covalab ANTIBODY FRAGMENTATION SERVICE

fragmentation

 

Distinct fragments of the immunoglobulin can be generated by cleaving the molecule using proteases. 

 

Targeting the hinge region allows to generate two types of fragments of interest depending on the final application: 

  • Antigen-binding fragments such as Fab and F(ab')2
  • Class-defining fragments such as Fc which do not bind antigen

Depending on which side of the hinge region (N- or C-terminal) is targeted, the proteolytic cleavage produces either two Fab and one Fc fragment or one F(ab')2 fragment.

 


 bubble PAPAÏN FRAGMENTATION

 

 When immunoglobulins are incubated with papain in the presence of a reducing agent, several peptide bonds above the hinge region are hydrolyzed, producing three fragments of similar size (50kDa):

 

 Papaine fragmentation

 

Fab fragment Fc fragment

Compared to whole IgG, Fab fragments can penetratecells and tissues and bind antigens more easily thanks to their lower steric hindrance. Moreover, the absence of Fc region prevents Fab fragments from nonspecific binding to Fc receptors.

When the Fc fragment is of interest, papain is consequently the enzyme of choice. Papain is primarily used to generate Fab fragments, but it can also be used to generate F(ab’)2 fragments when used in specific physico-chemical conditions.

 

 


 bubble PEPSIN FRAGMENTATION

 

 Digestion of immunoglobulins by pepsin occurs below the hinge region of the heavy chains, resulting in a F(ab’)2 fragment which is composed of two Fab fragments still linked together by the remaining portion of their respective heavy chains at the hinge region. The Fc fragment is extensively degraded, and its small fragments can be separated from F(ab')2 by dialysis, gel filtration or ion exchange chromatography.

 

 Pepsin fragmentation

F(ab')2 fragment
F(ab')2 fragments share many advantages with Fab fragments, but unlike the latter, F(ab’)2 fragments remain divalent and can be used to bind and precipitate antigens