The Thermo Scientific Pierce F(ab')2 Preparation Kits use immobilized pepsin protease to selectively digest whole human and other IgG antibodies to make F(ab')2 fragments that retain antigen binding activity.
These F(ab')2 Preparation Kits are suitable for human, rabbit, mouse and other species and subclasses of IgG except mouse IgG1. The antibody digestion reaction with pepsin agarose is performed in convenient disposable spin columns that allow efficient removal of the immobilized protease and maximum recovery of the IgG fragments. Also included in the kits are Thermo Scientific NAb Protein A Spin Columns and buffers to efficiently purify the resulting fragments. Protein A binds the Fc fragments and undigested IgG, allowing the pure F(ab')2 fragments to be recovered in the flow-through fraction. The kits also include Thermo Scientific Zeba Desalting Spin Columns for preparing the IgG sample quickly without dilution instead of utilizing time-consuming dialysis steps.
Highlights:
Enzyme-free digestion products – Immobilized Pepsin (beaded agarose resin) provides for control of the digestion reaction and complete removal of resulting antibody fragments from the proteolytic enzyme
Suitable for human and other species of IgG – the kit procedure is optimized for human, rabbit and mouse IgG, but pepsin-based digestion is also effective for other species and subclasses of IgG (Note: for best results with mouse IgG1, use Part No. 44980)
High capacity – kit provides for digestion and purification from as much as 4mg of IgG at a time. Use the standard size kit for fragmenting 0.25 to 4mg IgG, and the micro kit for 25 to 250µg IgG.
Complete – kits include all reagents needed to prepare and purify antibody fragments
Fast – spin format greatly reduces sample processing time
Flexible – protocols are included for multiple species and IgG subclasses, as well as sample size and concentration.
Efficient – enhanced yield and sample purity
Product Details:
The kits use pepsin, a nonspecific endopeptidase, to enzymatically digest the Fc portion of whole IgG to yield the fragment known as F(ab')2. This fragment is composed of a pair of Fab' units connected by two disulfide bonds. Because the pepsin protease is supplied in immobilized form as beaded agarose resin, the digestion reaction is easily stopped by removing the resin from the IgG solution; the result is digest products that are enzyme-free.
Digestion and purification scheme for preparing F(ab')2 fragments from IgG antibodies.
References:
Smith-Jones, P.M., et al. (2004). Nature Biotech. 22: 32-37.
Pruimboom, I.M., et al. (1999). Infect. Immun. 67(3): 1292-1296.
Wedrychowski, A., Kim, Y., and Chang, T.W. (1993) Immune enhancers composed of polyvalent binding sites of anti-CD3 antibodies. BioTechnology 11: 486.
Lamoyi, E. (1986). Preparation of F(ab')2 fragments from mouse IgG of various subclasses. Meth. Enzymol. 121: 652-663.
Kulkarni, P.N., et al. (1985). Conjugation of methotrexate to IgG antibodies and their F(ab')2 fragments and the effect of conjugated methotrexate on tumor growth in vivo. Cancer, Immunol., Immunotherapy 19: 211-214.
Rousseaux, J., et al. (1983). Optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses. J. Immunol. Meth. 64: 141-146.
