Hi,

What exactly do you mean by same antibodies? In general, it is always good to check the specificity and clonality of your antibodies. Mainly, (A) Some antibodies just aren't good quality. (B) depending on whether you are using a polyclonal or monoclonal antibody, this may also effect the binding specificity.

If you can give more detailed info on what type of antibodies you are using and what experiment you are trying to do, more help can be given.

Best wishes,

Omar

Hi Omar,

Thanks for taking interest.. i am trying to establish sandwich ELISA model. I am working on polyclonal antibodies which were raised in rabbit as well as goat.. ON first go i tried one for coating i.e goat and detection by rabbit HRP labelled.. But observed cross reactivity as in blank gave high reading.. because of that reading between sample of different concentration was too low even glipmse were thr of linearity.. In another set i tried to coat the rabbit antibodies , appllied antigen n then tried to detect also with same rabbit Ab which were labelled with HRP.. your input will be highly appreciated.. any more info u need..

Thanks

Hi Zulfaquar,

I'll try my best to help. First some more questions(sorry)!

1. When you say "blank", do you mean [capture antibody]+[no target]+[detection antibody], or do you mean [no capture]+[no target]+[detection antibody] or [no capture]+[no target]+[no detection antibody]+[chemiluminescent substrate]?

2. Have the antibodies been tested for specificity for your target using another technique such as western blotting?

3. What type of sample are you testing? Purified protein/cell lysate/blood sample etc.

4. Have you washed the plates well before application of the substrate?

I know its a lot of questions, but it will help narrow the problem and others with more experience than I might be better able to help.

Best wishes,

Omar

Hi Omar,

Thanks for keeping interest.. no problem for asking more q as this would help me in many ways

1. a) capture + no target + detection - high reading

b) no capture + no target + detection - no high background and same for third option also as u mentioned. for me blank is a).

2. Yes antibodies were tested for western blot and good expected good result. When i am doing direct ELISA where you need only one antibody there is no problem and assay working fine. I have checked for different blocking and different concentration of antibodies to minimize cross reaction. All other possibility or high background sorted out.

3. cell extract.

4. washed properly as no high background in other combination of 1 except when both antibodies were used.

hope this help you to provide some more feedback.

Thanks

zulfaquar

Hi Zulfaquar,

Yes it looks like the signal is due to cross Ab reactivity of some sort. All I can suggest is the following:

1. Can you try a monoclonal Ab instead? There might be an antibody subset (with less specificity the majority of the population) in your polyclonal prep that is reacting with the other antibody. You could try running one antibody on the gel (probably a native gel) and probing it with the other antibody. If the first antibody is detected using the second then you have non-specificity and need to change one of the antibodies.

2. You could try pre-adsorbing/cross adsorbing your antibodies against the other antibody and this might remove cross reactivity prior to use in ELISA systems. See http://www.pierce-antibodies.com/products/secondary-antibodies/ for info on why you would have to do this. Check if the IgG class is the same between the two antibodies.

I've pasted this from the link above:

"Because of the high degree of conservation in the structure of many immunoglobulin domains, class-specific secondary antibodies must be affinity purified and cross-adsorbed to achieve minimal cross-reaction with other immunoglobulins. Using the example described above, immobilized mouse IgG1 antibodies would be used to affinity purify all goat antibodies that bind to mouse IgG1. These anti-mouse IgG1 antibodies would then be further purified by passage through a chromatography column(s) containing mouse IgG2a, IgG2b, IgG3, IgM, etc., to remove any antibodies that cross-react with non-IgG1 isotypes. "

Hope this helps a bit and let us know how you progress.

Kind regards,

Omar

Hi Omar,

Thanks for your feedback.

1. No i dont have option to go for monoclonal.

2. Yes i am aware of this and plan. But i dont have so much time for going all through this, so this post was posted just to get any clue or short term solution.

Thanks for your time and noble advice.

regards,

Zulfaquar

Hi Zulfaquar,

Sorry I couldn't be more help, hope you find the answer soon :)

Best wishes,

Omar

Hi Nadia,

Selecting other animal may be the solution. like you can select mice .

But before doing this i will suggest you to do cross purification which might help you and will be economical too. Is you antibodies poly clonal or monoclonal ? In my case it was against the whole cell proteins so might be few reasons but in your case its just one antigen or protein which against antibodies was raised. I would also suggest to check the different plates and also to minimize error check if you blocking buffer doing any inteference with test.