Tonge Ebai1, Felipe Marques Souza de Oliveira1, Liza Löf1, Lotta Wik1, Caroline Schweiger2,3, Anders Larsson4, Ulrich Keilholtz3, Johannes Haybaeck2,5, Ulf Landegren6, Masood Kamali-Moghaddam6. 1. Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. 2. Charité Comprehensive Cancer Center, University of Berlin, Berlin, Germany. 3. Institute of Pathology, Medical University of Graz, Graz, Austria. 4. Department of Medical Sciences, Biochemical Structure and Function, Uppsala University, Uppsala, Sweden. 5. Department of Pathology, Otto von Guericke University Magdeburg, Magdeburg, Germany. 6. Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; masood.kamali@igp.uu.se ulf.landegren@igp.uu.se.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Authors: Axel Klaesson; Karin Grannas; Tonge Ebai; Johan Heldin; Björn Koos; Mattias Leino; Doroteya Raykova; Johan Oelrich; Linda Arngården; Ola Söderberg; Ulf Landegren Journal: Sci Rep Date: 2018-03-29 Impact factor: 4.379
Authors: I Hernández-Neuta; F Neumann; J Brightmeyer; T Ba Tis; N Madaboosi; Q Wei; A Ozcan; M Nilsson Journal: J Intern Med Date: 2018-09-12 Impact factor: 8.989