Analysis of labeling and comparison of the antigen binding potency of differently biotinylated antibody samples.

Two samples of a mouse anti-human interferon-gamma mIgG1 mAb were biotinylated using either a direct or a ZF5I-Q32C-MBP-BIO probe-mediated strategy and the resulting preparations compared with respect for their antigen binding potency. (A) Western blotting analysis of the samples and the probe, using a streptavidin horse radish peroxidase (HRP) conjugate. Lane 1: blank; lane 2: a sample of the anti-human interferon-gamma mIgG1 mAb biotinylated using a conventional amine reactive sulfo-NHS-ester-biotin reagent resulting in "global" biotinylation of both heavy (ca. 50 kDa) and light chains (ca. 25 kDa); lane 3: the ZF5I-Q32C-MBP-BIO probe alone, biotinylated using the same amine reactive sulfo-NHS-ester-biotin reagent; lane 4: a sample of the anti-human interferon-gamma mIgG1 mAb after photoconjugation to the ZF5I-Q32C-MBP-BIO probe (ca. 8 kDa) showing a selective biotinylation of only the heavy chains (ca. 50 + 8 kDa). A small amount of unreacted probe is also visible. (B) Biosensor analysis of the relative antigen binding potency of the biotinylated antibody preparations. Using a streptavidin coated biosensor chip, the biotinylated antibody proteins were selectively immobilized onto separate sensor chip surfaces, followed by injection over both surfaces of a common 7.5 nM solution of the antigen human interferon-gamma. This allowed for direct comparison of the effect on the antigen binding potency from the different biotinylation strategies. Sample (i): the anti-human interferon-gamma mIgG1 mAb biotinylated using a conventional amine reactive sulfo-NHS-ester-biotin reagent; sample (ii): the anti-human interferon-gamma mIgG1 mAb biotinylated via photoconjugation using the ZF5I-Q32C-MBP-BIO probe.