Antibody fragmentation, immobilization

Fig. 12 Microphotograph of an analyte concentrator fabricated with FAb antibody fragments immobilized to controlled-pore glass silica. The irregularly shaped beads were housed between two frit structures. The analyte concentrator device was connected to two separation capillaries by a Teflon sleeve. The plastic connector was glued to the separation capillaries by an epoxy resin. The entire fabrication process was monitored by an stereo microscope. (For details of experimental conditions, see Ref. 120.)...

The Protein Profiling Biochip is composed of six chips assembled in a flow cell cassette device. Each chip provides 200 data points (200 pillars per chip) for a total of 1200 data points per cassette. Pillars are 50 p in diameter. The mesa on each pillar is covered with a self-assembled monolayer (20 to 25 A thickness) of biotin-derivatized PLL-g-PEG groups. A constant grafting ratio of 3.5 parts Lys to 1 part PEG is maintained with variable biotin-PEG content. sAV antibody is immobilized at 0.5 to 2 pmole/cm and Fab fragments at 4 pmole/cm. ... 

In addition, 9E10 immobilized on protein A-Sepharose using dimethyl pimelim-idate can be used for purification of these antibody fragments (15)... 

One of the most important features in the immunosensor design is the proper choice of the immobilization method for keeping the affinity of the antibodies. As was previously demonstrated for Protein A, when the antibodies are immobilized through their Fc fragment to Protein A (or G), their Fab binding sites are mostly oriented away from the solid phase. As Protein A is able to link the Fc region of different antibodies, there is no need to modify the antibody with biotin. As an antecedent, we have previously demonstrated the utility of Protein A biocomposite (ProtA-GEB) for the universal attachment of antibodies with different specificities [54]. 

Those procedures that allow an oriented immobilization of intact antibodies, or antibody fragments, on solid supports have been the most applied in the fabrication of different immunosensors. 

Nevanen, T.K., Soderholm, L., Kukkonen, K., Suoitti, T., Teerinen, T., Linder, M., Soderlund, H., and Teeri, T.T. Efficient enantio-selective separation of drug enantiomers by immobilized antibody fragments. J. Chromatogr. A 925, 89-97, 2001. 

Immobilized papain and other components for antibody fragment preparation and Iodogen are available from Pierce (Rockford, IL). 

Figure 1. Capacity of model immunosorbent systems as a function of cycle number. (A) Goat anti-human IgC Fab fragment immobilized at 7 g/L. Column volume = 6 ml. (B) Same as above except that the initial antibody loading was 1 gL- and the immunosorbent volume was 4.2 ml.

---