A Highly Sensitive Immunoenzymometric Assay for the Determination of Angiogenin

A Highly Sensitive Immunoenzymometric Assay for the Determination of Angiogenin Introduction Materials and Methods Materials Nunc Immuno plates II were obtained from Nunc (Roskilde, Denmark). Horseradish peroxidase1) was obtained from Boehringer Mannheim (Mannheim, Germany) and 2,2'-azino-bis(3ethyl-benzthiazoline-6-sulphonic acid (ABTS) from Sigma (Deisenhofen, Germany). Human recombinant angiogenin was kindly donated by Behringwerke, Marburg. Blood samples were supplied by Prof. Kleesiek, Herzzentrum Bad Oeynhausen-(Bad Oeynhausen, Germany). 5-[2,3-bis(palmitoyloxy)propyl]-Npalmitoyl-cysteinyl-serinyl-glycine-succinimidyl-ester (Pam3 CysSerGlyOSu) and S-[(2J?S)-2,3-bis(palmiloyloxy)-propyl]-Npalmitoyl-cysteinyl-lysyl-lysyl-lysyl-lysine (Pam3Cys-Ser-Lys4) were kindly donated by Prof. Jung (Tübingen, Germany). Methods Preparation of the S-[2t3,bis(palmitoyloxy)propyl]-N-palmitoyl-cysfeinyl-sennyl-glycine/angiogenin (Pam3CysSerGIy/angiogenin) conjugate Recombinant angiogenin (0.5 mg) was mixed with 5mg Pam3CysSerGlyOSu in 500 freshly distilled dimethylformamide and incubated under continuous stirring for 15 h at room temperature. After removal of the dimethylformamide, the product was dissolved in /-butanol/water (3 + 1, by vol.) and lyophilised (10). Angiogenin is a single chain Afr 14100 protein, first isolated and characterised from a HT-29 human adenocarcinoma cell line (1). It has been shown to be an inducer of vascular growth. The protein has 35% identity with pancreatic ribonuclease (2) and has been shown to inhibit protein synthesis in vitro (3). Saxena et al. described angiogenin as a cytotoxic, t-RNAspecific ribonuclease iii the RNase A superfamily (4). Angiogenic and ribonucleolytic activities are blocked by a tight-binding placental ribonuclease inhibitor (5). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Chemistry and Laboratory Medicine de Gruyter

A Highly Sensitive Immunoenzymometric Assay for the Determination of Angiogenin

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Publisher
de Gruyter
Copyright
Copyright © 2009 Walter de Gruyter
ISSN
1434-6621
eISSN
1437-4331
DOI
10.1515/cclm.1993.31.8.513
Publisher site
See Article on Publisher Site

Abstract

Introduction Materials and Methods Materials Nunc Immuno plates II were obtained from Nunc (Roskilde, Denmark). Horseradish peroxidase1) was obtained from Boehringer Mannheim (Mannheim, Germany) and 2,2'-azino-bis(3ethyl-benzthiazoline-6-sulphonic acid (ABTS) from Sigma (Deisenhofen, Germany). Human recombinant angiogenin was kindly donated by Behringwerke, Marburg. Blood samples were supplied by Prof. Kleesiek, Herzzentrum Bad Oeynhausen-(Bad Oeynhausen, Germany). 5-[2,3-bis(palmitoyloxy)propyl]-Npalmitoyl-cysteinyl-serinyl-glycine-succinimidyl-ester (Pam3 CysSerGlyOSu) and S-[(2J?S)-2,3-bis(palmiloyloxy)-propyl]-Npalmitoyl-cysteinyl-lysyl-lysyl-lysyl-lysine (Pam3Cys-Ser-Lys4) were kindly donated by Prof. Jung (Tübingen, Germany). Methods Preparation of the S-[2t3,bis(palmitoyloxy)propyl]-N-palmitoyl-cysfeinyl-sennyl-glycine/angiogenin (Pam3CysSerGIy/angiogenin) conjugate Recombinant angiogenin (0.5 mg) was mixed with 5mg Pam3CysSerGlyOSu in 500 freshly distilled dimethylformamide and incubated under continuous stirring for 15 h at room temperature. After removal of the dimethylformamide, the product was dissolved in /-butanol/water (3 + 1, by vol.) and lyophilised (10). Angiogenin is a single chain Afr 14100 protein, first isolated and characterised from a HT-29 human adenocarcinoma cell line (1). It has been shown to be an inducer of vascular growth. The protein has 35% identity with pancreatic ribonuclease (2) and has been shown to inhibit protein synthesis in vitro (3). Saxena et al. described angiogenin as a cytotoxic, t-RNAspecific ribonuclease iii the RNase A superfamily (4). Angiogenic and ribonucleolytic activities are blocked by a tight-binding placental ribonuclease inhibitor (5).

Journal

Clinical Chemistry and Laboratory Medicinede Gruyter

Published: Jan 1, 1993

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