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Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicular soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) that has been implicated in neurite outgrowth. It has previously been reported that TI-VAMP is localised in the somatodendritic compartment of neurons indicating a role in membrane fusion events within dendrites. Using a newly produced monoclonal antibody to TI-VAMP that improves signal/noise immunodetection, we report that TI-VAMP is also present in subsets of axon terminals of the adult rat brain. Four distinctive populations of labelled axon terminals were identified: 1) the hippocampal mossy fibres of the dentate gyrus and of CA3, 2) the striatal peridendritic terminal plexuses in the globus pallidus (GP), substantia nigra pars reticulata (SNr), 3) peridendritic plexuses in the central nucleus of the amygdala, and 4) the primary sensory afferents in the dorsal horn of the spinal cord. The presynaptic localisation of TI-VAMP in these locations was demonstrated by co-localisation with synaptophysin. Ultrastructural studies showed TI-VAMP labelling over synaptic vesicles in the mossy fibres, whereas it was localised in tubulo-vesicular structures and multivesicular bodies in the pyramidal cell dendrites. The presynaptic localisation of TI-VAMP occurred by P15, so relatively late during development. In contrast, dendritic labelling was most prominent during the early post-natal period. Co-localisation with markers of neurotransmitters showed that TI-VAMP-positive terminals are GABAergic in the GP and SNr and glutamatergic in the mossy fibre system and in the dorsal root afferents. Most of these terminals are known to co-localise with neuropeptides. We found met-enkephalin-immunoreactivity in a sizeable fraction of the TI-VAMP positive terminals in the GP, amygdala, and dorsal horn, as well as in a few mossy fibre terminals. The function of TI-VAMP in subsets of mature axon terminals remains to be elucidated; it could participate in the exocytotic molecular machinery and/or be implicated in particular growth properties of the mature axon terminals. Thus, the presence of TI-VAMP in the mossy fibres may correspond to the high degree of plasticity that characterises this pathway throughout adult life.

Dengue virus infections are a growing public health concern and strategies to control the spread of the virus are urgently needed. The murine monoclonal antibody 4E11 might be of interest, since it neutralizes dengue viruses of all serotypes by binding to the 296-400 segment of the major dengue virus envelope glycoprotein (DE). When phage-displayed peptide libraries were screened by affinity for 4E11, phage clone C1 was selected with a 50% frequency. C1 shared three of nine residues with DE(306-314) and showed significant reactivity to 4E11 in ELISA. C1-induced antibodies cross-reacted with DE(296-400) in mice, suggesting that it was a structural equivalent of the native epitope of 4E11 on DE. Accordingly, 4E11 bound to the DE(306-314) synthetic peptide and this reaction was inhibited by DE(296-400). Moreover, DE(306-314) could block dengue virus infection of target cells in an in vitro assay. A three-dimensional model of DE revealed that the three amino acids shared by DE(296-400) and C1 were exposed to the solvent and suggested that most of the amino acids comprising the 4E11 epitope were located in the DE(306-314) region. Since 4E11 blocked the binding of DE(296-400) to heparin, which is a highly sulfated heparan sulfate (HSHS) molecule, 4E11 may act by neutralizing the interaction of DE(306-314) with target cell-displayed HSHS. Our data suggest that the DE(306-314) segment is critical for the infectivity of all dengue virus serotypes and that molecules that block the binding of DE(306-314) to HSHS may be antiviral reagents of therapeutic interest.

A partially purified nuclear inclusion (NI) fraction was obtained from tobacco plants infected by potato virus Y (PVY). Four monoclonal antibodies (MAbs) were produced and characterized using this semipurified fraction as antigen. Data showed that only one was directed against NIa whereas two were directed against cytoplasmic inclusion (CI) protein and the last one against coat protein (CP). These results were due to the fact that the semipurified NI fraction was usually contaminated with CI and CP proteins. When used on in situ immunofluorescence method the anti-NIa MAb showed accumulation of the NIa protein in both nucleus and cytoplasm. In vivo, this MAb was able to detect different forms of the NIa protein including precursors and cleavage products. It was also able to inhibit the cleavage of the polyprotein detected in the semipurified NI.

