Antibody titration - (May/18/2009 )

We have made a polyclonal antibody against whole cell that we would like to titer it. I need a protocol to do that. Thanks for your comments.

Winter

-winter-

Titrate as in see what dilution works best? or as in how much IgG there is?

-bob1-

bob1 on May 18 2009, 06:19 PM said:

Titrate as in see what dilution works best? or as in how much IgG there is?

What dilution works best. Thanks.

-winter-

You know how to make dilutions? So... make different dilutions of your antibody in the appropriate solution and apply them to your system, there is no need for a protocol other than the one you already have for western or ICC or IHC.

-bob1-

Things to do to optimize your ELISA:

1) optimize the amount of adsorbed antigen:
- Different dilutions of antigen
- Dilution of primary antibody (in excess)
- Dilution of secondary antibody (in excess)

2) optimize the secondary antibody dilution:
- Dilution of antigen (the best you found)
- Dilution of primary antibody (in excess)
- Different dilutions of secondary antibody (in excess).

3) optimize primary antibody dilution, is your antibody titration:
- Dilution of antigen (the best you found)
- Different dilutions of primary antibody
-dilution of secondary antibody. (to find the optimal)

If you change the lot of antigen or secondary antibody you must repeat these ELISA to ensure that you are working in the optimal conditions range.

You can express the results of the ELISA in a four parameters logistic curve function. For the antigen and secondary antibody dilution the optimal point are the most near to saturation, in your serum, takes the IC50 as an indicator of antibody titer in your serum.

For western blot you can do the same way. We use an Accutran-Cross blot system that is very useful to optimize the westerns in the lab.

I hope you serve

tonix37

-tonix37-

tonix37 on May 27 2009, 01:18 AM said:

tonix37

Thanks.

-winter-