Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification

滚动圆复制 微量滴定板 寡核苷酸 邻近连接试验 辣根过氧化物酶 结扎 抗体 分子生物学 化学 靶蛋白 蛋白质A 色谱法 生物 DNA 生物化学 免疫学 基因 受体 DNA复制
作者
Tonge Ebai,Felipe Marques Souza de Oliveira,Liza Löf,Lotta Wik,Caroline Schweiger,Anders Larsson,Ulrich Keilholtz,Johannes Haybaeck,Ulf Landegren,Masood Kamali‐Moghaddam
出处
期刊:Clinical Chemistry [Oxford University Press]
卷期号:63 (9): 1497-1505 被引量:25
标识
DOI:10.1373/clinchem.2017.271833
摘要

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.
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