9 replies to this topic

[PulmDoc]

Is there anyone out that that knows for a Sandwich ELISA, does it make a difference to use a polyclonal or monoclonal ab as the primary?  

I'm using monoclonal as primary and don't get the cleanest signal.

Thanks.

Edited by PulmDoc, 19 April 2004 - 09:59 AM.

[hula]

Hello,

Regarding mono- poly-antibody, either monoclonal or polyclonal antibodies may be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonals have an inherent monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. Then a monoclonal is used as the detecting antibody in the sandwich assay to provide improved specificity.

Hope it helps.

Hula

[PulmDoc]

Hula - Thanks for the reply.  What you say makes alot of sense.  After I posted my note, I tried both ways and find that my polyclonal coating followed by monoclonal secondary has given my a much cleaner control! Odd?

PD

[jadefalcon]

just wondering...

monoclonal coating -> less pulled down protein -> less signal -> longer incubation period to see something at all -> higher background ?

Mike

[Shubenok]

monoclonal coating -> less bounded unspecified protein (lacking epitope of interest)-> less signal-> more protein-> equal signal -> no longer incubation period to see something at all -> lower background of unspecified protein (lacking epitope of interest)

[masasasagawa]

Optimization of assay requires to use different conc of cAb (mono or poly) crossing with various conc of dAb (poly or mono). Incubation time, shaking, temperature, enzymatic process, and TMB incubation time, etc all influence S/N (signal to noise) ratio. Here is a guideline. First of all you want the noise (std 0) to be less than 0.2 for deltaOD. I do not know off hand if you use a single wavelength. Closer to 0 is better. Then you want at least 10 in S/N (highest conc of std to std of 0) on deltaOD (450-550nm) or m450 reading. Higher the better resolution. I am getting about 0.032 for background and S/N of 35.5 in IL12 btwn 1 to 1,000 pg/ml. If you need an optimization matrix, let me know.

[mohan joshi]

Hello There,
I am planning to do ChIP using polyclonal antibodies which works good in IF even at dilution of 1:20000.
But I have no idea how much specific would it be in pull down (magnetic bead method).
As monoclonal antibodies are not available, what would be the best strategies to proceed with these polyclonal antibodies.
Does purification of the polyclonal would be critical in ChIP result?
and if i proceed with qPCR than how would non-specific pulled down DNA will matter if i use only specific primer automatically (except my control primer).
Thanks
mohan

[PastaSauce]

I have heard opinions  from both camps. Rule of thumb does not always work in biology.

[rozer henry]

PulmDoc, on Apr 19 2004, 09:58 AM, said:

Is there anyone out that that knows for a Sandwich ELISA, does it make a difference to use a polyclonal or monoclonal ab as the primary?  

I'm using monoclonal as primary and don't get the cleanest signal.

Thanks.

well enzyme linked immunosorbent assay or Elisa is highly effective in detecting all type of antibody...there is no such diffrences except membrane potential....
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[fariasjose]

Hello,
I am developing a capture ELISA to quantify the glycoprotein of vesicular stomatitis virus, I've done some tests that have worked, which I have to apply statistics to validate this technique?