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Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylär och morfologisk patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. (Rudbeck Laboratory)
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. (Rudbeck Laboratory)
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylär och morfologisk patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. (Rudbeck Laboratory)
Vise andre og tillknytning
2013 (engelsk)Inngår i: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 61, nr 11, s. 773-784Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.

sted, utgiver, år, opplag, sider
2013. Vol. 61, nr 11, s. 773-784
Emneord [en]
antibody, biotin, conjugation, protein detection, tissue microarray
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-211012DOI: 10.1369/0022155413502360ISI: 000326066300001OAI: oai:DiVA.org:uu-211012DiVA, id: diva2:665486
Tilgjengelig fra: 2013-11-20 Laget: 2013-11-19 Sist oppdatert: 2017-12-06bibliografisk kontrollert
Inngår i avhandling
1. Validation of antibodies for tissue based immunoassays
Åpne denne publikasjonen i ny fane eller vindu >>Validation of antibodies for tissue based immunoassays
2015 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

In situ protein detection in human tissues using antibodies reveals the cellular protein localization, and affinity-based proteomic studies can help to discover proteins involved in the development of diseases. However, antibodies often suffer from cross-reactivity, and the lack of positive and negative tissue controls for uncharacterized proteins complicates the mapping of the proteome. The aim of this thesis is thus to improve the methodology for validating antibodies used for immunostaining on formalin-fixed paraffin-embedded tissues.

Two of the papers include comparisons between mRNA-expression and immunostaining of corresponding protein. In paper I, ISH and IHC staining patterns were compared on consecutive TMA-slides. The study of well-characterized genes showed that ISH could be used for validation of antibodies. ISH was further used for antibody evaluation, and could validate four out of nine antibodies showing potentially interesting staining patterns. In paper III, transcriptomic data generated by RNA-sequencing were used to identify tissue specific expression in lymphohematopoietic tissues. An increased expression in one or more of these tissues compared to other tissue types was seen for 693 genes, and these were further compared to the staining patterns of corresponding proteins in tissues.

Antibody labeling is necessary for many immunoassays. In paper II, two techniques for antibody-biotinylation were compared, aiming to find a stringent labeling method for antibodies used for immunostaining on TMAs. The ZBPA-method, binding specifically to Fc-part of antibodies, was found to be superior to the Lightning Link-biotinylation kit targeting amine groups, since labeling of amine groups on stabilizing proteins in the antibody buffer causes unspecific staining.

The localization of the estrogen receptor beta (ERβ) in human normal and cancer tissues was studied in paper IV. Thorough evaluation of 13 antibodies using positive and negative control cell lines showed that only one antibody, PPZ0506, is specific for ERβ in all three immunoassays used. Contradictory to previously published data, tissue profiling using PPZ0506 showed that ERβ is expressed in a limited number of normal and cancer tissues.

In conclusion, the present investigations present tools for validation of antibodies used for large-scale studies of protein expression in tissues.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2015. s. 45
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1109
Emneord
Antibody validation, Conjugation, Estrogen receptor-beta, IHC, ISH, Lymphohematopoietic tissues, Proteomic, RNAseq, TMA, Transcriptomic
HSV kategori
Forskningsprogram
Patologi
Identifikatorer
urn:nbn:se:uu:diva-251344 (URN)978-91-554-9258-8 (ISBN)
Disputas
2015-06-13, Fåhreussalen, Rudbecklaboratoriet, hus C5, Dag Hammarskjölds väg 20, Uppsala, 12:30 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2015-05-21 Laget: 2015-04-15 Sist oppdatert: 2015-07-07

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