BACKGROUND: Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. METHODS: Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. RESULTS: All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. CONCLUSIONS: With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc.

• Klíčová slova
• NAALADase, glutamate carboxypeptidase II, in vivo imaging, monoclonal antibody, prostate cancer,
• MeSH
• antigeny povrchové * imunologie   MeSH
• glutamátkarboxypeptidasa II * antagonisté a inhibitory   imunologie   MeSH
• lidé MeSH
• monoklonální protilátky imunologie   farmakologie   MeSH
• myší monoklonální protilátky imunologie   farmakologie   MeSH
• myši MeSH
• nádory prostaty * farmakoterapie   imunologie   MeSH
• prostata * imunologie   patologie   MeSH
• teranostická nanomedicína metody   MeSH
• xenogenní modely - testy protinádorové aktivity metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové * MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II * MeSH
• J591 monoclonal antibody MeSH Prohlížeč
• monoklonální protilátky MeSH
• myší monoklonální protilátky MeSH

BACKGROUND: Poor semen quality is one of the main causes of infertility. We have generated a set of monoclonal antibodies to human sperm and used them to investigate sperm quality. Some of these antibodies found differences in the expression of proteins between normal sperm and pathological sperm displaying severe defects. One of them was the Hs-14 antibody. The aim of this paper was to determine the target protein of the Hs-14 monoclonal antibody and to investigate the expression of the Hs-14-reacting protein on the sperm of asthenozoospermic men with sperm motility defect and of healthy normozoospermic men. METHODS: Indirect immunofluorescence, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry. RESULTS: The Hs-14 antibody binds fibronectin, β-tubulin and valosin-containing protein - new name for this protein is transitional endoplasmic reticulum ATPase (TERA). Since the Hs-14 reaction with TERA remained the strongest at the highest antibody dilution, and Hs-14 consistently labelled the same spot or band as the monospecific anti-TERA antibody on immunoblots, we assume that TERA is an Hs-14-specific protein. Binding of fibronectin and β-tubulin might represent nonspecific cross-reactivity or Hs-14 reaction with similar epitopes of these proteins. A significant difference (P < 0.001) in immunofluorescence staining with Hs-14 was found between the normozoospermic and asthenozoospermic men. CONCLUSION: The Hs-14 antibody enables discrimination between sterile or subfertile asthenozoospermic and fertile normozoospermic men. Decreased levels of TERA in men can be used as a biomarker of reduced fertility.

INTRODUCTION: La pauvre qualité de la semence est l’une des causes d’infertilité. Nous avons généré une série d’anticorps monoclonaux contre le sperme humain et nous l’avons utilisée pour examiner la qualité du sperme. Certains de ces anticorps ont montré des différences d’ expression des protéines entre le sperme normal et le sperme pathologique qui a des défauts sévères. L’un d’eux a été l’anticorps Hs-14. Le but de cet article était de déterminer la protéine cible de l’anticorps monoclonal Hs-14 et d’établir l’expression de la protéine réagissant avec Hs-14 sur le sperme des hommes asthénozoospermiques qui ont des défauts de la mobilité du sperme et sur celui des hommes normozoospermiques. MÉTHODES: Immunofluorescence indirecte, electrophorèse sur gel polyacrylamide à une ou deux dimensions, immunoblotting et spectrométrie de masse. RÉSULTATS: L’anticorps Hs-14 s’attache à la fibronectine, à la β-tubuline et à la protéine TERA (ATPase transitoire de réticulum endoplasmique). Etant donné que la réaction du Hs-14 avec TERA a été la plus forte à la dilution la plus grande de l’anticorps, et que Hs-14 marquait systématiquement la même tache ou bande que l’anticorps mono-spécifique anti-TERA sur les immunoblots, nous supposons que TERA est une protéine spécifique pour Hs-14. L’attachement à la fibronectine et à la β-tubuline pourrait représenter une réaction croisée non spécifique ou la réaction du Hs-14 avec des épitopes similaires de ces protéines. Une différence significative (P < 0.001) en immunofluorescence avec Hs-14 a été révélée entre hommes normozoospermiques et asthénozoospermiques. CONCLUSIONS: L’anticorps Hs-14 permet de différencier les hommes stériles ou subfertiles asthénozoospermiques des hommes fertiles normozoospermiques. Les niveaux de la TERA chez les hommes pourraient être utilisés comme un marqueur biologique d’une fertilité réduite.

• Klíčová slova
• Acrosome, Asthenozoospermia, Human spermatozoa, Monoclonal antibody, Transitional endoplasmic reticulum ATPase,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. METHODS: Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. RESULTS: Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. CONCLUSION: GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.

• MeSH
• akrozom metabolismus   MeSH
• energetický metabolismus MeSH
• flagella metabolismus   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy analýza   genetika   fyziologie   MeSH
• interakce spermie a vajíčka MeSH
• lidé MeSH
• motilita spermií MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• prasata metabolismus   MeSH
• spermatogeneze MeSH
• spermie metabolismus   MeSH
• zona pellucida metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glyceraldehyd-3-fosfátdehydrogenasy MeSH