Selections using biotinylated antigen

Selection of phage antibody libraries by selection on blotinytated antigen 

Make up an aliquot of the phage library (approx. 1 x 10 phage) to 500 (j.1 in PBS containing 3% skimmed milk and block for 30 min at room temperature. 

Add biotinylated antigen and incubate at room temperature for 1 h with end-overend rotation. The amount of biotinylated antigen you wish to add may vary. Antigen should be added to a concentration approximately equivalent to the desired ICa of the resulting selected antibodies and in molar excess over phage (1 x 10 phage/ml is 10 nM). 

Dispense 100 (il of streptavidin oated magnetic beads into a 1.5 ml Eppendorf tube and collect the beads at the bottom of the tube using the magnetic rack. Aspirate the supernatant and resuspend the beads in 1 ml PBS containing 3% skimmed milk. Block for at least 1 h with end-over-end rotation. 

Capture the blocked streptavidin beads using the magnetic rack, aspirate off the block solution, and resuspend the beads in the phage-antigen solution. Incubate at room temperature for 15 min with rotation. 

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Repeat the selection procedure another three to four times (use a 1 -mL aliquot for selection, and store the remaining phage at 4°C). For the second and subsequent rounds of selection, mix 1 mL phage from the previous round of selection with 0.5 mL of 6% MPBS, 10 pL Tween-20, and biotinylated antigen to give a final concentration of 50 nM Use only 300 pL of blocked streptavidin-coated Dynabeads... 

Armexin A1 emerged as a promising antigen for the radiolabeled antibody-based imaging and therapy of cancer [100]. In our laboratory, we use terminal perfusion protocols featuring active esters of biotin for the selective chemical labeling of accessible proteins in vascular structures. Biotinylated proteins are then purified from different organs (collected separately) and are submitted to a comparative proteomic analysis [106]. 

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