Precursors of molecules related to mammalian opioid peptides in brain of a marine worm.

Total mRNA were extracted from brain of Nereis diversicolor (Annelida, Polychaeta) and were translated in vitro or in ovo. The newly synthesized polypeptides were analyzed through electrophoresis of immunoprecipitated products or the Western blotting technique using polyclonal antibodies raised against mammalian dynorphin 1-17 and mammalian alpha-neo-endorphin. Among the products translated in vitro, only one class of polypeptide of 70 kDa was recognized by anti-dynorphin 1-17 antibodies. Furthermore, some in ovo translated products as well as proteins extracted from brain of worms showed identical immunoreactivity. These polypeptides, 60-70 kDa, reacted with anti-dynorphin 1-17 and anti alpha-neo-endorphin antibodies. These results suggest the existence of epitopes common to in ovo and in vitro translated products, to polypeptides extracted from the brain and to some mammalian opioid peptides of the prodynorphin family. We postulate the presence, in the brain of N. diversicolor, of precursors of peptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Data reported in this investigation do not allow us to propose or even postulate the presence, in the brain of the worm, of one precursor molecule common to polypeptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Furthermore, the Nereis precursor molecules exhibit a clear-cut difference in molecular mass with the mammalian prodynorphin: 70 kDa versus 30 kDa.     

Yolk protein immunoassays (YP-ELISA) to assess diet and reproductive quality of mass-reared Orius insidiosus (Heteroptera: Anthocoridae)

A yolk protein enzyme-linked immunosorbent assay (YP-ELISA) was developed for the predator Orius insidiosus (Say). The YP-ELISA is intended to assess reproductive response to dietary and other rearing conditions, and to assist in quality control and diet development for mass rearing. Hybridomas and monoclonal antibodies were produced against homogenates of eggs dissected from females. Hybridomas were selected for secretion of IgG that reacted with extracts of both females and their eggs, and that did not react with male extracts. Each cloned hybridoma produced a monoclonal antibody that specifically reacted on western blots against one of the two major yolk polypeptides, apoVn-I (180,000 molecular weight) or apoVn-II (40,000). Yolk protein ELISAs were developed with these antibodies to assess yolk protein content of female O. insidiosus as a measure of reproductive fitness and as a potential predictor of fecundity. Protocols for an indirect antigen ELISA and double antibody sandwich ELISA were developed to assess yolk protein contents of eggs and total contents in whole body homogenates. ELISA standards consisted of homogenates of eggs collected 0-24 h following oviposition. As determined with the sandwich ELISA, yolk protein contents of eggs declined with age before hatch, with a half-life of 32-34 h. Results were similar whether the detecting antibody-enzyme conjugate was anti-apoVn-I or anti-apoVn-II. Optimal conditions and sampling parameters were developed for the sandwich ELISA, which demonstrated minimal nonspecific interference in whole-insect extracts. In an initial application of the YP-ELISA, oviposition rates over a 10-d period were compared with yolk protein contents at the end of that period, dependent on diets of differing nutritional composition and quality. High and low yolk protein contents correlated with oviposition rates on respective diets, though oviposition showed more graded response to diets than did yolk protein. Improvements in sampling methods are discussed.     

Production and characterization of species-specific monoclonal antibodies against Leishmania donovani for immunodiagnosis

Sixteen species-specific monoclonal antibodies were produced against membranes of Leishmania donovani. These antibodies only reacted with determinants present on L. donovani. No cross-reactions were found with any other species of Leishmania or with membranes of Trypanosoma cruzi. An extensive analysis of the binding specificities of selected antibodies was carried out by using whole promastigote homogenates as antigen. Monoclonal antibodies D-1, D-2, D-3, and D-4 correctly identified all 44 L. donovani stocks from a cross-panel of 84 New and Old World Leishmania stocks. Antibodies D-1 and D-2 were also useful for species classification by immunofluorescence. No cross-reactions were observed with any other Leishmania species examined. Based on either Western blot and/or radioimmunoprecipitation analyses, five distinct groups of molecules associated with L. donovani-specific antigenic determinants were identified. These molecules range in m.w. from 18 to 84 kilodaltons. The antigenic molecules recognized by antibodies D-2, D-10, and D-13 are also recognized by antibodies present in sera from patients with visceral leishmaniasis (kala-azar). Kala-azar sera obtained from cases in both the Old and New World specifically compete with these monoclonal antibodies for the appropriate antigenic determinants in Western blot analysis. These monoclonal antibodies and/or the purified protein antigens may be useful in the development of a serologic assay for the clinical diagnosis of visceral leishmaniasis caused by L. donovani and in epidemiologic studies of leishmaniasis.     

