Affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats.
The importance of the ligand presentation format for the production of protein capture microarrays was evaluated using different Affibody molecules, produced either as single 6 kDa monomers or genetically linked head-to-tail multimers containing up to four domains. The performances in terms of selectivity and sensitivity of the monomeric and the multidomain Affibody molecules were compared by immobilization of the ligands on microarray slides, followed by incubation with fluorescent-labeled target protein. An increase in signal intensities for the multimers was demonstrated, with the most pronounced difference observed between monomers and dimers. A protein microarray containing six different dimeric Affibody ligands with specificity for IgA, IgE, IgG, TNF-alpha, insulin, or Taq DNA polymerase was characterized for direct detection of fluorescent-labeled analytes. No cross-reactivity was observed and the limits of detection were 600 fM for IgA, 20 pM for IgE, 70 fM for IgG, 20 pM for TNF-alpha, 60 pM for insulin, and 10 pM for Taq DNA polymerase. Also, different sandwich formats for detection of unlabeled protein were evaluated and used for selective detection of IgA or TNF-alpha in human serum or plasma samples, respectively. Finally, the presence of IgA was determined using detection of directly Cy5-labeled normal or IgA-deficient serum samples.
Renberg
Björn
B
School of Biotechnology, Division of Molecular Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE - 106 91 Stockholm, Sweden.
Nordin
Jon
J
Merca
Anna
A
Uhlén
Mathias
M
Feldwisch
Joachim
J
Nygren
Per-Ake
PA
Karlström
Amelie Eriksson
AE
D019921:Biotinylation; D001798:Blood Proteins / Q000378:metabolism; D019281:Dimerization; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry; D007073:Immunoglobulin E / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D007328:Insulin / Q000737:chemistry; D008024:Ligands; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry
D001798:Blood Proteins; D005456:Fluorescent Dyes; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007328:Insulin; D008024:Ligands; D014409:Tumor Necrosis Factor-alpha; D007073:Immunoglobulin E