Antibody titer protocol?

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alchemist147
alchemist147's picture
Antibody titer protocol?

Hi all,

      I am a grad student new to working with antibodies. I want to know if anyone can help me determine how best to find the optimum concentration for my primary and secondary antibodies. Ive noticed that some people perform titrations...

I have antigen that is 0.5mg/ml
primary antibody that is 0.5mg/mL and my secondary antibody is 0.5mg/ml as well. It was suggested to me to have a 1:50 dilution of the primary and 1:500 dilution of the secondary.

In order to determine if this is the best dilution would i set up a series of titrations in a well tray?

Also, can you tell me why you would use more primary than secondary? Is this bc of the bindng constants?

Thanks!
M

Sami Tuomivaara
Sami Tuomivaara's picture
alchemist147,

alchemist147,

Titration, or dilution series experiment is the way to go when optimizing immunoassay conditions. I'd first keep the secondary antibody concentrations constant and make a dilution series of the primary antibody. After finding out lowest dilution of the primary antibody where you still get a good signal to noise, do another dilution experiment with that constant amount of primary antibody and dilution series of the secondary. These two experiments give you the winning combination! Note that if the analyte (antigen) concentration can vary in different samples, you need to have some tolerance for samples that have low amount of antigen.

The most convenient way of doing this (microtubes, well trays, or something else) depends on your preference.

I think you are right about the reason for using more primary than secondary. I can't come up with other rational explanation except for that the secondary antibodies maybe have generally higher affinity for primary, than primary has for the antigen. All other things I can think of actually say the opposite...

Cheers,

alchemist147
alchemist147's picture
Thank you!

Thank you!

Right now only my secondary antibody is conjugated. I suppose I have to buy a primary antibody that is conjugated to do this titration exp?

alchemist147
alchemist147's picture
the primary antibody is not

the primary antibody is not available in conjugate form...(with fl. tag).

Help?!

Sami Tuomivaara
Sami Tuomivaara's picture
alchemist147,

alchemist147,

You don't need to conjugate the primary! The secondary antibody is conjugated and that's what you detect in your experiment...

Cheers,