Affinity purification of a rat-brain junctional protein, connexin 43.

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.     

Translation in a cell-free system, and in Xenopus oocytes, of mRNA which specify a cadmium-binding protein in Nereis diversicolor (Annelida, Polychaeta)

The presence in the marine worm Nereis diversicolor of a low molecular mass protein with the capacity to bind cadmium has been previously demonstrated. Poly(A)(+)-mRNA were extracted from coelomocytes of Nereis diversicolor and were translated either in vitro, using a rabbit reticulocyte lysate, or in vivo into Xenopus laevis oocytes. Analysis of synthesized polypeptides by enzyme-linked immunosorbent assay (ELISA) and by Western blotting, using a specific monoclonal anti-MP II antibody, showed that this metalloprotein was translated both in in vitro and in vivo translation systems, with an apparent molecular mass of 11-13 kDa. Two other products, with 26.5 and 28 kDa molecular mass, cross-reacted with the monoclonal anti-MP II antibodies. The present work confirms that coelomocytes are sites of important synthesis of MP II-mRNA.     

Preparation and characterization of monoclonal antibodies against native membrane-bound penicillin-binding protein 1B of Escherichia coli.

We prepared monoclonal antibodies against penicillin-binding protein 1B (PBP 1B) of Escherichia coli to study the membrane topology, spatial organization, and enzyme activities of this protein. The majority of the antibodies derived with PBP 1B as the immunogen reacted against the carboxy terminus. To obtain monoclonal antibodies recognizing other epitopes, we used PBP 1B lacking the immunodominant carboxy-terminal 65 amino acids as the immunogen. Eighteen monoclonal antibodies directed against membrane-bound PBP 1B were isolated and characterized. The epitopes recognized by those monoclonal antibodies were located with various truncated forms of PBP 1B. We could distinguish four different epitope areas located on different parts of the molecule. Interestingly, we could not isolate monoclonal antibodies against the amino terminus, although they were specifically selected for. This is attributed to its predicted extreme hydrophilicity and flexibility, which could make the amino terminus very sensitive to proteolytic degradation. All antibodies reacted against native PBP 1B in a dot-blot immunobinding assay. One monoclonal antibody also recognized PBP 1B in a completely sodium dodecyl sulfate-denatured form. This suggests that all the other monoclonal antibodies recognize conformational epitopes. These properties make the monoclonal antibodies suitable tools for further studies.     

Immunocytochemical studies of spectrin in hamster cardiac tissue

The spectrins are a family of cytoskeletal-membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight alpha-spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X-100-extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2-microns-thick; sections of adult. Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z-bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T-tubule-associated staining. Myofibrils isolated from cardiac myocytes using Triton X-100 show positive Z-band staining using all three antibodies. Double staining with Texas Red-labeled monoclonal desmin and FITC-labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z-line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate filaments in the mammalian heart.     

Phylogenetic conservation of epitopes in mammalian aldose reductase: application to immunoquantitation

Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific by Western blot analysis of kidney homogenates. In addition, the antiserum (alpha-BKALR2) reacts with a single electrophoretic species in homogenates from rabbit, porcine, and human kidney. ALR2 has been detected in homogenates of bovine kidney, heart, brain and lens, and estimation of the enzyme level in these tissues was accomplished by densitometric analysis of Western blots. Standard curves using highly purified bovine kidney ALR2 were linear in the range of 5-100 ng; a similar sensitivity was seen in tissue homogenates. The results presented here for the ALR2 level in bovine tissues (kidney greater than heart greater than brain greater than lens) are in agreement with literature values for those tissues from which the enzyme has previously been purified. The interspecies similarity in electrophoretic mobility and the retention of antibody reactivity suggest extensive phylogenetic epitope conservation in mammalian aldose reductase.     

