Antibody adsorption protocol - Immunology (Oct/27/2005 )

Hi all,

Does anyone have a good protocol for antibody asorption. How much antibody to antigen amount will be more appropriate for effective adsorption.

THanks!

hi ling,

there are several protocols that work well for antibody adsorption. coupling purified recombinant protein to a solid support like Affigel 15 or Affigel 10, or activated CNBr resin, or even nitrocellulose followed by incubation w/ crude serum diluted 1:5 in 1X PBS are common strategies. elution is then carried out by a short incubation of 100mM glycine, pH 2.0, and neutralization w/ 1:10 vol of 1M Tris, pH 8.0. the amount of antigen will vary, depending on the amount of antibody you wish to purify. if you're only looking to purify Abs from 1ml of serum, don't couple 30mg of protein to 5ml of resin. usually 2-3mg of purified protein is more than plenty, though the amount you need will vary slightly by the protocol you choose to use. if you have more questions about this, feel free to email me.

good luck,

jon

jonmike.reed@gmail.com