In vitro stimulation of antibody formation by peritoneal cells. I. Plaque technique of high sensitivity enabling access to the cells.

An improved method for the short-term culture of mouse peritoneal cells in a medium containing carboxymethylcellulose (CMC), sheep erythrocytes (SRBC), and guinea pig complement is described. It involves preparation of microcultures, of thickness 12-15 micro and volume 3.6 microl, under paraffin oil. With such cultures, peritoneal cells from normal, unimmunized young male CBA mice give about 3000 hemolytic plaques per million cells cultured, this figure being attained within 24 hr. The plaque detection method is about four times as sensitive as the Jerne technique. A method is described whereby such plaque-forming cells (PFC) can be transferred, by micromanipulation, to fresh monolayer cultures containing SRBC, CMC, and complement. In this fashion, the secretory capacity and susceptibility to inhibitors of peritoneal PFC can be tested in detail. Using this technique, evidence is presented that the hemolytic substance responsible for plaque formation is actually secreted by the cell at the center of the plaque, and is not a complement component but probably an antibody. Studies on the time of plaque appearance after cell transfer, and the subsequent growth rate of the zone of hemolysis, have been performed. They speak against the idea that the PFC is either a reservoir of cytophilic antibody or a "background" PFC. Rather they suggest that active antibody secretion is induced in the cell at some defined time point in culture. Detailed kinetics of the rate of appearance of plaques in peritoneal cell cultures revealed an exponential phase lasting from about 3 to about 13 hr with a doubling time of 2 hr. The reasons for this are not known. A greatly heightened reactivity was shown in peritoneal cells of mice that had been pregnant several times. Cultures of such cells showed more rapid plaque appearance and a peak activity about 20 times higher than with cells from young male mice. Cultures in which 1 cell in 10 formed a plaque were not infrequent. A series of experiments on germ-free mice showed reactivity similar to that of conventional mice from the same strain and source. The significance of the findings for cellular immunology are discussed.

In vitro stimulation of antibody formation by peritoneal cells. II. Cell interactions and effects of immunochemical or metabolic inhibitors.

Peritoneal cells (PC) from normal, unimmunized mice were placed in ultra-thin monolayer cultures containing carboxymethylcellulose (CMC), sheep red blood cells (SRBC), and complement, and tested for the appearance of plaques of lysis. The behavior of PC from young male mice and from female mice that had given birth to several litters (retired breeder mice) was studied. It was found that cells from spleen, mesenteric lymph node, thymus, bone marrow, thoracic duct lymph, or Peyer's patches could not form plaques in the CMC microcultures. Also, various combinations of these cells did not lead to plaque formation. When cells from any of these sources were mixed with PC, there was either no effect or an actual inhibition of plaque formation, the plaque counts being lower than would have been expected from the number of PC present in the mixture. Optimal plaque formation by peritoneal cells was found to be dependent on an optimal cell concentration, this optimum being around 5 x 10(6)/ml for young male mice and 0.5 x 10(6)/ml for retired breeders. Inhibition of plaque formation was found with either supra- or suboptimal cell concentrations. The inhibition by excess cell concentration may have been a simple nutritional or nonspecific overcrowding effect, as it could also be induced by an addition of an excess of spleen or lymph node cells. The failure of more dilute PC preparations to give adequate numbers of plaques appeared to be more specific, as plaque numbers could not be restored to normal by addition of spleen cells. The suggestion was that some cell to cell interaction between PC was involved. This dependence on cell concentration was not seen with immunized spleen PFC. Plaque appearance could be specifically and reversibly suppressed by placing PC in a medium containing rabbit anti-mouse IgM serum. Anti-IgG serum had no such effect. These experiments strengthened our view, expressed in the accompanying paper, that plaque formation was due to the formation of IgM, hemolytic antibody to SRBC by the PC. Metabolic inhibitors were incorporated into monolayer cultures and had different effects with the different types of PFC used. In the case of spleen cells from mice actively immunized against SRBC 4 days before killing, actinomycin D had no effect on plaque counts and puromycin reduced plaque numbers by a factor of 2. In the case of PC from young male mice, actinomycin D in concentrations above 0.01 microg/ml caused reductions down to < 2% of control values in plaque counts, and puromycin (10 microg/ml) had a similar effect. The PC from retired breeder mice occupied an intermediate position between the two cases just discussed. A compartment of cells, equal to about one-fifth of the total normal PFC compartment, was identified as resistant to high concentrations of either actinomycin D or puromycin, being similar in these respects to PFC from spleens of intentionally preimmunized mice. The mitotic poison, Colcemid, did not affect plaque counts in any situation tested. The theoretical implications of these results are briefly discussed.

Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ.

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.