A 50-kDa protein was purified as a potential receptor, using an affinity matrix containing biotinylated F14.6 or H9.3 anti-DNA mAbs derived from autoimmune (New Zealand Black x New Zealand White)F(1) mouse and membrane extracts from cells. This protein was identified as calreticulin (CRT) by microsequencing. Confocal microscopy and FACS analysis showed that CRT was present on the surface of various cells. CRT protein was recognized by a panel of anti-DNA mAbs in ELISA. The binding of F14.6 to lymphocytes and Chinese hamster ovary cells was inhibited by soluble CRT or SPA-600. Thus, the anti-DNA mAbs used in this study bound to CRT, suggesting that CRT may mediate their penetration into the cells and play an important role in lupus pathogenesis.

The three-dimensional structure of the major horse allergen Equ c 1 has been determined at 2.3 A resolution by x-ray crystallography. Equ c 1 displays the typical fold of lipocalins, a beta-barrel flanked by a C-terminal alpha-helix. The space between the two beta-sheets of the barrel defines an internal cavity that could serve, as in other lipocalins, for the binding and transport of small hydrophobic ligands. Equ c 1 crystallizes in a novel dimeric form, which is distinct from that observed in other lipocalin dimers and corresponds to the functional form of the allergen. Binding studies of point mutants of the allergen with specific monoclonal antibodies raised in mouse and IgE serum from horse allergic patients allowed to identify putative B cell antigenic determinants. In addition, total inhibition of IgE serum recognition by a single specific monoclonal antibody revealed the restricted nature of the IgE binding target on the molecular surface of Equ c 1.

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

Crotoxin is a potent presynaptic neurotoxin from the venom of the rattlesnake Crotalus durissus terrificus. It is composed of the noncovalent and synergistic association of a weakly toxic phospholipase A2, CB, and a nontoxic three-chain subunit, CA, which increases the lethal potency of CB. The A-56.36 mAb is able to dissociate the crotoxin complex by binding to the CA subunit, thereby neutralizing its toxicity. Because A-56.36 and CB show sequence homology and both compete for binding to CA, we postulated that A-56.36 and CB had overlapping binding sites on CA. By screening random phage-displayed libraries with the mAb, phagotopes bearing the (D/S)GY(A/G) or AAXI consensus motifs were selected. They all bound A-56.36 in ELISA and competed with CA for mAb binding, although with different reactivities. When mice were immunized with the selected clones, polyclonal sera reacting with CA were induced. Interestingly, the raised antibodies retained the crotoxin-dissociating effect of A-56.36, suggesting that the selected peptides may be used to produce neutralizing antibodies. By combining these data with the molecular modeling of CA, it appeared that the functional epitope of A-56.36 on CA was conformational, one subregion being discontinuous and corresponding to the first family of peptides, the other subregion being continuous and composed of amino acids of the second family. Phage-displayed peptides corresponding to fragments of the two identified regions on CA reacted with A-56.36 and with CB. Our data support the hypothesis that A-56.36 and CB interact with common regions of CA, and highlight residues which are likely to be critical for CA-CB complex formation.

Human tonsils were assessed for their ability to 7alpha-hydroxylate pregnenolone (PREG), dehydroepiandrosterone (DHEA) and 3-epiandrosterone (EPIA). Both 7alpha-hydroxy-DHEA and 7alpha-hydroxy-EPIA were produced by homogenates of either whole tonsils or of lymphocyte-depleted tonsil fractions. In contrast, isolated lymphocytes were found to be unable to carry out 7alpha-hydroxylation. When co-cultures of tonsil-derived T and B lymphocytes were set up under stimulatory conditions, IgGs were released in the supernatants and could be quantitated, and immunomodulating properties of different steroids were monitored. When PREG was added to a mixture of tonsil-derived B and T lymphocytes, a decrease of non-specific and specific IgG was observed. An increase in specific anti-tetanus toxoid and anti-Bordetella pertussis antigen IgGs was obtained with either 1 microM 7alpha-hydroxy-DHEA or 1 microM 7alpha-hydroxy-EPIA. In contrast, DHEA and EPIA were unable to trigger such an effect. When cultures of isolated tonsillar B cells were used, none of the steroids tested showed significant effects on specific IgG productions. These data led to the conclusion that human tonsillar cells transform DHEA and EPIA, but not PREG, into 7alpha-hydroxylated metabolites. These metabolites could act on target tonsillar T lymphocytes which in turn act upon B lymphocytes for increasing specific IgG production.

