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Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

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Published on: October 20, 2018

Detection of post-translational modifications using solid-phase proximity ligation assay.

Felipe Marques Souza de Oliveira¹, Stefan Mereiter², Peter Lönn¹

¹Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

New Biotechnology

|November 5, 2017

Summary

A new assay, solid-phase proximity ligation assay (SP-PLA), sensitively detects low-abundant post-translational modifications (PTMs) in biological samples. This method offers improved performance over ELISAs for PTM biomarker discovery.

Keywords:

CD44 E-cadherin EGFR Gastric cancer Glycosylation Phosphorylation Post-translational modifications Protein detection Solid-phase proximity ligation assay (SP-PLA)p53

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Area of Science:

Biochemistry
Molecular Biology
Biotechnology

Background:

Post-translational modifications (PTMs) are crucial for regulating protein function and cellular processes.
Aberrant PTMs are linked to various diseases, including malignancies, highlighting their potential as diagnostic biomarkers.
Sensitive detection methods for low-abundance PTMs are needed for advancing PTM-based diagnostics.

Purpose of the Study:

To investigate the utility of solid-phase proximity ligation assay (SP-PLA) for sensitive detection of diverse PTMs.
To evaluate SP-PLA's performance against established methods like ELISAs.
To establish SP-PLA as a viable tool for PTM biomarker development.

Main Methods:

Development and application of solid-phase proximity ligation assay (SP-PLA).
Utilized antibodies and lectins as affinity binders for target recognition.
Tested SP-PLA for the detection of glycosylated CD44/E-Cadherin and phosphorylated p53/EGFR in lysates and sera.

Main Results:

SP-PLA demonstrated a superior dynamic range and limit of detection compared to standard ELISAs.
Successfully detected various post-translational modifications, including glycosylation and phosphorylation.
The assay proved effective in both tissue lysates and serum samples.

Conclusions:

Solid-phase proximity ligation assay (SP-PLA) is an effective method for sensitive PTM detection.
SP-PLA offers advantages over traditional ELISAs for analyzing low-abundance PTMs.
This technique provides a foundation for developing novel diagnostic and prognostic biomarkers based on PTMs.