Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material.
A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 μl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.
Olink Bioscience, Dag Hammarskjölds väg 54A, 75183 Uppsala Sweden.
Martin
M
Lundberg
Stine Buch
SB
Thorsen
Erika
E
Assarsson
Andrea
A
Villablanca
Bonnie
B
Tran
Nick
N
Gee
Mick
M
Knowles
Birgitte Sander
BS
Nielsen
Eduardo
E
González Couto
Roberto
R
Martin
Olle
O
Nilsson
Christian
C
Fermer
Jörg
J
Schlingemann
Ib Jarle
IJ
Christensen
Hans-Jorgen
HJ
Nielsen
Björn
B
Ekström
Claes
C
Andersson
Mats
M
Gustafsson
Nils
N
Brunner
Jan
J
Stenvang
Simon
S
Fredriksson
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000097:blood; D014408:Biomarkers, Tumor / Q000097:blood*; D002272:Carcinoembryonic Antigen / Q000097:blood; D015179:Colorectal Neoplasms / Q000097:blood / Q000175:diagnosis*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D016209:Interleukin-8 / Q000097:blood; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D015999:Multivariate Analysis; D053481:Secretory Leukocyte Peptidase Inhibitor / Q000097:blood; D018709:Statistics, Nonparametric; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000097:blood; D042461:Vascular Endothelial Growth Factor A / Q000097:blood
D015295:Antigens, Tumor-Associated, Carbohydrate; D014408:Biomarkers, Tumor; C074601:CA 242 antigen; D002272:Carcinoembryonic Antigen; D016209:Interleukin-8; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D019715:Tissue Inhibitor of Metalloproteinase-1; D042461:Vascular Endothelial Growth Factor A
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