Development of homogeneous immunoassays based on protein fragment complementation.
We demonstrate a functional in vitro proof-of-principle homogeneous assay capable of detecting small (<1kDa) to large (150kDa) analytes using TEM-1 beta-lactamase protein fragment complementation. In the assays reported here, complementary components are added together in the presence of analyte and substrate resulting in colorimetric detection within 10-min. We demonstrate the use of functional mutations leading to either increased enzymatic activity, reduced fragment self-association or increased inhibitor resistance upon analyte driven fragment complementation. Kinetic characterization of the resulting reconstituted enzyme illustrates the importance of balancing increased enzyme activity with fragment self-association, producing diagnostically relevant signal-to-noise ratios. Complementation can be utilized as a homogeneous immunoassay platform for the potential detection of a range of analytes including, antibodies, antigens and biomarkers.
de las Heras
Rachel
R
Panbio Limited, Advanced Technologies Division, 532 Seventeen Mile Rocks Road, Sinnamon Park, Qld 4073, Australia.
Fry
Scott R
SR
Li
Jun
J
Arel
Ece
E
Kachab
Edward H
EH
Hazell
Stuart L
SL
Huang
Chang-Yi
CY
D002151:Calorimetry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007124:Immunoenzyme Techniques; D007700:Kinetics; D009154:Mutation; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D018440:beta-Lactam Resistance / Q000235:genetics; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1