Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Antibody titration


  • Please log in to reply
5 replies to this topic

#1 winter

winter

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 18 May 2009 - 11:03 AM

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

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,254 posts
339
Excellent

Posted 18 May 2009 - 05:19 PM

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

#3 winter

winter

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 26 May 2009 - 02:44 PM

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


What dilution works best. Thanks.

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,254 posts
339
Excellent

Posted 26 May 2009 - 05:35 PM

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.

#5 tonix37

tonix37

    member

  • Active Members
  • Pip
  • 14 posts
0
Neutral

Posted 27 May 2009 - 12:18 AM

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

#6 winter

winter

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 27 May 2009 - 03:46 PM

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 :rolleyes:

tonix37



Thanks.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.