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Fab Preparation

  • General scheme for Fab preparation
  • Example 1: Size exclusion HPLC and MALDI-TOF MS analysis of Fab produced from mouse monoclonal antibody (mAb) and rabbit polyclonal antibody (pAb) at CellMosaic
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In the preparation of antibody-enzyme conjugates there are a few advantages of using antibody fragments rather than intact antibodies. For example, antibody fragments lacking an Fc region have less interference with various Fc binding proteins, can easy penetrate cell membranes due to their lower molecule weight, and experience lower nonspecific binding to cell surfaces. Fab fragments are useful in immuno-histochemical studies. The procedure we use involves cleaving the IgG molecule in the hinge region above the interchain disulfides using papain to create two identical Fab portions and one intact Fc fragment, and then purification to afford the pure Fab.

Examples (see images):

  1. Fabs produced from mouse monoclonal antibody (mAb) and Rabbit polyclonal antibody (pAb) at CellMosaic (shown below): high quality Fab based on size exclusion HPLC, gel electrophoresis (not shown here), and MALDI-TOF MS analysis.bm0006-fab-copy.jpg

References:

  1. Rousseaux, J.; Biserte, G.; Bazin, H. The differential enzyme sensitivity of rat immunoglobulin G subclasses to papain and pepsin. Molecular Immunology 1980, 17, 469-482.
  2. Coulter, A.; Harris, R. Simplified preparation of rabbit Fab fragments. J. Immun. Methods 1983, 59, 199-203.
  3. Rousseaux, R.; Rousseaux-Prevost, R.; Bazin, H. Optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses. J. Immun. Methods 1983, 64, 141-146.

 

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