The Listeria monocytogenes InlB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB-coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3-kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine-rich repeat (LRR) region and/or the inter-repeat (IR) region. InlB-staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino-terminal 213-amino-acid LRR region conferred invasiveness to the normally non-invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties.

Crotoxin is a heterodimeric phospholipase A2 neurotoxin formed by the non-covalent association of an acidic and non-toxic subunit, CA, and a basic and weakly toxic phospholipase A2, CB. The two subunits behave in a synergistic manner. CA enhances the lethal potency of CB by increasing its selectivity of action. The mAb A-56.36, directed against the non-toxic subunit CA, was previously shown to neutralize crotoxin toxicity by dissociating the crotoxin complex. In the present report, a polypeptide sequence similarity was observed between some CDRs of mAb A-56.36 and two regions of CB (pos. 60-80 and 95-110). Phage displayed peptides corresponding to VH2 and VH3 of mAb A-56.36 and to their homologous sequences in CB bind CA to different extents. This observation shows that mAb A-56.36 interacts with a region of CA involved in its interaction with CB, therefore mimicking the binding of CB to CA. A similar approach was used to determine the regions of ammodytoxin A and of agkistrodotoxin, two phospholipase A2 neurotoxins similar to CB, which are involved in the formation of heterocomplexes with CA. The analysis of these data contributes to the determination of stretches of amino acids which could constitute the paratope of mAb A-56.36, as well as the region of association of CB with CA in crotoxin.

A recombinant Fab that recognizes a neutralizing epitope located in the (296-400) region of protein E of dengue virus was obtained from cloned hybridoma cells secreting the mouse monoclonal antibody (mAb) 4E11. The Fd and light chain antibody genes were amplified by polymerase chain reaction, cloned into the phagemid vector pMad, expressed in bacteria to produce Fab fragments and sequenced. The mAb 4E11, in particular its light chain complementary-determining regions, shared homologies with two other anti-viral mAbs. The affinity of the parental mAb and the cloned Fab to the MalE-E(296-400) fusion protein were shown to be of the same magnitude, i.e. nanomolar. Fab 4E11 neutralization capacity was found between 8 and 4-times or less lower than that of mAb 4E11, depending on serotypes, thus the Fab could have a smaller antiviral activity than the mAb in vitro.

Mono- and polyclonal antibodies directed against UMP kinase from Escherichia coli were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in whole E. coli cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.

We report the structure and antigenicity of the third variable region (V3) of the HIV2 envelope glycoprotein by the use of linear and cyclic peptides. To this end, a peptide mimicking this region was synthesized and purified, both as an iodoacetamidated linear peptide and a disulphide-bridged cyclic peptide. The cross-reactivity of three monoclonal antibodies (mAbs) produced against the envelope glycoprotein gp140 with the linear and cyclic peptides was tested with ELISA. The results showed that the cyclic peptide is a better ligand for the 3 mAbs 125-F, 125-J and 125-K. The avidity of the mAb/peptide interaction was further analysed by determining the concentration of linear or cyclic peptide leading to 50% inhibition of mAb-peptide complex formation (K0.5). The K0.5 value of mAb 125-F, which displayed the best reactivity with gp140, was estimated to be 5 times higher for the linear (K0.5 = 1.5 x 10(-6) M) than for the cyclic peptide (K0.5 = 3 x 10(-7) M). This indicates a higher affinity of mAb 125-F for the cyclic peptide. mAb 125-J, which exhibited a lower avidity for the gp140 compared to mAb 125-F, had a similar affinity for the cyclic and the linear peptides (K0.5 = 3 x 10(-7) M). mAb 125-K had the lowest reactivity with gp140 and its binding to adsorbed peptide could not be inhibited by the soluble linear or cyclic peptide used up to 10(-5) M. These results suggest that cyclic peptides may have a higher propensity for adopting a native-like structure for the peptide/antibody interaction. Nuclear magnetic resonance experiments at 25 degrees C in phosphate buffer pH 5.4, however, showed that neither peptide displayed a well-defined structure.

