Design and application of a polyclonal peptide antiserum for the universal detection of leptin protein

An epitope-specific polyclonal antiserum was produced in rabbits immunized against a synthetic 15 amino acid peptide (QRVTGLDFIPGLHPV) derived from the coding sequence reported for the porcine leptin gene (GenBank Accession No. U59894). This peptide contains a core sequence comprised of eight amino acids (GLDFIPGL) that is totally conserved in all leptin proteins studied to date. Purified recombinant human, mouse, rat, pig, and chicken leptin proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotted onto PVDF membranes. Western blots were developed employing the leptin-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The peptide antiserum was found to be highly specific for leptin which exhibited an estimated molecular weight of about 16 kDa for all species analyzed. The sensitivity of the Western blot assay was not sufficient to permit the direct detection of leptin in chicken serum or plasma. However, with this assay we were able to detect native leptin protein in an enriched fraction prepared from chicken plasma using a combination of gel filtration and ion exchange column chromatography. Slot blots indicated a potential application of the immunostaining technique for quantitative analysis of leptin protein. Finally, the peptide antiserum was successfully employed to localize leptin protein by immunohistochemical staining of thin sections prepared from adipose (chicken and pig) and liver (chicken) tissue samples. This study is the first to report a polyclonal peptide antiserum that apparently recognizes intact leptin protein, both native and recombinant, regardless of the species of origin.     

Production and application of a polyclonal peptide antiserum for universal detection of StAR protein

We report production of a polyclonal antibody against the StAR (steroidogenic acute regulatory) protein of steroidogenic cells and immunohistochemistry (IHC) staining of bovine adrenal gland tissue available in paraffin block. The epitope-specific polyclonal antibody was produced in a rabbit immunized against a synthetic 26 amino acid peptide (82AMQRALGILKDQEGWKKESRANGDE107) derived from the coding sequences reported for the bovine StAR gene (Gene Bank Accession No. Q28918). Western blots were developed using the StAR-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The antiserum was found to be highly specific for StAR, which exhibited an estimated molecular weight of about 30 KDa for all species analyzed. Finally, the peptide antiserum was successfully employed to localize StAR protein by immunohistochemical staining of thin sections prepared from bovine adrenal gland tissue. This study is the first to report a polyclonal peptide antiserum that apparently recognizes native StAR protein, regardless of the species of origin. The successful production of the antibody has provided a useful tool for studying regulation of StAR protein.     

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.     

Antibodies Raised Against Synthetic Peptides React with Choline Acetyltransferase in Various Immunoassays and in Immunohistochemistry

Abstract: Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity-purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity-purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168- 189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.     

Immunogold-EM localization of myrosinase in Brassicaceae

Summary Immunogold labelling has been used to study the cellular and subcellular localization of myrosinase (-thioglucosidase, EC 3.2.3.1), using LR-White acrylic resin and ultrahin sections from four different species of Brassicaceae;Brassica napus L.,Sinapis alba L.,Raphanus sativus L., and B.oleracea L. For immunolabelling, a polyclonal antibody raised in rabbit against a highly purified myrosinase fromSinapis alba was used. Western blots showed that the antiserum was specific against myrosinase from these species. Ultrathin sections were sequentially incubated with anti-myrosinase antiserum and with secondary antibodies conjugated with colloidal gold. Gold label was present in typical myrosin cells both in radicles and in cotyledons when observed in the electron microscope. The intracellular localization showed that myrosinase was present in myrosin grains in the myrosin cells in all four species of Brassicaceae.Abbreviations BSA bovine serum albumin - PBS phosphate buffered saline - PBS-T PBS with 0.5% v/v Tween-20     

Production of Antiserum to Recombinant Coat Protein for Detecting Lily mottle virus in Yunnan, China

