Purifying Chicken IgY

Curtis L. Ashendel ashendel at aclcb.purdue.edu
Mon Jan 10 12:33:55 EST 1994

On Mon, 10 Jan 1994 12:33:47 GMT, 
David M. Berman  <images at netcom.com> wrote:

>Has anybody tried the chloroform/PEG method of antibody purification
>from egg yolk?  Do these antibodies perform well?  The method (Polson,
>1990, J Imm Inv) is easy and gives good yields but the original
>article uses an Ouchterlony assay as the only proof of Ig function --
>I'm a little concerned about possible effects of chloroform on the Abs
>performance in sandwich assays.
>I'd appreciate any feedback from anyone with experience in these
>David Berman
>UT Southwestern Medical School
>Dallas, TX 

As for using chloroform in the isolation, I've never tried that.  We just 
used the old Polson method (a differential PEG cut) which gives very high 
apparent purity (>95%) and yield (grams of purified antibody per dozen eggs 
if I recall).  It is very easy.  I have not seen the newer method and do 
not know why it incorporates chloroform (except perhaps to get rid of all 
that cholesterol - which is normally lost in the first PEG precipitate).  
The IgY seemed rather resiliant since we could freeze-thaw repeatedly and 
lyophilize without noticable change in reactivity.  It is possible they are 
not affected by chloroform. 

As for the antibodies themselves, they work as well or better than rabbit 
antisera in IPs and westerns (with the added benefit of their ease of 
isolation and quantity), with two exceptions:  (A) They do not react with 
proteins A or G and require a second antibody, which must be made (I still 
can't find any commercial antibody that works very well). (B) The hens we 
immunized were vaccinated with something containing BSA and hence had 
strong anti-BSA titers in preimmune eggs.  Neither problem is 
insurmountable, but just inconvenient enough to keep me with rabbits 
whenever possible.
Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu

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