The single-chain antibody variable fragment (scFv), with a 15-residue polypeptide linker (Gly,Ser),, of monoclonal antibody NClO was expressed in Escherichia coli and purified to homogeneity. This scFv molecule, refolded from 6 M guanidine hydrochloride, was predominantly a monomer of 27 kDa and was stable on storage at 4" and 20°C. At higher protein concentrations (= 5 mg/ml) dimer and higher-molecular-mass multimers were formed and freezing enhanced this aggregation. The dimer was not stable and dissociated to monomer at 20°C with a half-life of approximately 8 days. The higher-molecular-mass multimers and dimer dissociated to monomer in 60% ethylene glycol. Both the monomer and dimer were active and with tern N9 sialidase yielded complexes of 276 kDa and 569 kDa, respectively, indicating that four scFv molecules boundkialidase tetramer and that the dimer was bivalent and cross-linked two sialidase tetramers. Binding studies at low concentrations and using radiolabelled scFv indicated that the binding affinity of the dimer was approximately twofold higher than that of the monomer, and the binding affinities of the scFv were similar to that of the parent NClO antigen-binding fragment (Fab) molecule. A complex between tern N9 sialidase and NClO scFv was crystallized and the structure of the complex was solved at 0.3-nm resolution by X-ray diffraction. Comparison of this scFv/sialidase structure with the parent Fabhalidase structure revealed that the modes of attachment of scFv and Fab to sialidase were very similar. There was no discernible electron density for the peptide linker joining the variable heavy (V,) and variable light (V,) chains. A close interaction between two symmetryrelated scFv suggests that they may have crystallized as dimers.Monoclonal antibodies with their unique specificity and affinity are used widely as immunodiagnostic and therapeutic reagents [I] A scFv fragment of antibody NC10, which recognizes an epitope on influenza virus sialidase, was recently cloned and expressed in Escherichia coli [14]. The V, and V, domains were linked with the 15-residue peptide described above and also contained a hydrophilic octapeptide (FLAG) [19] at the C-terminus of the V, domain as an affinity label to aid in detection of the scFv. The purified, monomeric scFv was found to form dimers and higher-molecular-mass aggregates at a protein concentration greater than 5 mg/ml. Recently, the presence of dimers and higher-molecular-mass multimers

The interactions between the Fab and single-chain Fv (scFv) fragments of an antibody (NC10) and its antigen, influenza virus neuraminidase, were analysed in the crystal structures of the Fab-neuraminidase and scFv-neuraminidase complexes. To investigate the contribution to binding made by cavities, salt links and hydrogen bonds in the antibody-antigen interface, 14 single amino acid replacements were made at six contact residues in the scFv fragment by site-directed mutagenesis. The binding affinity of each mutant scFv antibody for neuraminidase was determined with a BIAcore optical biosensor. Four of the mutations resulted in large changes in the free energy of binding to neuraminidase (deltadeltaG > 1 kcal/mol) and together may account for approximately 70% of the free energy of binding. Hence these data support the theory that a small number of residues form the 'functional epitope' and are most important for binding of NC10 to neuraminidase. The salt link between antibody residue (Asp)H56 and (Lys)N432 from neuraminidase was demonstrated to be important for affinity, since substitution of (Asp)H56 with Asn caused a large reduction in the free energy of binding (deltadeltaG = +2.8 kcal/mol). Hydrogen bonds provided by (Tyr)L32 and (Asp)H56 were also important for binding: mutation of (Tyr)L32 to Phe resulted in a significant reduction in binding affinity (deltadeltaG = +1.7 kcal/mol). Disruption of hydrophobic interactions (van der Waals contacts) led to significant reductions in affinity also ((Tyr)H99 to Ala, deltadeltaG = +1.5 kcal/mol; (Leu)L94 to Ala, deltadeltaG > +3.0 kcal/mol). An attempt to increase binding affinity by filling a cavity in the interface with a larger antibody side chain was unsuccessful, as the free energy gained by new antibody-antigen interactions did not compensate for the removal of cavity-bound water molecules.

Hydration equilibrium constants for acetaldehyde, propionaldehyde, nand iso-butyraldehyde, and chloral in H 2 0 and D20 have been measured in the temperature range 25-90'.The equilibrium constant for formaldehyde has been determined in H,O over the same temperature range. The effects of added electrolytes on the activity coefficients of the hydrates and free aldehydes have also been investigated.

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The reversible addition of molecular oxygen to the tetrasodium salt of cobalt phthalocyanine-4,4?,4?,4??-tetrasulphonic acid in aqueous solution has been confirmed. Visible absorption spectra of the monomeric and dimeric species and of the oxygen adduct have been determined. A monomer-dimer system prevails at neutral pH, low ionic strength, and low dye concentrations. The oxygen adduct and the dimeric form of the dye predominate in alkaline solution.