Introduction N d N N N l O 27 32 S S S S S S 5 23 37 23 2 5 10 22 2 5 10 22 24 28 4 24 6 7 23 1 12 17 Table 1 Characteristics of therapeutic heparinoids Heparinoid: generic name (brand name) Composition (and method of heparin modification) Molecular weight, kDa Heparin Heparin 5.0–30.0 Dalteparin (Fragmin) LMWH (deaminative cleavage with nitrous acid) 4.0–6.0 Nadroparin (Fraxiparine) LMWH (deaminative cleavage with nitrous acid) 2.4–7.2 Enoxaparin (Clexane, Lovenox) LMWH (β-eliminative cleavage by alkaline treatment) 3.5–5.5 Tinzaparin (Innohep, Logiparin) LMWH (β-eliminative cleavage by heparinase) 3.4–5.6 Sulodexide (Vessel Due F) a <8.0 Danaparoid (Orgaran) 84% HS, 12% DS, 4% CS 4.0–10.0 Fondaparinux (Arixtra) 31 43 3 10 8 1,728 All drugs are derived from porcine intestinal mucosa, with the exception of the chemically synthesized pentasaccharide fondaparinux. LMWH HS DS CS a 42 17 Materials and methods Heparinoids 1 Quantification of glycosaminoglycans by Farndale assay 13 Determination of glycosaminoglycan classes by agarose gel electrophoresis w v 2 2 w v 38 Enzymatic digestion of heparinoids 2 2 Flavobacterium heparinum Proteus vulgaris 11 11 Determination of glycosaminoglycan size by polyacrylamide gel electrophoresis 36 w v v v 38 Production of anti-heparin antibodies 2 41 8 43 39 et al. 15 8 41 Table 2 Characteristics of epitope-specific anti-heparin antibodies Antibody H H DP gene GAG used for selection Essential modifications Inhibitory modifications HS4C3 GRRLKD 3 38 41 N   O O O HS3A8 GMRPRL 3 38 8 N   C5-epimerization O O HS4E4 HAPLRNTRTNT 3 38 8 N O a O N C5-epimerization LKIV69 GSRSSR 3 38 43 N O C5-epimerization (likely) O EW3D10 GRTVGRN 3 38 39 b  EW4G2 GKVKLPN 3 38 39 b  NS4F5 SGRKGRMR 3 53 c N   C5-epimerization O O AO4B08 SLRMNGWRAHQ 3 47 15 N   C5-epimerization O d O RB4EA12 RRYALDY 3 32 15 N   N O MPB49 (control) WRNDRQ 3 38 – –  H H et al. GAG HS a O 16 b 8 39 c et al. d O 16 Enzyme-linked immunosorbent assay 41 w v v v w v v v p 2 43 Immunofluorescence staining 43 Results General biochemical analysis of heparinoids 1 1 42 Fig. 1 Agarose gel electrophoresis of heparinoids. The different classes of GAGs in the drugs were visualized by a combined azure A–silver staining procedure. The standard contains 40 ng HS, 20 ng DS, and 20 ng CS  2 O 2 Fig. 2 a b  Characterization of heparinoids using anti-heparin antibodies 3 17 N N Table 3 Reactivity of anti-heparin antibodies with heparinoids (ELISA)   HS4C3 HS3A8 HS4E4 LKIV69 EW3D10 EW4G2 NS4F5 AO4B08 RB4EA12 Heparin 100 100 100 100 100 100 100 100 100 Dalteparin 75 ± 6 44 ± 4 8 ± 5 3 ± 1 33 ± 6 49 ± 8 0 ± 0 16 ± 4 13 ± 2 Nadroparin 29 ± 3 5 ± 2 4 ± 3 0 ± 0 3 ± 1 7 ± 4 0 ± 0 1 ± 1 1 ± 1 Enoxaparin 80 ± 9 56 ± 8 7 ± 3 10 ± 2 36 ± 8 26 ± 7 1 ± 1 28 ± 7 21 ± 6 Tinzaparin 84 ± 1 55 ± 2 8 ± 3 44 ± 10 45 ± 12 74 ± 1 9 ± 2 20 ± 3 25 ± 8 Sulodexide 102 ± 5 71 ± 11 283 ± 21 51 ± 7 43 ± 10 100 ± 3 0 ± 0 102 ± 15 26 ± 8 Danaparoid 69 ± 11 22 ± 3 58 ± 17 2 ± 2 2 ± 1 1 ± 1 0 ± 0 63 ± 6 3 ± 1 n Heparinoids could be ranked according to their reactivity with antibodies. For instance, reactivity of antibody EW4G2 was highest with heparin and sulodexide, followed by tinzaparin, dalteparin, and enoxaparin. Reactivity with the other heparinoids was very low or absent. Antibody LKIV69 reacted strongly with heparin, followed by sulodexide and tinzaparin, whereas other heparinoids reacted poorly. Reactivity of antibody AO4B08 was highest with heparin and sulodexide, followed by danaparoid and enoxaparin/tinzaparin/dalteparin, whereas reactivity with nadroparin was absent. These data indicate that distinct heparin domain structures are present in different commercially available heparinoids. S S S S S S S 4 N N O O N O O O i.e. N O O N O Table 4 Reactivity of anti-heparin antibodies with heparin and fondaparinux (competition ELISA)   HS4C3 HS3A8 HS4E4 LKIV69 EW3D10 EW4G2 NS4F5 AO4B08 RB4EA12 Heparin 0.3 ± 0.1 0.4 ± 0.1 2.7 ± 0.1  0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 1.2 ± 0.2 <0.01 Fondaparinux 1.7 ± 0.2 1.3 ± 0.1  >200 10.3 ± 0.8 0.2 ± 0.0 1.5 ± 0.5  11.7 ± 0.8  12.3 ± 3.8 21.5 ± 0.5 Ratio fondaparinux/heparin 5.7 3.3 >74 51.5 2 15 117 10.3 >2150 50 n 3 e.g. e.g. e.g. Fig. 3 Bar G BC T  Discussion N O O et al. N O O O O i.e. N e.g. 2 23 3 4 4 23 O 2 23 6 31 6 19 7 23 1 14 21 25 30 33 34 40 3 20 29 18 e.g. 9 26 26 35 39 35 Finally, our antibodies may be used for fast industrial screening of heparinoids. A quality check of heparinoid batches may be performed using the antibodies since each heparinoid shows a specific immunoprofile. In conclusion, this study presents a novel way of characterizing heparinoids using immunoprofiling. Reactivity with antibodies was found indicative for chemical and biological aspects of the heparinoids, and may therefore be used for a fast and simple screening of industrial batches for defined characteristics.