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Analytically Sensit...
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Ebai, TongeUppsala universitet,Science for Life Laboratory, SciLifeLab,Molekylära verktyg
(författare)
Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification
- Artikel/kapitelEngelska2017
Förlag, utgivningsår, omfång ...
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2017-09-01
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Oxford University Press (OUP),2017
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printrdacarrier
Nummerbeteckningar
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LIBRIS-ID:oai:DiVA.org:uu-326672
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https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-326672URI
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https://doi.org/10.1373/clinchem.2017.271833DOI
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Språk:engelska
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Sammanfattning på:engelska
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Ämneskategori:ref swepub-contenttype
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Ämneskategori:art swepub-publicationtype
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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 by PLARCA and conventional sandwich ELISA or immuno RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared 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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Biuppslag (personer, institutioner, konferenser, titlar ...)
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de Oliveira, Felipe Marques SouzaUppsala universitet,Science for Life Laboratory, SciLifeLab,Molekylära verktyg(Swepub:uu)felma546
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Löf, LizaUppsala universitet,Science for Life Laboratory, SciLifeLab,Molekylära verktyg(Swepub:uu)lizlo931
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Wik, LottaUppsala universitet,Science for Life Laboratory, SciLifeLab,Molekylära verktyg(Swepub:uu)lowik226
(författare)
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Schweiger, CarolineCharité Comprehensive Cancer Center, University of Berlin, Berlin, Germany; Institute of Pathology, Medical University of Graz, Graz, Austria;
(författare)
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Larsson, AndersUppsala universitet,Biokemisk struktur och funktion(Swepub:uu)andlarss
(författare)
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Keilholtz, UlrichInstitute of Pathology, Medical University of Graz, Graz, Austria;
(författare)
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Haybaeck, JohannesCharité Comprehensive Cancer Center, University of Berlin, Berlin, Germany; Department of Pathology, Otto von Guericke University Magdeburg, Magdeburg, Germany.
(författare)
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Landegren, UlfUppsala universitet,Science for Life Laboratory, SciLifeLab,Molekylära verktyg(Swepub:uu)ulfland
(författare)
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Kamali-Moghaddam, MasoodUppsala universitet,Science for Life Laboratory, SciLifeLab,Molekylära verktyg(Swepub:uu)masookam
(författare)
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Uppsala universitetScience for Life Laboratory, SciLifeLab
(creator_code:org_t)
Sammanhörande titlar
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Ingår i:Clinical Chemistry: Oxford University Press (OUP)63:9, s. 1497-15050009-91471530-8561
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