Epitopic characterization of neuropeptide Y (NPY) by alanine-scanning mutagenesis.

Characterization of the epitopic structures of neuropeptide Y (NPY) has been studied by alanine-scanning mutagenesis, based on our previous investigation of a panel of six murine anti-NPY IgM monoclonal antibodies. To evaluate the structural requirement for these anti-NPY IgM antibodies, recognition variants of the native sequences of the NPY fragment (19-36) were prepared by single alanine substitutions in residues 22 and 25-36. Their binding to these antibodies was examined by competitive inhibition assays.

Primary B-cell response to neuropeptide Y and bovine pancreatic polypeptide.

An analysis of the murine primary response to protein epitopes has been made with two small highly structured proteins, neuropeptide Y (NPY) and bovine pancreatic polypeptide (BPP), both of 36-amino acid residue length and containing helical structures. A group of cell lines producing monoclonal IgM antibody have been prepared consisting of six anti-NPY and two anti-BPP. The VH nucleotide sequences have been determined and characterized as germ-line either by identity to established germ-line sequences or by inference from the germ-line character of the D and JH segments.

Probing the transglutaminase-mediated, posttranslational modification of proteins during development.

Sphaerechinus granularis eggs were fertilized in seawater in the presence of 0.2 mM dansylcadaverine, and development was allowed to take place with this compound in the medium. gamma-Glutamyldansylcadaverine, indicative of the utilization of the amine tracer by intrinsic transglutaminase, was isolated from the embryonic proteins, and identity of the product with the chemically synthesized gamma-glutamyl derivative of dansylcadaverine was confirmed.

Bacterial expression of immunoglobulin VH proteins.

A bacterial expression system in Escherichia coli has been developed that produces as much as 10 mg/l of culture of the VH protein associated with monoclonal antibodies specific for the 5-dimethylaminonaphthalene-2-sulfonyl (Dns) group. This system has been applied to the expression of the VH genes derived from a low-affinity, IgM-producing hybridoma and from a high-affinity, IgG-producing cell line. The plasmid vectors (contributed by Dr William F.

Organization of the middle RNA segment of snowshoe hare Bunyavirus.

The genetic organization of the M RNA segment of snowshoe hare (SSH) virus, a member of the Bunyavirus genus of the family Bunyaviridae, has been determined. The middle (M) RNA segment has a single open reading frame (ORF) of 1441 amino acids. We have used amino- and carboxy-terminus sequencing and synthetic peptides to map proteins within the ORF.

Transamidating activities of factor XIIIa and of transglutaminases, measured by an ELISA procedure.

The dansyl hapten in dansylcadaverine offers unique possibilities for measuring the incorporation of the monoamine into proteins (e.g. N,N'-dimethylcasein) by transamidating enzymes such as factor XIIIa and the transglutaminases.

Induction of polyclonal and monoclonal antibody responses to cholera toxin by the synthetic peptide approach.

The induction of an antibody response to cholera toxin (CT) was studied by using the synthetic peptide approach. Two peptides, corresponding to the amino acid sequences from residues 57 to 69 (CTBP1) and 47 to 60 (CTBP2) of the cholera toxin B subunit, were synthesized by the solid-phase method. These peptides were primarily chosen on the basis of their hydrophilicity and sequence identity with the B subunit of E.

Affinity of anti-peptide antibodies measured by resonance energy transfer.

Because of the increasing use of monoclonal anti-peptide antibodies we have undertaken to formulate a general method for the measurement of intrinsic association constants characterizing complex formation between peptide and antibody. The method is based on the phenomenon of resonance energy transfer between tryptophan-excited antibody and an appropriate fluorophor conjugated to the amino terminus of the peptide. The fluorophor we have employed is 8-(2-N-succinylaminoethylamino)-1-naphthalene-sulfonic acid with an absorption maximum at 344 nm and an emission maximum at 500 nm.

Germ-line affinity and germ-line variable-region genes in the B cell response.

The predominance of germ-line genes in IgM expression was evaluated from the nucleotide sequences of mRNA, derived from 10 hybridoma cell lines, coding for the VH and VL regions of anti-5-dimethylaminonaphthalene-1-sulfonyl (anti-Dns) IgM antibody. At least six germ-line VH gene segments distributed among four families are used in this response. Seven of the 10 independently rear-ranged VH genes were identified as germ line, with the other three possibly germ line.

Identification of transglutaminase substrates in inside-out vesicles from human erythrocytes: immunoblotting with anti-dansyl antibody.

An immunoblotting procedure, using anti-dansyl antibody, was employed to demonstrate that band 3 protein was the predominant substrate in inside-out vesicles from human erythrocytes reacting with transglutaminase.

Antibodies to the first constant-region domain of the murine mu-chain induced by a synthetic peptide.

A peptide corresponding to the N-terminal 13 amino acid residues of the murine C mu 1 domain was synthesized by the solid-phase method and was coupled to carrier proteins through an additional cysteine residue. Rabbit antisera to these peptide-carrier conjugates were found to react with intact mouse IgM as well as its Fab mu fragment. These antisera also reacted with the isolated mu-chain and the V mu fragment of the heavy chain.

