276 Abstracts sera. Libraries were enriched for U1 RNA-binding clones after several rounds of affinity selection against U1 RNA. Putative U1 RNA-binding clones were identified by colony lift and the corresponding Fab genes were expressed and purified from E. coli for binding studies. Results demonstrate that anti-U1 RNA-specific Fab can be isolated from combinatorial phage display libraries. The anti-U1 RNA Fab will provide an ideal model for the study of protein-RNA interactions. In addition, a more in depth look at the mechanism by which an Ab interacts with UI RNA will lend insight into immune disregu- lation and may have therapeutic potential in the treatment of autoimmune disease. Antigen-binding sites dominate the surface properties of antibod- ies. Ulla-Britt Hansson, Christer Wingren, Kerstin Andersson, Department of Biochemistry, Chemical Center, Lund University, S-22100 Lund, Sweden. We have found a remarkable relationship between the spe- cificity of antibodies and their chromatographic behaviour upon liquid-liquid partition chromatography (LLPC). Well- characterized human and murine monoclonal antibodies and Fab/Fc fragments thereof as well as mouse/human chimeric antibodies were employed. While, IgG 1, 2 and 4 antibodies with identical specificities (affinity constants) have identical partition properties, IgG antibodies with different partition properties reacted with dif- ferent epitopes or had different affinities against the same epitope. Hence, the surface properties of the antigen binding sites dominate over all other surfaces of the free antibody molecule. LLPC may also be used to detect conformational changes occuring upon binding of antigen by antibody. Antigen-anti- body complexes formed by different IgG antibodies against a large antigen like HSA all had similar surface properties, different from those of both antigen and antibody. In contrast, the surface properties of complexes formed by small antigens/ haptens are related to those of the IgG antibody. In addition, antigen-antibody complexes were found to have similar surface properties irrespective of the molar ratio of antigen to antibody at which the complexes had been formed. Structure of a humanized anti-lysozyme FV domain. [Abst. 1031 Margaret Holmes, Jefferson Foote, Program in Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. A humanized antibody is constructed by transplanting the complementarity determining regions from a rodent antibody onto the framework of a human variable domain. This creates an antibody which is mostly human in origin, thus reducing the potential for causing an immune reaction when used to treat human disease. In some cases, it has proved necessary to substitute additional rodent residues into the human frame- work region in order to attain high affinity binding of antigen. HuLys is a humanized antibody derived from the mouse anti-lysozyme antibody D1.3, the heavy chain framework of the myeloma protein NEW, and the light chain framework of the Bence-Jones protein REI. Several substitutions were also made to carefully selected framework residues. As the struc- tures of all the parent antibodies have been determined, HuLys is a model system to study how to most effectively humanize antibodies. We have solved the structure of the HuLys Fv domain in two space groups, P43212 and P6122, to a resolution of 2.9 ° using the technique of molecular replacement. We are currently in the midst of refining these structures. We will present the structures, compare them to their parent struc- tures, and present any lessons learned about effective human- ization. Affinity maturation and structural analysis of a humanized antibody. Hyo Jeong Hong a, Sung Sup Park a, Chun Jeih Ryu a, Kyoung Tai No b, Young Hun Kim b, Moon Hi Han a, aThe Protein Engineering Research Group, Korea Research Institute of Bio- science and Bioengineering, KIST, Taejon, bDepartment of Chemistry, Soong Sil University, Seoul, Korea. We have developed a humanized antibody with specificity for the preS2 surface antigen of hepatitis B virus (HBV) for the prevention and therapy of HBV infection. Initially, the complementarity determining regions (CDRs) of the murine heavy and light chain variable regions (VH and VL) were transplanted onto the framework regions (FRs) of a highly homologous human antibody, but the affinity of the resulting humanized antibody (Z6B) was lower than that of parental murine monoclonal antibody (mAb) H8 by about 10-fold. Only one amino acid change at the VH FR 3 residue (Thr) underlying the CDR3 of Z6B to Pro of the parental murine mAb resulted in the restoration of the antigen-binding affinity. This new version (ZP39) of humanized antibody has almost same antigen-binding affinity as murine mAb H8. Structural analysis of the antigen binding loops by molecular modelling and molecular dynamics simulation showed that the flexibility of CDR3 in ZP39 is higher compared with Z6B, suggesting that the Pro residue may play a crucial role in the antigen-an- tibody interaction by providing the flexibility for the CDR loops and, thus, fine-tuning the fit to the antigen. Design, expression and 3-D structure of bivalent single chain Ig-like molecules. [Abst. 105] Peter Husdon a, John Atwell a, Greg Coia a, Peter Coleman b, David Dougan a, Clem Gruen a, Peter Iliadesa, Alex Kortt a, Glenn Lilley a, Robyn Malby b, Airlie McCoy b, Lesley Pearce a, Barbara Powera, Peter Tullochb, aCSIRO Division of Biomolecular Engineering, bBiomolecular Research Institute, 343 Royal Parade, Parkville, Vic, 3052 Australia. We have determined the 3-D structure of scFvs in complex with target antigen (influenza NA) using EM and X-ray crystallography and a high-level bacterial expression system (pPOW). Binding studies using ELISA, sedimentation equi- librium and Biacore technique demonstrated that scFvs can be produced as stable monomers which retain similar affinity as the parent Fab to the target antigen, influenza neuraminidase (NA). A detailed structural comparsion of the NA/NC10 interface has been made using point mutations of key contact