IFNgamma ELISA standard curve - (Mar/14/2007 )

Hi everybody,

does anybody know how should the right standard curve of ELISA look like?

I've tried already many things including different BSAs, different Ab concentrations, but my standard curve doesn't look like in the manual.

We use the R&D DuoSet human IFNg ELISA kit.

It goes also only till 1 of optical density....

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looks fine to me, so what exactly is your problem?

mike

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The only thing I would say about your curve is that doesn't show any signs of saturation of the binding sites on the plate. I'd try a lower dilution of your samples, so you get an inflection point and a saturation of the curve.

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Thanks for your answer!

I don't have really much experience with ELISA, but for me it looks like I have very flat curve and no saturation at all... And the point is - i need the lower concentrations (15-250 pg/ml) for my assay. I think the sensivity of my ELISA is not so good...

what do you think?

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What are the steps in your assay? Could you try extending the incubation times, particularly for the colour reaction?

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Extending of the time of colour reaction or higher concentration of antibodies brought only higher ODs, but not better curve (the curve remain flat).

We are trying with 37°C incubation instead of RT.

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What is the top standard's concentration? If it's not saturating the coating, you're not going to get a maximum reading.

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1000pg/ml - it's recommended by R&D....

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If you followed every step as it is written in the manual, and the resulting curve is not what they say it should be, then something is wrong with either your technique or their reagents. Have you called them? Do you use a plate washer?

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I actually connected R&D and they sent me their BSA solution. So I'll try this and than we'll see....

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