Crotoxin is the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. It is a phospholipase A2 neurotoxin constituted by the association of two subunits: an acidic, non-toxic and non-enzymatic subunit (CA) and a basic, weakly toxic phospholipase A2 (CB). A murine monoclonal antibody directed to the non-toxic subunit CA, A-56.36, was shown to fully neutralize the toxicity of crotoxin. When the in vitro pharmacological properties of crotoxin were further tested, A-56.36 was shown to enhance the enzymatic activity on negatively-charged phospholipids and to increase the acetylcholine release triggered by crotoxin on Torpedo synaptosomes. These effects were explained by the fast dissociation of the crotoxin complex in the presence of the monoclonal antibody A-56.36 and the immunocomplexation of CA, with CB being released in solution. CB is less toxic than crotoxin, has a higher enzymatic activity and triggers a higher acetylcholine release than crotoxin, due to its strong enzymatic activity. A single-chain variable fragment antibody was prepared from monoclonal antibody A-56.36. It binds to CA with a similar affinity than the parental immunoglobulin and exhibits similar effects on the in vitro pharmacological properties of crotoxin.

Background: The display of repertoires of antibody fragments on the surface of filamentous bacteriophages offers a new way of making antibodies with predefined binding specificities., Objectives: Here we explored the use of this technology to find human antibodies with biological properties. Phage-scFv specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus, were isolated from a 'single pot' repertoire of more than 10(8) clones made in vitro from human V gene segments [1]. The crotoxin molecule is composed of two noncovalently linked subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component B (CB) and an acidic, nonenzymatic and nontoxic subunit called component A (CA). CA is able to increase the toxicity as well as the specificity of action of CB simultaneously reducing its enzymatic activity., Study Design: Two clones were isolated (4-21 and 5-3-1) which are specific of the basic subunit CB, but of a moderate affinity (about 10(-7) M). Clones 4-21 and 5-3-1 have different amino acid sequences and different effects on CB properties suggesting that they are raised against different CB epitopes. Purely cholinergic synaptosomes isolated from Torpedo electric organs provide a suitable model to study the presynaptic effects of crotoxin. In this model, CB was shown to induce a larger acetylcholine release than crotoxin., Results: A dose-dependent increase of acetylcholine release was observed when crotoxin was incubated with increasing amounts of phage-scFv 4-21. This clone was also shown to increase the enzymatic activity of crotoxin. These observations suggest that phage-scFv might dissociate the complex CA-CB. It could be therefore a neutralizing antibody since CB is much less toxic than crotoxin. This shows that 'single pot' libraries are capable of providing not only immunochemical reagents of high specificity but also biological reagents of high quality. The use of this library appears to open new possibilities for immune passive therapy.

A high-affinity anti-tenanus toxoid (TT) human monoclonal antibody showing neutralizing activity was isolated from a fusion between mouse myeloma and human splenic cells. Fab fragments from this antibody were obtained using a recombinant phage surface-display expression system. The parental antibody and the corresponding Fab had identical immunological activities, including specificity and affinity. These results confirm the feasibility of developing Escherichia coli expression of monoclonal human Fab from hybridoma cells.

