[Enriched cage systems for laying hens--legal minimum conditions and starting points for their scientific evaluation]. / Ausgestaltete Käfige in der Legehennenhaltung--rechtliche Rahmenbedingungen und Ansatzpunkte für ihre wissenschaftliche Beurteilung.

Recent developments in the European and national legislation regarding minimum standards for the keeping of laying hens will be causing changed conditions for egg production in the medium term. Enriched cages shall allow hens to better fulfill their needs than traditional battery cages. More space for the stretching of wings and legs, perches for resting, littered areas for scratching, pecking and dustbathing and nests for egg-laying behaviour shall allow layers to perform more of their normal behaviour patterns. However, regarding the sustainability of these systems there are different views and still a great lack of scientific data and practical experience. An overview is given on the development of European and national animal welfare legislation concerning laying hens, minimum requirements for enriched cages and the available evidence about animal behaviour, performance and health in these systems. Furthermore, a current project at the School of Veterinary Medicine Hannover on the animal welfare assessment of enriched cages, specifically on the Aviplus cage system, which was one of the first systems on the market and is in accordance with EU-directive, is introduced.

Rapid analytical tryptic mapping of a recombinant chimeric monoclonal antibody and method validation challenges.

A rapid and reproducible analytical tryptic mapping method was developed as an identity test for a recombinant chimeric monoclonal antibody for lot release testing. The unfolding, reduction, carboxymethylation, trypsin digestion, and reversed-phase (RP) HPLC steps were optimized to provide a reproducible method. The optimized method requires 30 min for unfolding the protein, 30 min for carboxymethylation, 4 h for digestion with TPCK-trypsin and 140 min for RPHPLC analysis. The total time required is less than 8 h compared to conventional procedures, which must be performed over several days. The optimized method was validated for its precision, recovery, specificity, and robustness. The precision of the method was determined by repeatability and intermediate precision experiments. Relative standard deviation (RSD) values were < or = 10% for the relative peak areas of marker peaks. The mean recovery of these marker peaks was 88.4%. The specificity was demonstrated by the unique tryptic mapping patterns obtained compared with several other monoclonal antibodies. Robustness was demonstrated by the relative insensitivity of the tryptic map to small deliberate changes in key method parameters. Excessive relative peak area variability observed for one peak (RSD 52%) was traced to adsorption to glass autosampler vials. This variability was substantially reduced (RSD 11%) by substituting polypropylene autosampler vials. The data demonstrate that this method may be applicable to a wide range of pharmaceutically relevant monoclonal antibodies.

Factor VIII procoagulant protein interacts with phospholipid vesicles via its 80 kDa light chain.

In a previous report, we detailed fractionation of polyclonal human anti-Factor VIII:C into a component directed exclusively against the phospholipid-binding site on Factor VIII (PL-site antibody) and another directed at other sites (non-PL-site antibody). The location on the F.VIII molecule of its PL-binding site has now been studied by two different methods using this fractionated 125I-labelled anti-F.VIII:C Fab'. The first method was modified from that of Weinstein et al. (Proc Natl Acad Sci USA 1981; 78: 5137-41), involving electrophoresis of F.VIII peptide-125I-Fab' A/F.VIII immunocomplexes in SDS-polyacrylamide gels. PL-site antibody reacted with F.VIII peptides of apparent Mr approximately 80 kDa and sometimes 160 kDa in plasma and concentrate, but not with larger peptides. Non-PL-site antibody, however, reacted with a range of peptides of apparent Mr 90 kDa to 280 kDa. In addition, when purified F.VIII containing heavy and light chains (HC + LC), and isolated LC peptides were analysed, PL-site antibody bound to LC peptides whereas non-PL-site antibody did not. The second method used the antibody pools in immunoradiometric assays (IRMA's) of purified F.VIII peptides. Both labels measured similar amounts of F.VIII:Ag in a sample of purified F.VIII containing both HC and LC; on assaying an HC preparation, however, PL-site label measured only 2% of F.VIII:Ag found by non-PL-site label, indicating that PL-binding sites are absent in HC preparations. These results indicate that F.VIII binds to PL via its 80 kDa light chain.

Localization of a factor VIII binding domain on a 34 kilodalton fragment of the N-terminal portion of von Willebrand factor.

Factor VIII (F.VIII) was tested for its ability to bind in solid phase system to von Willebrand Factor (vWF) or fragments obtained with Staphylococcus aureus V-8 protease, ie, SpIII (N-terminal), SpI (central), and SpII (C-terminal). Bound F.VIII was estimated in situ by clotting and chromogenic assays. F.VIII bound in a dose-dependent manner to immobilized vWF and SpIII but not to SpII or SpI. Binding was inhibited by 0.25 mol/L CaCl2 as well as by an excess of vWF or SpIII. Accordingly, immobilized F.VIII specifically bound 125I-vWF and SpIII but not SpII or SpI. Twelve monoclonal antibodies (MoAbs) directed towards SpIII, specifically blocking binding of F.VIII to vWF or SpIII, were used for the mapping of plasmic or tryptic fragments of vWF or SpIII. We thus established that a F.VIII binding domain of vWF is located on a 34 kilodalton (kd) fragment of the N-terminal portion of vWF, between residues 1 and 910, and that it is distinct from the GPIb and collagen binding domains.

Isolation and characterization of human factor VIII: molecular forms in commercial factor VIII concentrate, cryoprecipitate, and plasma.

Human factor VIII has been isolated from a high purity factor VIII concentrate by immunoaffinity chromatography and HPLC on Mono Q gel. Two fractions of factor VIII were obtained with a specific activity of approximately equal to 7000 units/mg. The major fraction contained eight peptide chains of 200, 180, 160, 150, 135, 130, 115, and 105 kDa plus one doublet chain of 80 kDa. The minor fraction contained one peptide chain of 90 kDa plus the chain of 80 kDa. Both fractions were activated by thrombin to the same extent. Amino-terminal amino acid sequence analysis was performed on the 180-kDa, 130-kDa, and 90-kDa chains and showed an identical amino-terminal sequence in these chains. Each chain from 200 kDa to 90 kDa was linked to one 80-kDa chain by a metal-ion bridge(s). Studies on factor VIII in plasma and cryoprecipitate, prepared and gel filtered in the presence of protease inhibitors, showed that one 200-kDa plus one 80-kDa chain were the only or dominating chains in the materials and may represent native factor VIII. The results indicated that all chains from 180 kDa to 90 kDa are fragments of the 200-kDa chain. All of these more or less fragmented chains form active factor VIII complexes with the 80-kDa chain.

Binding of native and thrombin activated factor VIII to platelets.

The interaction of human Factor VIII with platelets has been studied using discontinuous albumin gradient centrifugation. When purified Factor VIII complex was mixed with released platelets about 10% of the recovered Factor VIII activity became associated with the platelets. In the corresponding experiment with thrombin-activated Factor VIII about half of the Factor VIII activity was bound to the platelets. This binding was reversible; dilution of the platelet suspension containing bound Factor VIII resulted in dissociation of part of the Factor VIII activity. With non-released platelets very little binding of Factor VIII activity occurred. A mechanism for the in vivo formation of the Factor X activator complex is suggested.