title: TaqManR proximity ligation technology for the detection of heterodimeric adhesion receptors on lymphocytes.
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.
authors.affiliation: Department of Geriatric Medicine, Christian-Doppler-Klinik, Paracelsus Medical University, Ignaz-Harrer-Str. 79, 5020 Salzburg, Austria; Institute of Tendon and Bone Regeneration, Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Strubergasse 22, 5020 Salzburg, Austria. Electronic address: renate.gehwolf@pmu.ac.at.
authors.forename: Renate
authors.initials: R
authors.lastname: Gehwolf
authors.affiliation: Department of Neurology, Christian-Doppler-Klinik, Paracelsus Medical University, Ignaz-Harrer-Str. 79, 5020 Salzburg, Austria. Electronic address: elisabeth.band@gmail.com.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Band
authors.affiliation: Department of Ophthalmology, St. Johanns Spital, Paracelsus Medical University, Müllner Hauptstrasse 48, 5020 Salzburg, Austria. Electronic address: a.zurl@salk.at.
authors.forename: Andrea
authors.initials: A
authors.lastname: Trost
authors.affiliation: Department of Geriatric Medicine, Christian-Doppler-Klinik, Paracelsus Medical University, Ignaz-Harrer-Str. 79, 5020 Salzburg, Austria. Electronic address: b.iglseder@salk.at.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Iglseder
authors.affiliation: Department of Neurology, Christian-Doppler-Klinik, Paracelsus Medical University, Ignaz-Harrer-Str. 79, 5020 Salzburg, Austria. Electronic address: e.trinka@salk.at.
authors.forename: Eugen
authors.initials: E
authors.lastname: Trinka
authors.affiliation: Central Laboratory, Christian-Doppler-Klinik, Paracelsus Medical University, Ignaz-Harrer-Str. 79, 5020 Salzburg, Austria. Electronic address: e.haschke-becher@salk.at.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Haschke-Becher
authors.affiliation: Department of Neurology, Christian-Doppler-Klinik, Paracelsus Medical University, Ignaz-Harrer-Str. 79, 5020 Salzburg, Austria. Electronic address: joerg.kraus@salk.at.
authors.forename: Jörg
authors.initials: J
authors.lastname: Kraus
authors.affiliation: Department of Neurology, Christian-Doppler-Klinik, Paracelsus Medical University, Ignaz-Harrer-Str. 79, 5020 Salzburg, Austria. Electronic address: a.harrer@salk.at.
authors.forename: Andrea
authors.initials: A
authors.lastname: Harrer
mesh_terms: D000906:Antibodies / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D002448:Cell Adhesion; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D039441:Integrin alpha4 / Q000032:analysis* / Q000276:immunology; D019012:Integrin beta1 / Q000032:analysis* / Q000276:immunology; D015345:Oligonucleotide Probes / Q000737:chemistry; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000032:analysis* / Q000276:immunology; D061251:Primary Cell Culture; D055503:Protein Multimerization; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D019012:Integrin beta1; D015345:Oligonucleotide Probes; D019038:Platelet Glycoprotein GPIb-IX Complex; C094990:adhesion receptor; D039441:Integrin alpha4
keywords: Alpha-4/beta-1 integrin; Immuno-PCR; Lymphocytes; Proximity ligation technology; Receptor heterodimers