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Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification
Tonge Ebai1, Felipe Marques Souza de Oliveira1, Liza Löf1
1Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Clinical Chemistry
|July 2, 2017
Summary
Proximity Ligation Assay with Rolling Circle Amplification (PLARCA) enables sensitive protein detection at lower concentrations than ELISA. This method is adaptable for clinical use, improving early disease diagnosis.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunotechnology
Background:
- Detecting low-concentration plasma proteins is vital for early disease diagnosis.
- Current methods like sandwich ELISA have limitations in sensitivity for ultra-low concentrations.
- Proximity Ligation Assay with Rolling Circle Amplification (PLARCA) offers enhanced sensitivity and specificity.
Purpose of the Study:
- To evaluate PLARCA's performance in detecting proteins at low concentrations.
- To compare PLARCA with conventional sandwich ELISA and immuno-rolling circle amplification (iRCA).
- To assess PLARCA's adaptability for clinical instrumentation.
Main Methods:
- Utilized oligonucleotide-conjugated antibodies for target protein recognition.
- Employed rolling circle amplification (RCA) for signal amplification.
- Detected amplified products via colorimetric readout using a microplate reader.
Main Results:
- PLARCA demonstrated superior sensitivity, detecting lower protein concentrations than ELISA and iRCA.
- Achieved broader dynamic ranges for protein detection compared to ELISA.
- Successfully detected Interleukin-4 (IL-4) and Interleukin-6 (IL-6) at femtomolar concentrations in clinical samples.
Conclusions:
- PLARCA provides enhanced sensitivity and dynamic range for protein detection over ELISA.
- The PLARCA assay is adaptable to standard hospital and research laboratory instrumentation.
- PLARCA holds promise for improved early diagnosis through sensitive biomarker detection.