The 7alpha-hydroxysteroids produced in human tonsils enhance the immune response to tetanus toxoid and Bordetella pertussis antigens.

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.

Preliminary crystallographic study of the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen.

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.

Reproducing the immune response against the Plasmodium vivax merozoite surface protein 1 with mimotopes selected from a phage-displayed peptide library.

We have used phage display technology to identify peptides binding D14-3, a monoclonal antibody raised against the M(r) 42,000 C-terminal fragment of Plasmodium vivax merozoite surface protein 1 (PvMSP1). By screening a constrained hexapeptide library, seven independent clones binding D14-3 were isolated. The reactivity of D14-3 for these peptides was lower than for the natural antigen and the antibody binding was strictly associated with the viral context and the peptide conformation. Sequence analysis showed that five of them shared homology with the M(r) 42,000 C-terminal fragment (Pv42) and therefore appears to identify the D14-3 epitope. However, the other two peptides, while related to each other, did not correspond to any sequence in the Pv42 molecules. To evaluate their immunological interest, these phagotopes were injected into mice belonging to Balb/c, IC57BI/6 and Biozzi strains. All animals developed a strong immune response against phage particles but only Biozzi mice produced antibodies cross-reacting with Pv42. All phagotopes in Biozzi mice elicited a specific response against Pv42, even those sharing no sequence similarity with the antigen. Moreover, the avidities of these immune sera and the polyclonal response against Pv42 were comparable, suggesting phagotopes could be used as components of a subunit vaccine based on the C-terminal fragment of MSP1.

Study of the antigenic structure of human serum albumin with monoclonal antibodies.

Analysis of the antigenic structure of human serum albumin was undertaken using monoclonal antibodies. Nineteen antibodies were prepared and their specificities were studied using fragments which encompass the whole sequence of the albumin molecule. These antibodies recognized 13 different epitopes which are different from the one previously identified with two other monoclonal antibodies [Doyen et al., Immun. Lett. 3, 365-370 (1981)]. Among those 13 different epitopes, six were overlapping. Four epitopes were located on the N-terminal half of the albumin molecule. One of these required integrity of methionine 87 and the other three were overlapping and located around methionine 123. Eight epitopes were located on the C-terminal half of the albumin. Two of them were within the sequence, 330-422 and 299-496 respectively; the other six appeared to be topographic determinants which were altered or lost in the albumin fragments. A last epitope could not be located on any region of albumin. Four monoclonal antibodies directed against a given portion of the albumin molecule reacted slightly with another part of albumin, thus confirming the existence of an intramolecular cross-reactivity between the different domains of human albumin.

Monoclonal anti-GAT antibodies with different fine specificities express the same public idiotype.

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.

Immunochemical analysis of a snake venom phospholipase A2 neurotoxin, crotoxin, with monoclonal antibodies.

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.

Analysis of a major rat idiotype associated with Anti-GAT antibodies.

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.

Identification of allergenic epitopes on Der f I, a major allergen of Dermatophagoides farinae, using monoclonal antibodies.

The antigenic and allergenic structure of Der f I, a major allergen of the house dust mite Dermatophagoides farinae (Df) was investigated by means of a panel of 11 selected monoclonal antibodies (mAb) obtained from BALB/c mice immunized with purified Der f I. The species specificity of these mAb, tested with Der f I and Der p I--the homologous allergen from Dermatophagoides pteronyssinus--was generally restricted to Der f I since 10 out of 11 mAb reacted only with this allergen. Epitope specificity of the mAb was determined by both competitive inhibition and sandwich ELISA experiments. The results indicated the presence of at least four non-overlapping, non-repeated antigenic sites on Der f I, which were recognized by one or several mAb (sites A, B, C and D). Comparative epitope specificity studies between human IgE antibodies and mice mAb were performed, on sera and basophils of Df sensitive patients, using different inhibition assays (ELISA and histamine release experiments). The degree of inhibition varied between the patients and upon the assay design. Most of the mAb tested were found to significantly inhibit the binding of human IgE to Der f I (p less than 0.01) when compared with Der p I specific mAb as a control. The mAb reacting with site A was found to be the most potent inhibitor, presenting a mean inhibition of up to 56% in ELISA as well as in histamine release experiments. The results show that both human IgE antibodies and mAb can be directed against identical or closely related epitopes of Der f I. Therefore anti-Der f I mAb constitute immunologic probes in further allergenic epitope and peptide analysis of this major mite allergen.

Contribution of the H- and L-chains and of the binding site to the idiotypic specificities of mouse anti-GAT antibodies.