Interaction of monoclonal anti-peptide antibodies with lysozyme.

The interaction of monoclonal anti-peptide antibodies with the free peptide and its protein counterpart has been evaluated for hen egg white lysozyme and the peptide constituting residues 38 to 45. Fluorescence methodology has been developed for the measurement of association constants based on resonance energy transfer between the excited tryptophan of antibody and bound peptide ligand conjugated to a fluorescent probe. Five antibodies, four IgM and one IgG, have been assayed by ELISA, and have demonstrated binding to the adsorbed peptide alone, to the adsorbed lysozyme alone, or to both.

Restriction in IgM expression--VI. Affinity analysis of monoclonal anti-dansyl antibodies.

Affinity restriction in the IgM response to the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group was studied with 56 monoclonal IgG and IgM affinity-purified antibodies. These were generated by immunization with dansyl-Ficoll or dansyl-B gamma G. Association constants for N epsilon-dansyl-lysine were determined by fluorescence titration based on resonance energy transfer.

Restriction in IgM expression--V. Fine structure analysis in the anti-lactose system.

A methodology for the analysis of the fine specificity of monoclonal anti-lactose IgM and IgG antibodies is described using structural variants of the homologous lactoside epitope. These variants are used as inhibitors of the binding of a reference ligand N-(5-dimethylaminonaphthalene-1-sulfonyl)-p-aminophenyl-beta-lactoside. Excitation of the antibody with bound ligand at 295 nm leads to resonance energy transfer to and fluorescence emission by the ligand.

Attachment of immunoglobulin to liposomal membrane via protein carbohydrate.

A general method has been developed for the covalent attachment of immunoglobulin molecules to the outer layer of liposomal membranes. Aldehyde groups are generated by the mild oxidation with periodate or galactose oxidase of the carbohydrate groups on the constant region of the heavy chain. The oxidized protein is then reacted with a hydrazide group linked to a membrane component.

Antibody interaction with a membrane-bound fluorescent ligand on synthetic lipid vesicles.

The interaction of membrane-bound ligand with bivalent and monovalent fragments of monoclonal antibody was studied by fluorescence and precipitation analysis using synthetic lipid vesicles. The ligand N epsilon-[5-(dimethylamino)-naphthyl-1-sulfonyl]lysine was linked to the hydrophobic anchor dipalmitoylphosphatidylethanolamine and ranged between 0.01 and 1 mol% of the membrane components.

Specific reactivity of lipid vesicles conjugated with oriented anti-lactose antibody fragments.

The method previously described (Sinha, D. and Karush, F. (1979) Biochem.

Restriction in IgM expression. III. Affinity analysis of monoclonal anti-lactose antibodies.

A clonal analysis has been made of the murine BALB/C response to lactose-containing immunogens with respect to the affinity restriction in IgM expression. Monoclonal IgM and IgG antibodies were prepared from antilactosyl hybridomas generated from mice immunized with p-aminophenyl-beta-lactoside (PAPL) coupled to BGG or with a vaccine of Streptococcus faecalis (Strain N). Association constants for the binding of monovalent derivatives of PAPL were measured by quenching fluorescence.

Proximity relationships within the Fc segment of rabbit immunoglobulin G analyzed by resonance energy transfer.

Resonance energy transfer analysis has been carried out with a noncovalent rabbit hybrid of immunoglobulin G (IgG) composed of normal rabbit IgG and rabbit anti-lactose IgG. The hybrid IgG was prepared from proteins in which the single inter-heavy-chain disulfide linkage was specifically reduced and alkylated. Normal rabbit IgG was alkylated with the iodoacetyl derivative of N-(aminoethyl)-5-naphthylamine-1-sulfonic acid while the rabbit anti-lactose IgG was alkylated with either iodoacetamide or the iodoacetyl derivative of p-[[p-(dimethylamino)phenyl]azo]aniline.

Surface component of primate thymus-derived lymphocytes related to a heavy chain variable region.

In a study designed to determine whether T cells of man and higher primates express a surface component related to the variable region of immunoglobulin heavy chain (VH), chickens were immunized with the purified VH fragment of a monoclonal Waldenström macroglobulin. The antibody preparation reacted with a mu chain determinant contained in the Fd fragment and with individual determinants characteristic of the orginal Waldenström protein. As estimated by immunofluorescence analysis, a subpopulation of normal human peripheral T cells (approximately 30%) bound the anti-VH antibody.

Proximity of antibody binding sites studied by fluorescence energy transfer.

Fluorescence energy transfer experiments by steady-state and nanosecond monophoton techniques were carried out with a covalently linked hybrid rabbit IgG antibody containing one antilactose site and one anti-Dns [5-(dimethylamino)-1-naphthalenesulfonyl] site. The hybrid antibody was prepared from antilactose and anti-Dns antibody by mild reduction, dissociation into half-molecules in acid, and random reassociation with re-formation, to the extent of 80%, of the single disulfide bond between the heavy chains. Fractionation with an antilactose-specific immunoadsorbent yielded a population in which each IgG molecule contained no more than one anti-Dns site per antibody.