Huddinge, Sweden
Huddinge, Sweden

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Diswall M.,Sahlgrenska University Hospital | Gustafsson A.,Recopharma AB | Holgersson J.,Sahlgrenska University Hospital | Sandrin M.S.,University of Melbourne | Breimer M.E.,Sahlgrenska University Hospital
Xenotransplantation | Year: 2011

Background: αGal-specific lectins, monoclonal and polyclonal antibodies (Abs) are widely used in xenotransplantation research. Immunological assays such as immunohistochemistry, flow cytometry, Western blot and thin layer chromatography are often the only applicable characterization procedures when limited amount of tissue is available and biochemical characterization is impossible. Hence, detailed knowledge of the Ab/lectin carbohydrate-binding specificity is essential. Methods: The binding specificity of human blood group AB serum, three different affinity-purified human polyclonal anti-Gal Ab batches, and two anti-Gal mAb clones (TH5 and 15.101) as well as Griffonia simplicifolia isolectin B4 and Marasmius oreades agglutinin were examined for reactivity with glycolipid fractions isolated from human and pig (wild-type and α1,3GalT-KO) tissues using thin layer chromatogram and microtiter well binding assays. Results: All anti-Gal-specific reagents reacted with the pentaglycosylceramide Galα1,3nLc4, and several 6-12 sugar compounds in wild-type pig kidneys. However, their staining intensity with different αGal antigens varied considerably. Some, but not all, anti-Gal reagents cross-reacted with a pure iGb3 glycolipid reference compound. No reactivity with glycolipids isolated from α1,3GalT-KO pig small intestine or human tissues was found, confirming the specificity of the anti-Gal reagents in those tissues for α1,3Gal-epitopes produced by the α1,3GalT (GGTA1). Conclusions: Different anti-Gal reagents vary in their carbohydrate epitope specificity. Mono-/polyclonal Abs and lectins have different carbohydrate epitope fine specificity toward pig glycolipids as well as purified Galα1,3nLc4, and iGb3. Despite the difference in αGal specificity, all reagents were completely non-reactive with glycolipids isolated from α1,3GalT-KO pig small intestine. © 2011 John Wiley & Sons A/S.


Lindberg L.,AbSorber AB | Liu J.,AbSorber AB | Gaunitz S.,Karolinska University Hospital | Nilsson A.,Recopharma AB | And 3 more authors.
Glycobiology | Year: 2013

Assays for quantification, and methods for removal, of anti-A and anti-B antibodies are the key for the success of ABO incompatible organ transplantation programs. In order to produce tools that can be used as substrates in tests for anti-A/anti-B quantification and specificity determination or as affinity matrices in extracorporeal immunoadsorption (IA) columns, we engineered Chinese hamster ovary (CHO) cells secreting mucin-type fusion proteins carrying blood group A or B determinants on defined O-glycan core saccharide chains. Besides the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIgG2b) cDNA, CHO cells were transfected with plasmids encoding core 2 (β1,6GlcNAc-T1) or core 3 (β1,3GlcNAc-T6 and β1,3Gal-T5) enzymes together with α1,2Fuc-T1 or α1,2Fuc-T2 and the A or B gene-encoded α1,3GalNAcT or α1,3Gal-T, respectively. Selected clones with the correct glycophenotype were expanded and cultured in shaker flasks and Wave bioreactors. Western blotting was used to characterize purified fusion protein and liquid chromatography-mass spectrometry was used to characterize the released O-glycans. Clones producing PSGL-1/mIgG2b carrying O-glycans with A and B determinants on type 1 (Galβ3GlcNAc), type 2 (Galβ4GlcNAc) and type 3 (Galβ3GalNAcα) outer core saccharide chains were established. The conversion of CHO cells from exclusive inner core 1 (Galβ3GalNAc) to core 3 (GlcNAcβ3GalNAc) O-glycan producers was almost complete, whereas conversion to inner core 2 (GlcNAcβ6GalNAc) O-glycans was incomplete as was the α2-fucosylation of the core 1 chain. Sialylation may prevent these biosynthetic steps. The clinical utility of the blood group A and B substituted mucin-type fusion proteins as substrates in enzyme-linked immunosorbent assay or as affinity matrices in IA columns is explored. © 2012 The Author.


Gaunitz S.,Karolinska University Hospital | Liu J.,Gothenburg University | Nilsson A.,Recopharma AB | Karlsson N.,Gothenburg University | Holgersson J.,Gothenburg University
Glycoconjugate Journal | Year: 2014

The interaction between P-selectin glycoprotein ligand-1/mouse IgG 2b (PSGL-1/mIgG2b) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Siaα2-3Gal on different O-glycan chains and recombinant human influenza H5N1 A/Vietnam/1203/04 hemagglutinin was investigated with a Biacore biosensor. The fusion protein was produced by stable cell lines in large scale cultures and purified with affinity- and gel filtration chromatography. The C-P55 and 293-P cell lines were established by transfecting the Chinese hamster ovary (CHO)-K1 and Human embryonic kidney (HEK)-293 cell lines with plasmids encoding the PSGL-1/mIgG2b fusion protein, while the C-PSLex cell line was engineered by transfecting CHO-K1 cells with the plasmids encoding the core 2 β1,6GnT-I and FUT-VII glycosyltransferases. Glycosylation was characterized by lectin Western blotting of the proteins and liquid chromatography - mass spectrometry of released non-derivatized O-glycans. Biacore experiments revealed that PSGL-1/mIgG 2b is a good binding partner of H5. The binding curves displayed a slow dissociation indicating a multivalent binding. The H5 hemagglutinin binds with similar strength to PSGL-1/mIgG2b carrying mostly sialylated core 1 (clone C-P55), a mix of sialylated core 1 and sialylated lactosamine (clone 293-P) or mainly sialylated lactosamine (clone C-PSLex) O-glycans, indicating that this hemagglutinin is unable to discriminate between these structures. The potential use of the large, flexible PSGL-1/mIgG2b mucin-type fusion protein carrying Siaα2-3Gal as a multivalent inhibitor of influenza virus is discussed. © 2013 Springer Science+Business Media New York.


Patent
Recopharma AB | Date: 2014-09-24

The present invention provides compositions and methods for treating or preventing viral infections.


Patent
Recopharma AB | Date: 2011-09-29

The present invention provides compositions and methods for treating or preventing viral infections.


Patent
Recopharma AB | Date: 2011-12-21

The invention features ophthalmic formulations of mucin polypeptides to treat or prevent dry eye.


Patent
Recopharma AB | Date: 2010-12-22

The present invention provides compositions and methods for treating or preventing viral infections.


Patent
Recopharma AB | Date: 2010-12-22

The present invention provides compositions and methods for treating or preventing viral infections.


The present invention provides compositions and methods for augmenting vaccine immunogenicity using mucin-immunoglobulin fusion proteins.


Patent
Recopharma Ab | Date: 2014-03-04

The present invention provides compositions and methods for treating or preventing viral infections.

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