How to detect if you've got antibody in your tube? - I want to know if my sample is really a monoclonal antibody! (Jul/05/2008 )

Hi,

I've got an antibody microtube from my friend and I want to make sure that it really contains monoclonal antibody.

I ran Western blot with it but got no bands. so I suspect it.

how can I do a simple test to see if I really have antibody and it is not water or PBS or whatever?

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Spec it to see if it contains protein?

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a simple immunostain on a positive control?

you'd have to know what would be positive of course

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don't have positive control.
for protein detection using a spec do I need to do bradfor assay? I just want to know it exists.

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You only need to do a bradford if you want to determine the concentration of your sample. If you spec at 320nm it will tell you if there is protein

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You will need to do some type of an immunological reaction. But, you said you need to know if it is monoclonal. You could also have Mouse IgG polyclonal and you have to differentiate this from the Mab.

Only way would be to detect one IgG subclass and one class of light chain. Radial immunodiffusion with anti mouse abs against the subclasses and also abs against the light chains. You would be looking for detection of only one heavy chain and one light chain. If you get any other type of reaction it is NOT monoclonal.

All those other tests mentioned will not differentiate mono from poly and some can not distinguish one protein from another or two proteins of the same MW.

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I'm a bit confused about what you're really asking.

Are you saying you know there are antibodies there but you want to see if they are monoclonal?

or

Are you saying you want to know if there are antibodies present?

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yes, actually I just want to know if there is antibody in there or not, with the quickest method.

Not necessarily I want to know if it is mono.

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Hi curtis,
the simple way to find if there is mouse antibody in the tube is just to coat it in ELISA plate, block with BSA and add anti-mouse IgG-HRP or ALP conjugate and then develop it in the normal way like ELISA
that would also solve why you couldn't see band in page
all the best
Leelaram

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Another easy way...

I'm not familiar with the tubes you're using... but if they have antibodies inside them... probably coated to the walls?

Then simply use a fluorescently labeled antibody.

IF they are mouse antibodies use something like Alexa fluor 488 goat anti-mouse IgG (Invitrogen A11029)

If they are rabbit antibodies use something like Aleza fluor 488 goat anti-rabbit igG (Invitrogen A11034)

Just incubat with the antibody (5 to 10 micro grams per ml in PBS) and then wash... voila, it's labled. Then measure the fluorescence the way you normallly would.

The whole proceedure could be done in about 45 minutes.. most of that being incubation time.

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