Monoclonal antibodies against Mycoplasma hyorhinis: A secondary effect of immunization with cultured cells

Monoclonal antibodies were prepared against two different human tumour cell lines, the melanoma cell line SK-Mel-25 and the acute lymphoblastic leukemia T cell line CCRF-CEM. Presence of antibodies against human tumour cells in the supernatants of hybridoma cultures was tested by binding of ¹²⁵I-F(ab′)₂ anti-mouse IgG. On two occasions a hybridoma culture, initially selected for subsequent cloning as it seemingly produced antibodies against tumour cells, was later found to produce monoclonal antibodies specific for Mycoplasma hyorhinis. In immunofluorescent staining patchy structures were visible which seemed to be attached to the cell surface. By combined staining with FITC-conjugated anti-mouse immunoglobulin for monoclonal antibody, Evans blue for cytoplasm and Hoechst compound no. 33258 for DNA, the reaction against mycoplasma could be recognized. These results demonstrate that if cultured cells are used for preparation of monoclonal antibodies, there is a good chance that the selected hybridomas may produce antibodies against ‘culture artifacts’ such as mycoplasmas, in addition to the target antigens. Thus mycoplasma contamination of cell cultures poses a serious problem in the hybridoma research and the testing system for antibody specificity should be carefully monitored.     

Characterization of Mono- and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.     

A Conformation- and Phosphorylation-Dependent Antibody Recognizing the Paired Helical Filaments of Alzheimer's Disease

Abstract: Hyperphosphorylated tau (PHF-tau) is the major constituent of paired helical filaments (PHFs) from Alzheimer's disease (AD) brains. This conclusion has been based largely on the creation and characterization of monoclonal antibodies raised against PHFs, which can be classified in three categories: (a) those recognizing unmodified primary sequences of tau, (b) those recognizing phosphorylation-dependent epitopes on tau, and (c) those recognizing conformation-dependent epitopes on tau. Recent studies have suggested that the antibodies recognizing primary sequence and phosphorylation-dependent epitopes on tau are unable to distinguish between normal adult biopsy tau and PHF-tau. We now present evidence for a new fourth class of monoclonal antibodies recognizing conformation-dependent phosphoepitopes on tau, typified by TG-3, a monoclonal antibody raised to PHFs from AD brain homogenates. Studies using a series of deletional tau mutants, site-directed tau mutants, and synthetic peptides enable the precise epitope mapping of TG-3. Additional studies demonstrate that TG-3 reacts with neonatal mouse tau and PHF-tau but does not recognize adult mouse tau or tau derived from normal human autopsy or biopsy tissue. Further investigation reveals that TG-3 recognizes a unique conformation of tau found almost exclusively in PHFs from AD brains.     

Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits: advantages over antisera and technical limitations

Four monoclonal antibodies against guinea pig myelin basic protein (MBP), and four against subunits of bovine neurofilament triplet proteins (NF) were produced and their activity determined by enzyme-linked immunosorbant assay. The specificity and cross-reactivity of these eight monoclonal antibodies and one heterologous antiserum against each of the two central nervous system (CNS) antigens were examined in a histological study using the immunoperoxidase, antibody sandwich technique in rat and human brain tissue. Tissue sections were prepared from paraffin-embedded or fresh brain tissue that had been fixed with one of five different fixatives. The resulting immunoperoxidase labeling was then graded for intensity and examined for artifacts. One monoclonal antibody against MBP and one against NF resulted in labeling that was superior to that given by each of the antisera against their respective antigens. Of the five fixatives tested, a mercuric chloride-formalin solution gave the best preservation of these two antigens in rat and human brain tissue. The mercuric chloride-formalin solution was found to be superior to the other fixatives when immersion fixation was used, and was especially optimal when brains were perfused fixed. Three artifacts were encountered among the various antibody-fixative combinations that produced erroneous, but seemingly specific staining of Purkinje cells, neurons and axons, or astrocytes.     

Brain-associated glycoproteins expressed in bovine heart purkinje fibres

Summary The Purkinje fibres of bovine heart were investigated immunohistochemically by use of monoclonal antibodies with specificity against the glycoproteins Thy-1 and Gp120, expressed in human brain. The existence and expression in bovine tissues (brain and thymus) of antigens displaying similar properties and immunochemical crossreactivity with monoclonal antibodies against the human antigens were confirmed.Both these antigens, as identified by use of anti Thy-1 and anti-Gp120 monoclonal antibodies were found to be associated with the membranes of the impulse conduction system. The presence of the antigens was seen in areas facing other conduction cells. No parts of the cells facing the basal membrane of the fibres were stained. The continuous staining between the cells was different from that of desmosome related proteins. These findings may have physiological and functional implications and are interesting in relation to recent evidences suggesting that the conduction tissue might be a derivative of the neural crest.     

Brain-associated glycoproteins expressed in bovine heart Purkinje fibres.

