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Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification.
Ebai, Tonge; Souza de Oliveira, Felipe Marques; Löf, Liza; Wik, Lotta; Schweiger, Caroline; Larsson, Anders; Keilholtz, Ulrich; Haybaeck, Johannes; Landegren, Ulf; Kamali-Moghaddam, Masood.
Afiliación
  • Ebai T; Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
  • Souza de Oliveira FM; Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
  • Löf L; Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
  • Wik L; Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
  • Schweiger C; Charité Comprehensive Cancer Center, University of Berlin, Berlin, Germany.
  • Larsson A; Institute of Pathology, Medical University of Graz, Graz, Austria.
  • Keilholtz U; Department of Medical Sciences, Biochemical Structure and Function, Uppsala University, Uppsala, Sweden.
  • Haybaeck J; Institute of Pathology, Medical University of Graz, Graz, Austria.
  • Landegren U; Charité Comprehensive Cancer Center, University of Berlin, Berlin, Germany.
  • Kamali-Moghaddam M; Department of Pathology, Otto von Guericke University Magdeburg, Magdeburg, Germany.
Clin Chem ; 63(9): 1497-1505, 2017 Sep.
Article en En | MEDLINE | ID: mdl-28667186
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas / Técnicas de Amplificación de Ácido Nucleico / Detección Precoz del Cáncer Tipo de estudio: Diagnostic_studies / Screening_studies Límite: Humans / Male Idioma: En Revista: Clin Chem Asunto de la revista: QUIMICA CLINICA Año: 2017 Tipo del documento: Article País de afiliación: Suecia Pais de publicación: Reino Unido
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas / Técnicas de Amplificación de Ácido Nucleico / Detección Precoz del Cáncer Tipo de estudio: Diagnostic_studies / Screening_studies Límite: Humans / Male Idioma: En Revista: Clin Chem Asunto de la revista: QUIMICA CLINICA Año: 2017 Tipo del documento: Article País de afiliación: Suecia Pais de publicación: Reino Unido