Medizinische Universität Graz - Research portal
Selected Publication:
SHR Neuro Cancer Cardio Lipid Metab Microb
Ebai, T; Souza de Oliveira, FM; Löf, L; Wik, L; Schweiger, C; Larsson, A; Keilholtz, U; Haybaeck, J; Landegren, U; Kamali-Moghaddam, M.
Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification.
Clin Chem. 2017; 63(9):1497-1505
Doi: 10.1373/clinchem.2017.271833
[OPEN ACCESS]
Web of Science
PubMed
FullText
FullText_MUG
- Co-authors Med Uni Graz
- Haybäck Johannes
- Schweiger Caroline
- Altmetrics:
- Dimensions Citations:
- Plum Analytics:
- Scite (citation analytics):
- Abstract:
- Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals. Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader. We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor 15 (GDF-15) by PLARCA and conventional sandwich ELISA or immuno-RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared to ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA. PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories. © 2017 American Association for Clinical Chemistry.
- Find related publications in this database (using NLM MeSH Indexing)
- Antibodies, Immobilized - chemistry
- Early Detection of Cancer - methods
- Enzyme-Linked Immunosorbent Assay -
- Growth Differentiation Factor 1 - blood
- Growth Differentiation Factor 1 - chemistry
- Humans -
- Interleukins - blood
- Interleukins - classification
- Limit of Detection -
- Male -
- Neoplasms - blood
- Neoplasms - immunology
- Nucleic Acid Amplification Techniques -
- Prostatic Neoplasms - blood
- Prostatic Neoplasms - immunology
- Proteins - analysis
- Proteins - chemistry