Twelve murine monoclonal antibodies (MAbs) to human immunodeficiency virus type 2 (isolate ROD) envelope glycoproteins have been generated and characterized. Nine MAbs were specific to the external gp125 and three reacted with the transmembrane gp36. A large majority of MAbs displayed a significant affinity for the native gp140 precursor and were shown to bind to viral antigens on the surface of fixed HIV-2-infected cells. In Western blot analysis, the 12 MAbs showed varying profiles of cross-reactivity, but none of the MAbs cross-reacted with the HIV-1LAI envelope. Six MAbs reacted exclusively with the homologous HIV-2ROD isolate whereas only two MAbs displayed cross-reactivity with HIV-2ROD, HIV-2EHO, and SIVmac251. The four other MAbs cross-reacted with either HIV-2EHO or SIVmac251. Results of competitive binding assays indicated that the three anti-gp36 MAbs shared the same competition group, whereas at least eight competition groups were defined with the nine anti-gp125 MAbs. The epitopes of the three anti-gp36 and four anti-gp125 MAbs have been delineated using synthetic peptides or by immunological screening of an SIVmac251 peptide library expressed in yeast. The anti-gp36 MAbs are directed against the same domain of the transmembrane gp36 corresponding to the major antigenic determinant of HIV-2 and HIV-1. The four anti-gp125 MAbs recognize four distinct epitopes localized in the V2, V3, and C1 domains. None of the 12 MAbs displayed neutralizing activity against HIV-2ROD, including the 2 MAbs directed against the V2 and V3 domains.(ABSTRACT TRUNCATED AT 250 WORDS)

A mouse hybrid hybridoma (tetradoma) was prepared by fusing hybridomas producing monoclonal antibody to acetyl-aminofluorene with hybridomas producing antibody against calf intestine alkaline phosphatase. The tetradoma line established secreted immunoglobulin manifesting parental and bispecific binding characteristics. Bispecific monoclonal antibody was purified and used for a one-step immunodetection assay of non-radioactive DNA and RNA probes. The immunoassay developed was able to detect 5 pg DNA within 2 h and gave low background noise.

Crotoxin is the major neurotoxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus. The crotoxin molecule is composed of two subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component-B (CB), and an acidic, nonenzymatic and nontoxic subunit called component-A (CA). Crotoxin exists as a mixture of several isoforms (or variants) resulting from the association of several subunit isoforms. We prepared monoclonal antibodies (MAbs) against each isolated subunit. Six anti-CA MAbs and eight anti-CB MAbs were tested for their cross-reactivities with each subunit and with other toxic and nontoxic PLA2s. Four of the six anti-CA MAbs cross-reacted with CB, whereas only one of the eight anti-CB MAbs cross-reacted with CA. Two anti-CB MAbs were found to cross-react with agkistrodotoxin, a single chain neurotoxic PLA2 purified from the venom of Agkistrodon blomhoffii brevicaudus. We determined the dissociation constants of each MAb for CA and CB isoforms and their capacities to neutralize the lethality and to inhibit the catalytic activity of crotoxin. We defined three epitopic regions on CA and four on CB, and used a schematic representation of the two subunits to characterize these epitopic regions with respect to: (1) the "toxic" and the "catalytic" sites of CB, and (2) the zone of interaction between the two subunits. We propose three-dimensional structures of the crotoxin subunits in which we localize amino acid residues that might be involved in the epitopic regions described here.

