Chemokine biotinylation

HS is a GAG naturally expressed by mammalian cells and is therefore more representative of physiological GAGs that immobilize chemokines than heparin. It is commercially available as a sodium salt isolated from kidney extract. We report here the biotinylation of HS and its use in a biolayer interferometry (BLI) assay to monitor the binding of monoclonal antibodies to their chemokine target displayed on HS (Fig. 2). This assay has also been used successfully with chemokine-binding proteins. 

Dilute biotinylated HS (see paragraph above) 1 2000 in KB. Dilute the chemokine to be tested to 100 nM in KB. Dilute mAb to 1 pg/mL in KB. Transfer 200 pL/well of the adequate solution to a black flat bottom 96-weU microplate to have the following solution in each well ... 

During the experiment, maintain a constant shaking (1000 rpm) and temperature (30 °C). To determine whether the chemokine or the antibody interact nonspecificaUy with the biosensors, a control experiment should be performed, by replacing the biotinylated HS with an irrelevant biotinylated protein. 

Dilute biotinylated HS 1 2000 in KB. Dilute the chemokine to be tested to 60 nM in KB. Dilute mAbl to 50 pg/mL in the 60 nM chemokine solution (tube 1, final volume 700 pL). Add 300 pL of 60 nM chemokine solution in seven test tubes labeled 2—7. Mix the solution in tube 1 and serially transfer 300 pL (dilution 1 2) from tube 1 to tube 7 to obtain a dose—response of antibody in presence of 30 nM chemokine. Incubate these solutions for 30 min at 30 °C. 

Chemokines were produced as previously described (in E. coli, either as native proteins or as NusA fusions (MagistreUi et al., 2005 Proudfoot Borlat, 2000) or purchased from Peprotech (Rocky Hill, NJ). Biotinylated chemokines were obtained from Almac (Craigavon, Northern Ireland) and oligonucleotides from Thermo Fischer Scientific. 

Add 100 pL/well of 1 pg/mL biotinylated chemokine in PBS and incubate at room temperature for 1 h on a horizontal orbital shaker with gende agitation (450 rpm). 

Chemokine stimulation is blocked by adding to each well 15 pi of Streptavidin DB/biotinylated cAMP detection mix to each well. Plate is incubated for at least 1 h at RT in the dark. 

With the ELISA-Hke competition assay, it is possible to determine and compare the competitive potential of different chemokines for the same GAG ligand. Instead of a capturing antibody, as it is used in a classical ELISA, GAGs are coated on specially prepared GAG-binding plates (Iduron) that adsorb GAGs without further chemical modifications while retaining their natural protein-binding characteristics. Biotinylated chemokines are incubated on these plates and can be fiirther competed off with other nonlabeled chemokines, proteins, or mutants. Therefore, biotinylation of the chemokines is an essential first step. After competition, the nondisplaced biotinylated chemokine is detected with streptavidin horseradish peroxidase... 

Exchange the buffer of your sample to 0.1 M MES using centricons with a cutoff of 3 kDa to provide optimum reaction conditions for biotinylation. Incubate the chemokine with 20 molar excess of EZ link... 

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