An immunoradiometric assay for squirrel monkey prolactin.

Determination of immunoreactive prolactin in squirrel monkeys has been hampered by the lack of specific antibodies. We investigated the adaptability of a commercially available immunoradiometric assay for human prolactin, which employs two separate monoclonal antibodies (MAb I and II) to human prolactin, to determine the presence of squirrel monkey prolactin. We found that immunoreactivity curves for prolactin in squirrel monkey pituitary homogenates and serum were parallel to human prolactin standards, suggesting that the epitopes recognized by these antibodies were common to both human and squirrel monkey prolactin. Both nonglycosylated (23 kD) and glycosylated (26 kD) forms of squirrel monkey prolactin were detected in squirrel monkey pituitary homogenates by Western blot analysis using [125I]-MAb II. Neither sheep nor rat prolactin was recognized by Western blot analysis, indicating that the assay may be specific for primate prolactins. We examined the effect of ketamine HCl, an anesthetic that has been shown to elevate serum prolactin levels in other primates, on prolactin secretion in squirrel monkeys. Serum prolactin levels increased greater than fourfold after the administration of ketamine HCl (30 mg/kg b.w., i.m.) compared with control levels. Serum prolactin levels were unaffected by anesthesia with pentobarbital sodium (15 mg/kg b.w., i.v.). This assay provides a reliable and sensitive method for determining immunoreactive squirrel monkey prolactin.     

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

Stimulatory effects of prolactin and anti-prolactin receptor serum on prolactin binding sites in rat liver cells in suspension culture

The effects of prolactin and a serum containing anti-prolactin receptor antibodies on prolactin binding sites were investigated in a suspension culture of rat liver cells. In this model, prolactin binding sites decline rapidly with time, with 90% of the sites lost at 24–48 h of culture. The inclusion of 10 to 100 nM ovine prolactin in the incubation medium, results in a 6-fold increase in prolactin binding compared to control cultures. Anti-prolactin receptor serum is capable of preventing this PRL-induced increase in its receptors. However, when incubated alone, these antibodies at lower concentrations (0.5 to 5%) mimic the up-regulatory effect of prolactin on its own binding site. These findings suggest that in rat liver cells, as has been observed for rabbit mammary gland, that the prolactin molecule is not required beyond the initial binding to its receptors for its action to be attained.     

Antigenic analysis of potato virus A particles and coat protein

Five monoclonal antibodies (MAbs) were prepared to particles of potato virus A (PVA), isolate B11. In immunoblots, MAbs A1D8 and A5B6 reacted only with full length molecules of PVA coat protein (CP). Pepscan tests with overlapping octapeptides representing the whole sequence of PVA CP showed that the epitope detected by MAb A5B6 is contained in its N-terminal octapeptide. MAbs A9A4, A3H4 and A6B8 reacted with CP molecules that lacked about 5 kD of sequence at their end(s) and detected epitopes at residues 52 to 62, 64 to 73 and 75 to 82 respectively, all of which lie in the protease-resistant core of the CP. The epitope which reacts with MAb A3H4 is in a region predicted to be hydrophobic and is not detected in intact virus particles, indicating it is a cryptotope. In contrast, MAbs A6B8 and A9A4 reacted with freshly purified PVA particles but more strongly with partially degraded ones. Pepscan tests with polyclonal antibodies to PVA isolate B11 identified five additional immunogenic sequences in PVA CP and showed that regions at the N-termini of the intact and core molecules are immunodominant. PVA isolate B11 was not transmitted by aphids, and its CP N-terminal octapeptide contains the sequence DAS, which is associated with aphid-non-transmissibility in other potyviruses. MAb A5B6, which detects this region, reacted strongly in ELISA with three out of four other aphid-non-transmissible PVA isolates but only weakly with three aphid-transmissible ones, suggesting that differences in N-terminal sequence may underlie most of the differences in aphid transmissibility.     

Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.     

Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation.

Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.     

Monoclonal antibodies against bovine growth hormone.