Lily mottle virus (LMoV), Potyvirus genus, is very difficult to purify for the preparation of diagnostic antisera. The coat protein (CP) gene of LMoV was amplified by RT-PCR from infected plants and cloned into the prokaryotic pET-30a vector to generate the recombinant plasmid pET-CP. The resulting carboxy-terminal His-tagged CP was over-expressed in Escherichia coli BL 21 cells by Isopropyl-β- d -thiogalactoside (IPTG) induction and purified over Ni-NTA affinity columns. The purified CP was used to elicit a polyclonal antiserum in rabbits. The antiserum had a titre of 1 : 128 in double diffusion tests, and specifically recognized LMoV in Western blots, Enzyme-Linked Immunosorbent Assays and Immuno-Electron Microscopy. The CP antiserum was also used to evaluate LMoV infection of lily bulbs used for commercial production in Yunnan, China. Substantial levels of infection were found in both imported and native bulbs, and provide the basis to implement flexible, rapid and large-scale virus indexing of lily plants for use in propagation and to meet virus-free quarantine regulations.     

A polyclonal antiserum against the rabbit progesterone receptor recognizes the human receptor: biochemical characterization

Polyclonal antiserum was generated in guinea pigs immunized with the 116,000 Mr rabbit uterine progesterone receptor (PR). The PR antigen was partially purified by DEAE-cellulose chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and the 116,000 Mr band excised and injected into guinea pigs. The antiserum recognized on protein blots rabbit uterine PR of Mr 116,000 and 81,000. The antiserum was judged to be specific for PR from normal and malignant human tissues as determined by sedimentation shift on sucrose gradients, immunoprecipitation studies, protein blotting, and fluorographic analysis using photolabelled samples. Comparison of protein blots probed with this polyclonal antiserum or with a recently obtained monoclonal antibody to human PR indicated that similar PR structures were recognized in rabbit and human samples by both antisera. Characterization of the polyclonal antiserum has demonstrated its suitability for investigating the immunolocalization or PR in normal and malignant human tissues as well as the receptor structure detected on protein blots.     

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydrolase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Cloning and expression of a region of vesicle associated membrane protein2 (VAMP2) gene and its use as a recombinant peptide substrate for assaying clostridial neurotoxins in contaminated biologicals

An assay for the endopeptidase activities of clostridial neurotoxins in contaminated biotherapeutic products has been developed. Based on a synthetic peptide substrate representing amino acid residues 60–94 of the intracellular vesicle associated membrane protein2 (VAMP2), RT-PCR was used to amplify the VAMP2 sequence. The extended insert was digested with EcoRI and SalI and ligated into pGEX4T-1 vector for construction of the pGEX4T-1/VAMP plasmid for expressing in Escherichia coli a fusion protein linked to glutathione S-transferase (GST). The fusion protein was purified by affinity chromatography and used in an ELISA assay for comparison with the commercially available synthetic VAMP peptide and rabbit polyclonal antiserum. The identity of the immunoreactivity of recombinant VAMP2 protein with the chemically synthesized peptide was demonstrated by western blot. Our results indicated that recombinant VAMP2 peptide not only reacted with specific polyclonal antibody in a dose-dependent manner, without any remarkable difference observed between the reactivity of the fusion protein and commercial VAMP2 segment peptide, but also cleaved by botulinum neurotoxin type B (BONT/B) after endopeptidase assay. Thus, recombinant VAMP2 could serve as a replacement for VAMP2 synthetic peptide, potentially useful in endopeptidase assays for replacement of the currently used mouse bioassay for clostridial neurotoxins contaminating biotherapeutic products.     

Purification of the Phytoplasma Associated with China-tree (Melia azedarach L.) Decline and the Production of a Polyclonal Antiserum for its Detection

China-tree or paraiso ( Melia azedarach ) decline, caused by a phytoplasma, is a disease in Argentina for which hitherto no diagnostic reagents were available. The production of a polyclonal antiserum using a purification protocol involving a double Percoll gradient is reported. The antiserum was used successfully in immuno-dot blot assay to discriminate between infected and healthy plants. It also recognized, although less reliably, other phytoplasmas present in garlic ( Allium sativum ), peach ( Prunus persica ) and tona ( Tona cilliata ). Organisms that by their size and morphology resembled phytoplasmas were detected by immuno-electron microscopy and immunogold labelling. The ability of the antiserum to discriminate between healthy and infected plants was confirmed by PCR amplification of fragments only from infected plants.     

