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Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc.

ABSTRACT: Studies of post-translational modification by beta-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc-specific antibodies that can be used for detection, isolation and site localization. We have obtained a large panel of O-GlcNAc-specific IgG monoclonal antibodies having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc-modified proteins, including a large number of new glycoproteins. A substantial number of the glycoproteins were enriched by only one of the antibodies. This observation, combined with the results of inhibition ELISAs, suggests that the antibodies, in addition to their O-GlcNAc dependence, also appear to have different but overlapping local peptide determinants. The monoclonal antibodies made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model.

SUBMITTER: Teo CF

PROVIDER: S-EPMC2857662 | biostudies-other | 2010 May

REPOSITORIES: biostudies-other

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Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc.

Teo Chin Fen CF Ingale Sampat S Wolfert Margreet A MA Elsayed Galal A GA Nöt Laszlo G LG Chatham John C JC Wells Lance L Boons Geert-Jan GJ

Nature chemical biology 20100321 5

Studies of post-translational modification by beta-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc-specific antibodies that can be used for detection, isolation and site localization. We have obtained a large panel of O-GlcNAc-specific IgG monoclonal antibodies having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to t ...[more]

PMID: 20305658

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Project description:BackgroundOrthopedia homeobox (OTP) has shown to be a useful prognostic marker to predict outcome in pulmonary carcinoids, which is also supported by the World Health Organization. However, the discontinuation of the initially used polyclonal antibody and absence of a reliable routinely applicable monoclonal OTP antibody hampers implementation in routine diagnostics. Here, new monoclonal antibodies directed against OTP were developed and verified on formalin-fixed paraffin-embedded tissue of pulmonary neuroendocrine tumors (NETs) for clinical diagnostics.MethodsOTP specific monoclonal antibodies were produced from mice immunised with a recombinant human OTP protein fragment. Enzyme-linked immunosorbent assay (ELISA) positive hybridomas were evaluated using immunohistochemistry (IHC). Following epitope-mapping and isotyping, purified monoclonal antibodies were validated for IHC in formalin-fixed paraffin-embedded tissues, the optimal dilution was determined, and results were cross validated with the OTP polyclonal antibody (HPA039365, Atlas Antibodies). Staining protocols were optimized on two automated staining platforms and performance was harmonized using a tissue microarray (TMA).ResultsTwo clones (CL11222 and CL11225) were selected for purified monoclonal antibody (mAb) production. Intratumor heterogeneity assessment revealed similar performance for both clones. While clone CL11225 displayed a unique epitope compared to those present in the polyclonal antibody, this clone performed most similar to the polyclonal antibody. Cross-platform assessment revealed an excellent agreement for clone CL11225 while clone CL11222 showed somewhat discordant results on Dako.ConclusionsNew monoclonal OTP specific antibodies have been developed and verified on different automated immunohistochemical staining platforms. The OTP specific monoclonal antibodies showed excellent agreement with the often-used polyclonal antibody allowing application in routine diagnostics.

Dataset Information

Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc.

Publications

Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc.

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