Marie-Madeleine Riottot

A monoclonal antibody enzyme immunoassay for the detection of epitopes encoded by the pre-S2 region of the hepatitis B virus genome.

Pre-S2-coded sequences of the hepatitis B virus (HBV) represent important serological markers of HBV infection and elicit the antibodies essential for recovery from type B hepatitis. Monoclonal antibodies (McAbs) directed against two non-overlapping epitopes (pre-S2a and pre-S2b) of the pre-S2 protein of HBV were used to develop an enzyme immunosorbent assay (EIA). The assay was based on the solid-phase sandwich principle in which two different epitope-specific antibodies were used as immunadsorbents and as enzyme-labelled probes. The assay sensitivity was in the pg range and permitted precise quantitation of the pre-S2 sequences in sera. Using the site-specific monoclonal assay we demonstrated that pre-S2a and pre-S2b epitopes are expressed on HBsAg particles of both ay and ad subtypes. The assay is the most sensitive currently available method for the detection of pre-S2 epitopes and may be used for routine immunodiagnosis of hepatitis B.

Crystallization and preliminary X-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes☆

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.

Structural and functional characterization of a monoclonal antibody specific for the preS1 region of hepatitis B virus

The monoclonal antibody 5a19, raised against the ay serotype of hepatitis B virus, binds to the segment of the preS1 region comprising residues 37–43, which is implicated in attachment of the virus to hepatocytes. The dissociation constant, derived from kinetic studies using surface plasmon resonance techniques, is in the low nanomolar range. The nucleotide sequence of the variable domains has been determined and the corresponding germ‐line genes have been identified. The three‐dimensional structure of the Fab fragment has been determined by X‐ray crystallography to 2.6 Å resolution.

Three-dimensional structure and antigen binding specificity of antibodies

A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site.

Sequence analysis of a monoclonal antibody specific for the preS2 region of hepatitis B surface antigen, and the cloning, expression and characterisation of its single-chain Fv construction

The nucleotide sequence of the monoclonal antibody F124, specific for the preS2 region of the surface antigen of hepatitis B virus, has been determined and an single‐chain Fv fragment (scFv) recombinant construction has been cloned and expressed into the periplasmic region of Escherichia coli. The recombinant antibody fragment contains a (Gly4Ser)3 linker connecting the C‐terminus of the heavy chain variable region (VH) domain to the N‐terminus of the light chain variable region (VL) domain. A 23‐residue peptide segment, containing a c‐myc marker for immunochemical detection of the scFv and hexahistidine tag to facilitate its purification, was added C‐terminal to the VL domain. The scFv mimics the antibody in binding to the native antigen in the form of recombinant hepatitis B surface antigen (HBsAg) particles as well as to peptide fragments carrying the viral epitope.

Crystallization of antibody fragments and their complexes with antigen

Abstract Immunoglobulins, myeloma light chains and their fragments, and Fab fragments from monoclonal antibodies of predefined specificity have been crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab and Fc fragments separately. Intrasegmental mobility in Fabs has not been an obstacle to their crystallization, although this has been a low frequency event, occuring in about 1 in 25 to 1 in 50 trials with different Fabs. However, the immune system provides a large functional and structural diversity of antibody molecules so that an active search may eventually reveal antibodies of the desired specificity suitable for crystallization and X-ray diffraction studies.

Structure of the Fab fragment from F124, a monoclonal antibody specific for hepatitis B surface antigen

The crystal structure of the Fab fragment from the monoclonal anti-preS2 antibody F124 (IgG1,kappa) has been solved by molecular replacement and refined at 3.0 A resolution. The Fab crystallizes with two independent molecules in the asymmetric unit. F124 recognizes an epitope contained within the preS2 segment between residues 120 and 132 of the surface antigen of hepatitis B virus. The antibody shows a high affinity for the glycan N-linked to Asn123, but it also cross-reacts with the non-glycosylated peptide fragment 120-132. Although crystallization was performed in the presence of an eightfold excess of the cross-reactive peptide, no evidence for the ligand was found in the antigen-binding site, which is close to a neighbouring molecule in the crystal lattice. The antigen-binding site has a groove-like topology which is modulated with pocket-like cavities. It is characterized by a large number of tyrosine and aspartate residues. The importance of germ-line mutations at the binding site is discussed.

Cloning, bacterial expression and crystallization of Fv antibody fragments

Abstract The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

Nucleotide sequence of the VH,VL regions of an anti-idiotopic antibody reacting with a private idiotope of the anti-lysozyme D1.3 antibody

Antibody E225 reacts with a private idiotope of the anti-lysozyme antibody D1.3. A complex between the Fab fragments from these BALB/c monoclonal antibodies has been crystallized and the determination of the three-dimensional structure of this idiotope-anti-idiotope complex is under way. The nucleotide VH and VL sequences of E225 presented here have been determined to provide the amino acid sequence information necessary for the interpretation of the high resolution electron density maps of the complex, obtained by X-ray crystallography. The cDNAs synthesized from the Vkappa and VH mRNAs were cloned in E. coli. Both cDNA strands were sequenced by the dideoxy termination method. The translated amino acid sequence shows that Vkappa, VH correspond to groups five (V) and II(b) of mouse immunoglobulin light and heavy chains, respectively. Sequence alignments between the complementarity determining regions of E225 and the antigenic determinant of lysozyme recognized by D1.3 do not indicate whether or not the anti-idiotopic antibody structurally mimics the external antigen.