A brain adenylyl cyclase was shown to contain an epitope closely related to that specified by a conserved sequence containing a nucleotide-binding consensus sequence GXXXXGKS and located in the catalytic sites of bacterial, calmodulin-dependent adenylyl cyclases [Goyard, S., Orlando, C., Sabatier, J.-M., Labruyere, E., d'Alayer, J., Fontan, G., van Rietschoten, J., Mock, M., Danchin, A., Ullmann, A., & Monneron, A. (1989) Biochemistry 28, 1964-1967]. A monoclonal antibody, mab 164, produced against a peptide corresponding to this conserved sequence specifically inhibited the Bordetella pertussis adenylyl cyclase. It also specifically inhibited rat and rabbit brain synaptosomal adenylyl cyclases. The extent of inhibition depended upon the type of enzyme purification, reaching 90% for the calmodulin-sensitive species of enzyme and 20-35% for the forskolin-agarose-retained species. The extent of inhibition in a given fraction also depended upon the effector present. mab 164 reacted on Western blots of forskolin-agarose-retained fractions with a 175-kDa component and did not recognize the Gs alpha stimulatory subunit. Consequently, the 175-kDa protein was considered as a good candidate for an adenylyl cyclase catalyst. The adenylyl cyclase activity contained in forskolin-agarose-retained fractions was further purified on calmodulin-Sepharose. On Western blots of such fractions, mab 164 reacted with a 140-kDa protein, a component that appeared to derive from the 175-kDa protein enriched in the previous step. The kcat of this 140-kDa presumptive adenylyl cyclase was estimated to be of the order of 600 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

After the recent demonstration of the facilitatory effect exerted by corticotropin-like intermediate lobe peptide (CLIP or adrenocorticotropic hormone (ACTH) 18-39) on paradoxical sleep in the rat (Chastrette and Cespuglio, 1985), we undertook the production of monoclonal antibodies against this peptide. Wistar rats were immunized against CLIP and their spleen cells fused with mouse myeloma cells. After recloning, 25 supernatants were found to give positive immunohistochemical reactions in the rat brain. In immunohistochemical tests performed by preabsorption, the 25 supernatants presented similar properties, i.e. recognized CLIP, ACTH (1-39) and ACTH (25-39), but not ACTH (1-24) and the C-terminal fragment (34-39). We assume that the epitope(s) recognized by the 25 supernatants is (are) located between the amino-acids Asn25 and Ala34 of the CLIP molecule. The immunoreactivity observed in the rat brain and hypophysis with this antibody was distributed with a pattern quite similar to that described for anti-ACTH antibodies. A main group of immunoreactive cell bodies was located in the mediobasal hypothalamus and a small group in the nucleus of the solitary tract. Immunoreactive fibres were distributed from the olfactory nucleus to the spinal cord and formed particularly rich networks in the hypothalamus and preoptic area. Among other locations, immunoreactive axons were also present in the brainstem centres involved in the control of the sleep-waking cycle, which is in accordance with the influence of CLIP on paradoxical sleep. Using Abercrombie's formula, the number of immunoreactive cells in the mediobasal hypothalamus was estimated at about 3000 neurons. We conclude that our monoclonal anti-CLIP antibody can be considered as a good marker of proopiomelanocortin neurons.

In the studies presented here, age-related natural antibody specificities have been investigated in the autoimmune NZB mouse strain by cell fusion. The monoclonal immunoglobulins (MIg) secreted by productive hybridoma clones were examined for their antibody activities against a panel of antigens, including single- and double-stranded DNA, actin, tubulin, myosin, bromelain-treated mouse red blood cells (BrMRBC) and TNP-BSA, employing both direct and competitive enzyme immunoassays. The antibody specificities against this panel of antigens were strikingly frequent among hybridoma clones from neonatal NZB (49%) mice, compared to normal BALB/c neonates (8.8%) shown earlier. Among neonatal hybridomas with known antigen reactivities, 73% of the clones exhibited polyspecific binding. In contrast, the majority of hybridomas from 5- and 7-month-old NZB spleen reacted monospecifically (76%) with the antigens tested. Such a characteristic reactivity pattern reflects an age-related evolution of B-cell repertoire expression. Unlike normal BALB/c mice, a high frequency of monospecific TNP-hapten-reactive clones (75%) was noticed among hybridomas of known antigen reactivities from 5- and 7-month-old NZB-strain mice, an age when autoimmune haemolytic anaemia sets in. In conclusion, an elevated frequency of autoreactive clones among neonates (49%) and an aberrant expression of TNP-reactive clones in adults seem to be an outward signal of certain discrepancies at the level of B-cell repertoire expression in autoimmune NZB-strain mice.

