Biotinylation, heparin

Plasma was separated from heparinized blood samples, ahquoted and stored at — 80°C. All samples were then analyzed simultaneously with identical hatches of antibodies and other reagents. ELISAs for the detection of sICAM-1, sE-selectin and IL-12 were performed with ELI-Pairs (Diaclone, France distributed by Holzel, Germany). 96-well Maxisorp plates (Nunc) were coated overnight with capture antibodies at 4°C. After 1 h sample incubation, detection was performed with biotinylated anti-ICAM-1, anti-sE-selectin or anti IL-12 detection antibodies followed by streptavidin-horse raddish peroxidase and a TMB color reaction. Light absorption was detected with a Spectra ELISA reader at 450 nm. 

Fig. 33.5. The plot of the series resonance frequency of AT-cut crystal covered by neutravidin and with immobilized biotinylated 32-mer DNA aptamer selective to heparin binding site of thrombin as a function of thrombin and HSA concentration, respectively. The fundamental frequency of the crystal was 9MHz. The frequency was determined by HP4395A Network-Spectrum analyzer (Hewlet Packard, Colorado Springs, CO, USA). The crystal was placed in a flow cell developed by Thompson et al. [37] (experiment was performed by I. Grman in M. Thompson laboratory [75]).

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. 

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