The Purkinje fibres of bovine heart were investigated immunohistochemically by use of monoclonal antibodies with specificity against the glycoproteins Thy-1 and Gp120, expressed in human brain. The existence and expression in bovine tissues (brain and thymus) of antigens displaying similar properties and immunochemical crossreactivity with monoclonal antibodies against the human antigens were confirmed. Both these antigens, as identified by use of anti Thy-1 and anti-Gp120 monoclonal antibodies were found to be associated with the membranes of the impulse conduction system. The presence of the antigens was seen in areas facing other conduction cells. No parts of the cells facing the basal membrane of the fibres were stained. The continuous staining between the cells was different from that of desmosome related proteins. These findings may have physiological and functional implications and are interesting in relation to recent evidences suggesting that the conduction tissue might be a derivative of the neural crest.     

Precursors of neuropeptides in the marine worm Nereis diversicolor. In vitro translation of a precursor related to human prepro-cholecystokinin and immunolocalization of this precursor in the nervous system

Total mRNA extracted from the brain of a marine worm, Nereis diversicolor, were in vitro translated using 2 cell-free systems: rabbit reticulocyte lysate and wheat germ extract. Among numerous products newly translated in both systems, only one class of 70 kDa polypeptides immunoprecipitated when we used a mixture of 3 well defined antibodies raised against known sequences of the human prepro-CCK. At the cellular level, using immunocytochemistry techniques, strong and moderate immunoreactivities were seen in perikarya located in various ganglionic nuclei of the worm brain. Immunoreactive nerve fibres were visible in the neuropile but not in the infracerebral region, a neurohemal area. Immunoreactions also appeared on perikarya located in the anterior and medial groups of the ventral nerve cord. Furthermore, immunolabeled cells were observed in the midgut. Interestingly, several co-localizations of materials immunologically related to human prepro-CCK and CCK/gastrin were observed in the brain and the ventral nerve cord both in perikarya and in nerve fibres. We propose that, in Nereis a polypeptide (molecular mass 70 kDa) is the large precursor of molecules related to those of the CCK/gastrin peptide family.     

Human monoclonal antibodies to a genus-specific chlamydial antigen, produced by EBV-transformed B cells

Stable B cell lines producing human monoclonal antibodies to Chlamydia were established from salpingitis patients in the early convalescence phase. The antibody-producing cells were immortalized by Epstein Barr virus (EBV) transformation. Specific antibody-secreting clones were enriched by a stepwise microtiter plate cloning procedure. The selected B cell clones showed stable antibody production for more than 1 yr in continuous culture. Serologic specificity was demonstrated by micro-immunofluorescence (micro-IF) tests against a panel of Chlamydia reference strains. The antibodies were of the IgG1 subclass, and complement fixation could be demonstrated for one clone. There was no cross-reactivity against a large number of other bacteria. The monoclonal antibodies are directed against a common genus-specific surface antigen of the Chlamydia organism. Infected McCoy cells showed a brilliant, punctuated fluorescence surrounded by an inclusion membrane. Compared with conventional antisera, the monoclonal antibodies showed a clearer fluorescence pattern with very low background.     

Production and characterization of monoclonal antibodies to porcine brain pyridoxal kinase.

Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.     

Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.     

Fasciola hepatica: development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.     

Sharing of antigenic epitopes between synaptophysin and granulophysin.

The immunological crossreactivity between the two granule-specific membrane glycoproteins, synaptophysin and granulophysin, was studied using a series of site-specific monoclonal and polyclonal antibodies. The epitope relatedness of six monoclonal antibodies against granulophysin was examined by competitive ELISA. The antibodies are shown to recognize distinct, but overlapping epitopes within a compact region that is constructed by the three-dimensional configuration of the molecule. All these antibody clones also recognize rat neuronal synaptophysin. Two monoclonal antibodies against synaptophysin, of which one is the well-characterized SY38 antibody, directed against the carboxy terminal of the molecule, are also shown to react with granulophysin. Characterized polyclonal antibodies against different peptide antigens of synaptophysin failed to recognize granulophysin. Synaptophysin and granulophysin are distinctly recognized in brain cell (white matter) and the pituitary both qualitatively and quantitatively. Based on these and other observations, it is suggested that the repeat motif in the cytoplasmic tail of synaptophysin represents an immunodominant construct that is the target for the observed crossreactive antibodies and that a similar tertiary construct has been preserved in granulophysin and in other transmembrane proteins.