2006
1TrademarksAffibody is a trademark owned and used by Affibody AB. Affibody is a registered trademark in Sweden, EU and USA. Trademark application is pending in Japan.
Affibody® Molecules – A Robust Scaffold
Engineered on a three-helix bundle Protein A domain scaffold
Small (58 aa, 6 kDa)
Stable (temperature, pH, proteases)
Simple structure (no S-S)
Highly soluble
Bacterial production or peptide synthesis
Ig-binding domains of Protein A
E AD CB
Affibody® Molecule Diversity
Randomization of 13 selected positions
Protein A-domainscaffold
3x109 Affibody®
library members
-20
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20
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60
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0 60 120 180 240 300 360 420 480 540 600
Time s
Resp. D
iff.
RU
Nanomolar Affinities to a Variety of Targets
n Target origin• Human proteins
TNFα: <1 nMHER2: 22 pM
• Viral proteinsgp120: 30–70 nM
• Bacterial proteinsTaq Polymerase: 25 nM
n Target size• From 4 to 800 kDa One Target Protein
- Different Binding Properties
Flexibility – Engineering of Affibody® Molecules
n
α-X α-Y
SH
Multimeric
Bi-specificSite-specific modifications
Fusion protein
Biotechnology Applications
ELISA
Affibody® molecule as the capture reagentEasily coated on plasticsWorks well in pair with antibodySensitivity in the pg/ml range
HRP HRP
Immunoprecipitation
Affibody® molecule as capture reagent on solid phase - pull out desired molecule from a complex mixSingle step precipitationNo interference by the 50 kDa heavy and 25 kDa light chains of an immuno-precipitating antibody in the Western blot
n1 bead
Conjugated Affibody®
molecule as ”one step reagent”Unique C-terminal cysteine for directed modification
• Fluorescence labeling• Biotinylation• HRP conjugation
Low background staining and selective binding
Immunofluorescence Cell and Tissue Staining
Fluorescein conjugatedAnti-HER2 Affibody®
molecule staining HER2 expressing human SKOV-3 cell line
HRP conjugatedAnti-HER2 Affibody®
molecule staining tumor tissue
Fluorescein conjugatedAnti-HER2 Affibody®
molecule staining tumor tissue
Immunohistochemistry - Xenograft TumorsHER-2 expression in frozen tissue sections of SK-OV-3 xenograft tumors
Immunohistochemistry – Paraffin Embedded Tissue Sections
HRP conjugated Anti-Insulin Affibody® molecule staining staining pparaffin embedded normal human pancreas
HRP conjugatedAffibody® molecule as convenient ”one step reagent”Low background staining and selective binding
Flow Cytometry
Red line: Oregon® Green-conjugated Anti-EGFR Affibody® moleculeBlack: Negative control
Robust alternative to antibodies
Protein Microarrays
Affibody® molecule as capture reagentEasily coated on most types of commercially available slides for proteins
Affinity Separation
n Isolation of pure target protein
n Removal of the target protein
n Recovery of the flow through fraction for further analysis
Depletion of HSA from Serum
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ElutionSample injection Equilibration
FT fraction Eluted fraction
Overlay chromatograms of repeated affinity removal of HSA from serum. The chromatograms represent run number 1, 50, and 300 after consecutive injections of 300 µl of five times diluted human serum on an 0.37 ml Anti-HSA Affibody® column.
1 2 3 4 5 6 7 8 9
SDS-PAGE analysis of flow-through fractions (FT) and eluted fractions after repeated affinity removal of HSA from serum. Lane 1: Untreated 5x diluted serum sample; lane 2: FT run 1; lane 3: FT run 75; lane 4: FT run 150; lane 5: FT run 225; lane 6: FT run 300; lane 7: eluate run 1; lane 8: eluate run 300;lane 9: HSA standard.
n Extreme stability• Reversible folding
n High specificity• Binding surface large fraction of total surface
n Engineering flexibilityn Cost-efficient production
• Bacterial expression or chemical synthesis
n Unique patent position• No overlap with conventional aptamer,
monoclonal and domain antibody patents
Affibody® Molecules - Key Competitive Features
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