Polyclonal vs. Monoclonal antibody for Sandwich ELISA? - (Apr/19/2004 )

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.

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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

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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

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just wondering...

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

Mike

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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)

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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.

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Its best to use monoclonal antibody as the primary antibody......try using low conc of polyclonal antibodies as secondary antibody..this may help to reduce high back ground

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If you are measuring proteins like growth factors and such like then you can try replacing the capture monoclonal antibody with a receptor-Fc chimera. This way you are measuring biologically active ligand rather than immunologically active ligand. Some companies now sell these for a range of proteins. You need to ensure the receptor is of an appropriate affinity, but I know off-hand that this approach works for quite a few proteins, eg. VEGF, TGF beta, TNF and EPO. I get my receptor Fc chimeras from apollo cytokine research. The thing is that mammalian proteins consist of a range of isoform species, not all of which are biologically active. So you may be better off measuring only the species that bind to their cognate receptors. An unconventional ELISA approach, but probably a more valid measurement method.

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Hi I have the opposite advice, I work at a biotech company and all our ELISA matched antibody pairs work on the principal that capture antibody is monoclonal while the detection is polyclonal, this is because you want specificity during the binding of your analyte (thus a monoclonal) then once the plate is washed your a left with only the analyte of interest, the detection is usually a polyclonal as this various configurations during the initial capture and the blocking proteins mean that having a polyclonal maximise your ability to detect the bound analyte.

In the above way if you use a polyclonal first you run the risk of getting an increase in non-specific binding with is not washed away and can give you false positives

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i agree with the concept given where u use the monoclones as the capture abs... even so the use of polyclonals may not be totally not suitable for use. It largely depends on the sensitivity and type of antigen you want to capture. Of course the polyclonal being poly-specific may tend to capture other structurally similar molecules that may have some small resemblance to your antigen of interest which would lead to flase positive results. If it is possible use two monoclonal antibodies with different epitopes for ur assay. Would help increase ur specificity.

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