Classification of protein profiles from antibody microarrays using heat and detergent treatment

Science for Life Laboratory Stockholm, School of Biotechnology, KTH - Royal Institute of Technology, Box 1031, SE-171 21 Solna, Sweden.
New Biotechnology (Impact Factor: 2.9). 10/2011; 29(5):564-70. DOI: 10.1016/j.nbt.2011.10.005
Source: PubMed


Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.

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Available from: Maja Neiman
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    • "Only anti-C1QC capture revealed bands that indicate off-target binding. Even though it remains to be further understood whether denaturation (conformational changes) or changes in protein assemblies (dissolving complexes) altered epitope accessibility, this finding supports that the enhanced or reduced effects of heat treatment detected by single binder assays [6] for these antibodies relates to more or less capture of the intended target protein. "
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    • "Coupling of antibodies to beads was performed as previously described [25] and 30 µl of each bead identity was coupled to 1.6 µg of a different antibody. First, beads were washed twice on a magnet with activation buffer (AB) (0.1 M NaH2PO4 (Merck), pH 6.2) and subsequently beads were resuspended in 50 µl of AB. "
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