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