Fab production

SBehar sbehar at aol.com
Mon Feb 27 23:48:42 EST 1995


I just tried making Fab fragments for the first time from a mAb IgG1.  I
used a kit from Pierce, which works by using immobilized ficin, which is
preferable for IgG1 digestions.  The principle was very simple.  The
immobilized ficin is on a matrix (?sepharose), and comes as a prepacked
column.  The purified antibody is diluted in a digestion buffer containing
cysteine to activate the enzyme, and then loaded onto the column.  Once it
is loaded onto the column, one incubates the column at 37C for 3-5 hours. 
Then, the column is washed and fractions are collected (the antibody never
actually binds to the column).  Subsequently, the fractions containing the
digested products are run over a ProteinA column to remove undigested
antibody and the Fc fragment.  Perhaps this will give you some idea for
using the immobilized papain.

Good luck,
Samuel M. Behar, MD PhD
Seeley G. Mudd Bldg.-- Rm 508
Department of Rheumatology
Brigham and Women's Hospital
250 Longwood Avenue
Boston, MA 02115
FAX: 617-432-2799



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