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Forschungsdatenbank PMU-SQQUID

TaqManR proximity ligation technology for the detection of heterodimeric adhesion receptors on lymphocytes.
Gehwolf, R; Band, E; Trost, A; Iglseder, B; Trinka, E; Haschke-Becher, E; Kraus, J; Harrer, A;
J Immunol Methods. 2014; 404:81-86
Originalarbeiten (Zeitschrift)

PMU-Autor/inn/en

Gehwolf Renate
Harrer Andrea
Haschke-Becher Elisabeth
Iglseder Bernhard
Kraus Jörg
Trinka Eugen
Zurl Andrea

Abstract

TaqMan(R) proximity ligation technology (TaqMan(R) PLA) is an innovative advancement of immuno PCR. It allows a fast and quantitative detection of vicinal proteins or protein-protein interactions from cell lysates by combining antibody-antigen binding with a real-time PCR detection. We tested if TaqMan(R) PLA also was applicable to investigate and relatively quantitate adhesion receptor heterodimers such as the alpha-4/beta-1 integrin on the surface of intact cells. Both, alpha-4, beta-1 and the alpha-4/beta-1 heterodimer were detected on the surface of lymphocytes by TaqMan(R) PLA. Results were specific, reproducible and comparable to flow cytometric data. However, preciseness of reactions varied dependent on the antibody pairs used. Co-detection of proximate identical subunits suggested clusters of alpha-4 and/or beta-1 on the cell surface which we confirmed by microscopy. We conclude that real-time PCR-based TaqMan(R) PLA is of limited applicability to investigate heterodimeric receptor molecules such as the alpha-4/beta-1 integrin. Determination of an abundance ratio of alpha-4/beta-1 in relation to total alpha-4 or beta-1 was not possible and real-time detection did not allow conclusions on the surface distribution of molecules. The related in situ PLA developed for microscopy allows visualizing proximate protein interactions and might be an interesting alternative for research into receptor heterodimers and their surface distribution on immune cells.


Useful keywords (using NLM MeSH Indexing)

Antibodies/chemistry

Antigen-Antibody Reactions

Antigens, CD29/analysis*

Antigens, CD29/immunology

Cell Adhesion

Flow Cytometry

Fluorescent Dyes

Humans

Integrin alpha4/analysis*

Integrin alpha4/immunology

Oligonucleotide Probes/chemistry

Platelet Glycoprotein GPIb-IX Complex/analysis*

Platelet Glycoprotein GPIb-IX Complex/immunology

Primary Cell Culture

Protein Multimerization

Real-Time Polymerase Chain Reaction/methods*

Reproducibility of Results

Sensitivity and Specificity

T-Lymphocytes/cytology*

T-Lymphocytes/immunology


Find related publications in this database (Keywords)

Immuno-PCR
Proximity ligation technology
Lymphocytes
Receptor heterodimers
Alpha-4/beta-1 integrin