Preliminary crystallographic data are given for the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen. This crystalline complex was found by screening a number of Fab-lysozyme complexes prepared from monoclonal anti-lysozyme antibodies produced by hybrids of BALB/c immune spleen cells with a non-secreting mouse hybrid myeloma line. The complex crystallizes in the monoclinic space group P21 with a = 55.5 (+/- 0.1) A, b = 143.5 (+/- 0.3) A, c = 49.1 (+/- 0.1) A, beta = 120 degrees 20' (+/- 10'). X-ray photographs show reflections extending to a resolution of 2.7 A. The crystals are suitable for high-resolution X-ray diffraction studies.

Spleen cells from Balb C mice immunized with purified Yu human myeloma IgE were fused with NS-1 mouse myeloma cells. After initial EIA screening for antibody-secreting cells, 20 hybrids were further characterized for cell growth, ascites production, antibody titer, specificity and affinity. Immunoglobulins purified from ascites fluid obtained from selected clones were labelled with beta-galactosidase. Combinations were made using either antibodies as capture and as conjugate against calibrated human IgE plasma samples. The combination of monoclonal anti IgE X b 10-22 as a capture antibody and X b 6-16 as a conjugate gave the best sensitivity and slope in EIA. It was successfully used in a sensitive two-step-enzyme-immunoassay for total IgE. The X b 6-16 conjugate was also assayed for the detection of allergen specific IgE antibodies. The results presented and discussed indicate that monoclonal antibodies could favourably substitute for polyclonal anti IgE antibodies in such assays.

Spleen cells from nonimmunized BALB/c mice were fused with two nonsecreting myeloma lines. The hybrids were selected in HAT medium and screened for Ig production and for antibody activity against actin, tubulin, myosin, thyroglobulin, myoglobin, spectrin, dsDNA, fetuin, and transferrin. Among 161 hybrids secreting Ig, three were found to react with DNA, one with thyroglobulin, and one mainly with myosin. Two of these hybrids could be propagated and further characterized. On the basis of inhibition experiments, one was found to be directed against dsDNA; the other was directed mainly against myosin but at the same time reacted significantly with actin, tubulin, spectrin, and dsDNA. Reactivity with myosin seemed to be concentrated in the light meromyosin subfragment, known to be rich in alpha-helical structure. These results indicate: 1) There are reactive B cell clones directed against self antigens. 2) The antibody specificities found for these antibodies are very similar to those found for natural antibodies in normal human serum and for human monoclonal Ig. 3) The widespread reactivity found for the clone mainly reacting with myosin raises the possibility that the determinant recognized by this antibody is a conformational structure that possibly is associated with alpha-helical structures.

This paper reports a competitive solid-phase enzyme immunoassay for measuring histamine in various biological samples. In this assay, the histamine to be quantified is chemically modified by 1,4-benzoquinone treatment and allowed to compete with a histamine-peroxidase conjugate for binding to a limited amount of an anti-histamine monoclonal antibody which was used to coat the wells of a microtitration plate. After incubation and washing, peroxidase activity associated with the solid phase is measured. With this method the histamine concentration in blood or various tissues may be determined easily, safely and reproducibly. Histamine concentrations from 0.3 to 20 ng/ml may be measured with the procedure reported here.

From BALB/c mice immunized with BALB/c polyclonal anti-GAT antibodies, we have obtained monoclonal antiidiotypic antibodies directed against public idiotopes expressed following GAT (Glu60Ala30Tyr10) immunization in all individuals of all mouse strains tested. Nine monoclonal anti-Id antibodies were studied in detail. One of these monoclonals recognizes only polyclonal anti-GAT antibodies; the other eight recognize polyclonal anti-GAT antibodies and monoclonal anti-GAT antibodies. The distribution of the structures recognized by the different monoclonal anti-Id on a battery of twenty monoclonal anti-GAT antibodies allows a definition of two families of public idiotopes. The significance of the existence of these two families is discussed in light of the hypothesis recently proposed by Jerne et al. (EMBO J., 1982, 1, 243-247) who consider that the induction of internal images of antigen during immunization leading to the production of antiidiotypic reagents is responsible for the existence of public idiotopes. Our present results may be interpreted to indicate that even though certain monoclonal anti-Id reagents could be internal images of the antigen, the others do, in fact, recognize public idiotopes. Therefore, the notion of internal image does not exclude the existence of public idiotypic specificities.