A number of mouse x mouse hybridomas producing monoclonal antibodies (MAbs) against bovine growth hormone (bGH) were prepared by fusion of spleen cells from bGH-primed mice (Balb/c) with non-secretory mouse myeloma cells (PAIOP3) and characterized. MAbs obtained from three fusion experiments belonged to IgM, IgG1 and IgG2b class/subclass of antibodies. Cross-reaction studies indicated that generated antibodies were against three different epitopes of bGH. VIA6E8 (IgG1) and VIIB2E11C9 (IgM) did not cross-react with ovine prolactin (oPRL), ovine leutinizing hormone (oLH) and porcine follicle stimulating hormone. Antibody VIB3C9E8 (IgM) exhibited cross-reaction with oPRL and oLH. Antibody VIC1F9 (IgG2b) cross reacted with oPRL. All MAbs were against conformational epitopes of bGH.     

Antibodies to newly recognized murine 13-18 KDa pituitary peptides crossreact with growth hormone and prolactin from several species, including man

Recently we identified five novel peptides of Mr 13,000 to 18,000 (designated P13, P14, P16, P17, and P18 according to approximate Mr) in the anterior pituitary gland of rat and man that appeared related to GH and PRL in regulation and structure. We have now raised polyclonal antibodies in the rabbit to four of these peptides--P13, P14, P17 and P18--isolated from rat anterior pituitary; the rabbit injected with P16 did not produce antibodies. Besides reacting with their respective immunogens, antisera to all four peptides crossreacted, quite unexpectedly, with human GH and with human, porcine, and ovine PRL. Antiserum to P17, in addition, crossreacted very strongly with rat PRL, while P18 antiserum crossreacted not only with human GH but also with its 20K and cleaved variants. These results provide strong evidence for the structural relatedness of these peptides to GH and PRL, and raise the possibility that they may be related functionally as well.     

Lack of a direct effect of homocysteic acid on growth hormone and prolactin receptors.

The effects of homoystine derivatives upon GH and prolactin receptors which are reported to stimulate growth in the rat were investigated. Several concentrations of homocysteic acid, homocysteine, homocysteinethiolactone, homoystine as well as other amino-acids were used in GH and prolactin receptor assay systems to determine their growth promoting or the lactogenic activities. None of the aminoacids used displaced labelled hGH or ovine PRL. It os concluded that derivatives of homoystine stimulate growth by an indirect mechanism and exhibit a lack of a direct stimulation of GH and prolactin receptors.     

Immunoreactive prolactin in the pituitary gland of cyprinodont fish at the time of hatching

In the developing pituitary gland of embryos of the annual fish Cynolebias whitei and the medaka, Oryzias latipes, prolactin cells have been identified before hatching by means of a light-microscopic immunocytochemical method with antiserum against ovine prolactin. At the time of hatching, changes in the intensity of the immunoperoxidase staining occur. Histological staining by Cleveland and Wolfe's trichrome shows differentiation of cell types in the adenohypophysis only later in ontogeny. Our results indicate that, in teleosts, differentiated prolactin cells are present before hatching and that prolactin may be involved in the endocrine control of the hatching process.     

Mechanisms of membrane estrogen receptor-alpha-mediated rapid stimulation of Ca2+ levels and prolactin release in a pituitary cell line

The role of membrane estrogen receptor-alpha (mERalpha) in rapid nongenomic responses to 17beta-estradiol (E(2)) was tested in sublines of GH3/B6 rat prolactinoma cells selected for high (GH3/B6/F10) and low (GH3/B6/D9) mERalpha expression. E(2) elicited rapid, concentration-dependent intracellular Ca(2+) concentration ([Ca(2+)](i)) increases in the F10 subline. Lack of inhibition by thapsigargin depletion of intracellular Ca(2+) pools, together with abrogation of the response in Ca(2+)-free medium, suggested an extracellular source of Ca(2+) for this response. The participation of voltage-dependent channels in the E(2)-induced [Ca(2+)](i) increase was confirmed by the specific L-type Ca(2+) channel inhibitor nifedipine. For comparison, the D9 mERalpha-depleted subline was insensitive to steroid action via this signaling mechanism. [Ca(2+)](i) elevation was correlated with prolactin (PRL) release in the F10 cell line in as little as 3 min. E(2) caused a much higher PRL release than KCl treatment (which caused maximal Ca(2+) elevation), suggesting that secretion was also controlled by additional mechanisms. Participation of mERalpha in these effects was confirmed by the ability of E(2)-peroxidase (a cell-impermeable analog of E(2)) to cause these responses, blockage of the responses with the ER antagonist ICI 182 780, and the inability of the E(2) stereoisomer 17alpha-E(2) to elicit a response. Thus rapid exocytosis of PRL is regulated in these cells by mERalpha signaling to specific Ca(2+) channels utilizing extracellular Ca(2+) sources and additional signaling mechanisms.     