The contribution of the H- and L-chains to the structure of the main idiotype of anti-poly (Glu60-Ala30-Tyr10) (GAT) antibodies has been studied. This idiotype has been previously divided into four types of specificity: (1) the highly conserved idiotypic specificity (h.c. GAT) is expressed by anti-GAT antibodies from the guinea-pig, rat and mice; (2) the public specificity (p. GAT) is expressed in an identical form by all anti-GAT antibodies from all strains of mice tested and by all hybridoma products (HP) with anti-GAT activity; (3) the strain-restricted specificity (s.r. GAT-1) is only expressed by anti-GAT antibodies from strains with Ig-1a, Ig-1c and Ig-1c allotypic markers; and finally (4) the individual specificity i1-GAT defined on HP G5 is also expressed by most of the hybridoma protein with anti-poly (Glu50-Tyr50) (GT) activity. In this paper we show that h.c.GAT, p.GAT and i1-GAT require the interaction of H- and L-chains to be expressed: (1) isolated H- and L-chains from HP G5 did not express these specificities; and (2) recombinant molecules composed of H- and L-chains from HP with anti-GAT activity and an irrelevant myeloma protein (MOPC21) never expressed h.c.GAT, p.GAT and i1-GAT. We next investigated the relationship between the GAT binding site and the p.GAT, h.c.GAT and s.r.GAT-1 idiotypic specificities. GAT and GT were not able to inhibit the binding to s.r.GAT-1 while they inhibit the idiotypic binding to p.GAT and h.c.GAT. A GAT fragment of mol. wt 3000 was also shown to inhibit the binding of p.GAT and h.c.GAT to the appropriate sera. Thus p.GAT and h.c.GAT are very close to the GAT combining site while s.r.GAT-1 represents an idiotypic specificity located outside the GAT binding site.

Alzheimer's disease: microtubule-associated proteins 2 (MAP 2) are not components of paired helical filaments.

In Alzheimer's disease, the most characteristic neuropathological changes are the formation of neurofibrillary tangles (NFT) and neuritic plaques (NP) characterized by the presence of bundles of paired helical filaments (PHF) that accumulate in the degenerating neurites and neuronal cell bodies. Although the protein composition of the PHF is ill-defined, a number of microtubule-associated proteins have been implicated in these lesions. Here we report results with an antiserum monospecific for the microtubule-associated protein MAP 2 which does not cross-react with any other microtubular protein. Immunostaining with this antibody of sections from an Alzheimer's brain show a strong reactivity with NFT but no reactivity at the level of the NP. On the other hand, immunostaining of Alzheimer's brain sections with another antibody specific for the microtubule-associated protein tau shows strong staining of PHF on both NFT and NP. These findings confirm the presence of the tau proteins in the PHF and strongly suggest that MAP 2 may not be a main structural component of the PHF. Labelling of NFT with the anti-MAP 2 antiserum suggests a non-specific binding of MAP 2 to the PHF during the process of NFT formation.

Enzyme immunoassay for the measurement of histamine.

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.

Monoclonal anti-nucleoside antibodies. Characterization and application in an enzyme immunoassay of single-stranded DNA.

Two mouse monoclonal antibodies were raised against adenosine and guanosine coupled to bovine serum albumin (BSA) by periodate oxidation. They were named A-16 and G-K21 respectively and selected for their ability to recognize single-stranded DNA. Their epitope specificities were assessed and their dissociation constants determined by an indirect ELISA method. The KD values for adenosine and guanosine coupled to BSA were 9.9 X 10(-7) M and 1.1 X 10(-10) M for G-K21 respectively, and 2.5 X 10(-8) M and 1.0 X 10(-6) M for A-16. These monoclonal anti-nucleoside antibodies were used to develop a sandwich enzyme immunoassay for single-stranded DNA. The purified IgG antibodies were coupled to beta-galactosidase and alkaline phosphatase by the one-step glutaraldehyde method and used in a test optimized for pH, saturating proteins, coating antibody, nature of the conjugate and protein concentrations. Less than 100 pg/well of single-stranded DNA could be detected, and the detection was linear over a DNA concentration ranging from 0.34 to 34 ng/ml. The assay could quantitate single-stranded DNAs of differing origin, but not RNAs. The test was compared to another titration method, and used to calibrate target DNA amounts in non-radioactive hybridization experiments.

Development of a bispecific monoclonal antibody for use in molecular hybridisation.

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.

Monoclonal anti-histamine antibody. Preparation, characterization and application to enzyme immunoassay of histamine.

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).

Tetanus neurotoxin-insensitive vesicle-associated membrane protein localizes to a presynaptic membrane compartment in selected terminal subsets of the rat brain.

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.

Characterization of monoclonal antibodies to human immunodeficiency virus type 2 envelope glycoproteins.

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 monoclonal antibody directed against CLIP (ACTH 18-39). Anatomical distribution of immunoreactivity in the rat brain and hypophysis with quantification of the hypothalamic cell group.

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.