An anti-idiotypic antiserum was raised in a rabbit against a pool of purified F.344 rat anti-GAT antibodies. GAT-13, the idiotype defined by this serum, is present in all F.344 anti-GAT sera from primary and secondary anti-GAT responses. Anti-GAT sera of 13 inbred rat strains, with different RT1 haplotypes and with different heavy- and light-chain allotypes, all express idiotypic determinants cross-reacting with GAT-13. Thus, like in mice anti-GAT antibodies from rats express public idiotypic determinants. The anti-idiotypic serum also recognizes a highly conserved idiotypic specificity present on mouse and guinea-pig anti-GAT antibodies. The mouse, rat and guinea-pig express a similar highly conserved idiotypic specificity after immunization with GAT. All anti-GAT antibodies from the mouse and guinea-pig bear this idiotypic specificity. These results confirm the existence in the anti-GAT response of interspecies cross-reactive idiotypic determinants.

We have studied the idiotypic specificities expressed by 22 anti-GAT hybridoma products (HP). These antibodies, although derived from cells of mice with three distinct heavy-chain linkage groups (BALB/c, Igh-1a, DBA/2, Igh-1c and C57BL/6, Igh-1b) all express the same public idiotypic specificity, p. GAT, defined by the heterologous binding of anti-idiotypic serum 715 to C57BL/6 anti-GAT antibodies. None of these antibodies expressed the strain-restricted idiotypic specificity, s.r. GAT-1, defined by the binding of anti-idiotypic serum JL 122 to BALB/c anti-GAT antibodies. BALB/c anti-GAT HP could be shown to fall into three subsets with respect to their fine antigenic specificity for GAT, GT and GA. An individual idiotypic specificity, i1-GAT (defined by syngeneic anti-idiotypic sera raised against one of the BALB/c HP), was also found on a group of BALB/c HP which all shared a similar fine antigenic specificity pattern. Taken together, these observations suggest that the expressed mouse anti-GAT repertoire derives from a very few V-germ-line genes (VH-GAT and VK-GAT) which are highly conserved in the species, and which determine the structure resulting in the p. GAT idiotypic specificity. The variations in fine specificity and individual idiotype are likely therefore to reflect somatic variations affecting these germ-line genes.

An enzyme immunoassay to measure histamine has been developed. A histamine-bovine serum albumin conjugate was prepared using 1,4-benzoquinone as the coupling agent and was employed to immunize mice for the preparation of monoclonal antibodies against histamine. After an initial screening to identify antigen-binding monoclonal antibodies the clones were isolated by limiting dilution cloning, grown in ascites and antibodies which had been secreted into the ascitic fluid were precipitated by ammonium sulphate at 50% saturation. A systematic approach for the determination of epitope specificities of monoclonal antibodies was performed. It was found that for the most specific antibody the main epitope encompassed the 2-histaminyl-1,4-benzoquinone moiety and that the KD value determined by indirect ELISA was 1.5 X 10(-8) M for the hapten part of the immunogen and 4.6 X 10(-10) M for a histamine-Bq-ovalbumin conjugate. The selected monoclonal antibody could not recognize histidine or methyl-histamine. Using this antibody, we developed an enzyme immunoassay for histamine and pg amounts could be detected. The same assay was used to quantify the allergic release of histamine from guinea pig lung mast cells. Results obtained either by the present enzyme immunoassay or by a fluorometric assay were closely correlated (correlation coefficient r = 0.9702, n = 37).