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.     

Monoclonal antibody specific for Listeria monocytogenes, Listeria innocua, and Listeria welshimeri

Eight hundred fifty-nine murine hybridomas were produced from eight fusions, and 27 were characterized for secretion of antibodies reactive to Listeria monocytogenes. One monoclonal antibody (MAb), P5C9, reacted with all test strains of L. monocytogenes (31 of 31), L. innocua (3 of 3), and L. welshimeri (1 of 1) but not with any strains of the other four Listeria species or with any of 22 gram-positive or 11 gram-negative species of bacteria when tested in microtiter and dot blot enzyme immunoassays. Of the other 26 antibodies, 20 reacted with either L. monocytogenes Scott A or V7 and with some or all of the other six Listeria species but also cross-reacted with some or all of the non-Listeria bacteria tested. MAb P5C9 is of the immunoglobulin G1 murine subclass. In Western blot (immunoblot) analyses, this MAb reacted with a single antigen with a molecular weight of 18,500, and it is shared in common with all three reactive species, L. monocytogenes, L. innocua, and L. welshimeri. This antigen was extracted with detergent and appeared to be cell bound.     

Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary

Summary Using the peroxidase-antiperoxidase immunocytochemical staining technique, prolactin and growth hormone cells have been identified and described in the ovine pituitary. The image analysing computer, Quantimet 720, was used to assess accurately the size range of the secretory granules in these cell types. The area size distributions of the prolactin and growth hormone granules are similar. An increased proportion of larger granules was observed in the prolactin cells post-partum. Serial sections stained alternately for prolactin or growth hormone confirmed that the cells contain either prolactin or growth hormone but not both.     

Absence of pituitary prolactin epitopes in immunoreactive prolactin of rat brain.

Immunoreactive prolactin (ir-PRL) in rat brain has been consistently documented. However, the identity of this ir-PRL is controversial. Ir-PRL is defined by its ability to bind to PRL antibodies. All previous studies of brain ir-PRL have used polyclonal antibodies, at least one of which apparently crossreacts with a portion of the proopiomelanocortin molecule. To begin to define the epitopes comprising ir-PRL in the brain, we utilized two monoclonal antibodies (MAb) that recognize pituitary PRL in a variety of species, including rat. Immunocytochemistry was performed on rat brains and pituitary glands using two monoclonal and one polyclonal PRL antibody. Although both MAb immunostained lactotrophs of the rat pituitary gland, neither antibody immunostained cell bodies or neuronal processes in the brain. However, the polyclonal antiserum immunostained lactotrophs and a system of neuronal cell bodies and processes in the brain. Thus, epitopes found in pituitary PRL from several species are not found in ir-PRL in rat brain.     

Characterization of antisera to glutamate and aspartate

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.     

Production and characterization of monoclonal antibodies to N-acetyl-aspartyl-glutamate

N-acetyl-aspartyl-glutamate (NAAG) is a putative neuromodulator/neurotransmitter in the mammalian nervous system. Immunohistochemical studies with polyclonal NAAG antisera have revealed immunoreactive neurons and processes in several brain regions. However, these antisera crossreact to some degree with N-acetyl-aspartate (NAA), which is present in mM concentrations in brain, prompting the development of monoclonal antibodies (MAb) more specific for NAAG. By fusing spleen lymphocytes obtained from BALB/c mice pre-immunized with NAAG covalently linked to bovine serum albumin by carbodiimide with SP2/0-Ag 14 mouse myeloma cells, we produced three IgG2a (kappa) MAb which specifically reacted with NAAG. These MAb exhibited negligible crossreactivity with NAA or with structurally similar peptides, as shown by solid-phase radioimmunoassay. Antibody activity was absorbed out selectively by both NAAG-thyroglobulin conjugate and free NAAG. These MAb stained many nuclei of the medulla-pons and midbrain, mitral cells in the olfactory bulb, pyramidal neurons in sensorimotor cortex, locus ceruleus, and several cholinergic cranial nuclei. The staining pattern strongly correlated with NAAG levels determined by HPLC. Monoclonal antibodies significantly enhanced sensitivity of staining, allowing visualization of dorsal horn neurons in spinal cord, which were not readily detectable with polyclonal antiserum. Availability of these MAb now facilitates further clarification of the role of NAAG in the brain.