Antiserum to a novel peptide sequence reacts selectively with epithelial subpopulations

A synthetic peptide corresponding to a novel protein sequence isolated from bovine kidney was used to immunize rabbits. When applied to Western blots of bovine kidney extracts, antiserum to this peptide recognizes proteins with molecular weights of 23 and 18 KD. Immunohistochemical examination of a variety of bovine and rat tissues with this antiserum revealed a unique distribution of immunoreactivity with the intermediate layers of a variety of stratified epithelia, in addition to renal glomeruli. The pattern of reactivity differed from previously described epithelial markers such as cytokeratins. These results indicate that this antiserum may be useful as a tool for the identification of cells of the intermediate layer of stratified epithelia and, as such, may aid in the study of this differentiating/proliferating tissue compartment.     

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.     

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher’s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59–66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.     

An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum.

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.     

Immunolocalization of CC10 in Clara cells in mouse and human lung

Two antisera, denoted R41 and R42, were raised against a synthetic peptide from the murine Clara cell-specific protein CC10, and one antiserum, denoted R40, was raised against human recombinant uteroglobin, the human homolog of murine CC10. Purified antigen-specific antisera, denoted R40AP, R41AP, and R42AP were prepared using peptide columns. The purified antisera were characterized by dot blots, immunohistochemistry, and immunoblots. Immunohistochemistry of mouse lung showed specific labeling of Clara cells in distal bronchioles by all three antisera. In human lung, the antiuteroglobin antiserum specifically labeled Clara cells, while the anti-mouse peptide antisera had weak crossreactivity and higher background staining. Electron microscopy revealed immunogold labeling of CC10 granules in Clara cells of mouse lung with all antisera. All antisera also labeled a 5-kDa protein on immunoblots of mouse lung homogenates. The surface epithelium of the alveolar air spaces around the distal bronchioles were CC10 positive suggesting a functional activity for CC10 in the lung parenchyma distal to Clara cells. R40AP immunohistochemical staining of sections of normal human lungs and lungs from patients with surfactant protein B deficiency, bronchopneumonia, and idiopathic alveolar proteinosis illustrate the utility of the anti-human CC10 antibody for diagnostic pathology.     

The use of constitutive nuclear oncoproteins to count neurons in the enteric nervous system of the guinea pig

Immunohistochemical double labelling of the enteric nervous system of the guinea pig ileum was performed with a monoclonal antibody (anti-MYC 033) directed against a peptide sequence of the human c-Myc protein together with antibodies directed against either the neuron-specific antigens neuron-specific enolase or PGP 9.5 or the glia-specific marker S-100 to demonstrate that anti-MYC 033 labelled the nuclei of all enteric neurons but not glia. This strategy was also employed to demonstrate that another anti-c-Myc monoclonal anti-body, anti-MYC 070, labelled the nuclei of all neurons and glia, as well as perhaps all other cells in these preparations. A polyclonal antiserum raised against a peptide sequence of the human c-Fos protein (anti-FOS 4) was shown to label the identical nuclei as anti-MYC 033. The ganglionic density of nuclei labelled by anti-FOS 4 was found to be similar to previous measures of the ganglionic density of neurons. Double labelling with anti-MYC 033 and an antiserum directed against vasoactive intestinal polypeptide was performed to reexamine the ganglionic density of neurons that express this neuropeptide. Our results suggest that the ganglionic density of these neurons might be less than previously determined.     

Tick paralysis: development of a vaccine.

The paralysis tick of Australia, Ixodes holocyclus, causes a severe toxicosis in domestic animals such as dogs and cats, livestock, and in some cases, humans. It is characterised by a rapidly ascending flaccid paralysis. The causative agent of the toxicosis is a neurotoxin(s) produced in the tick salivary glands. The current treatment for tick paralysis is in the form of a polyclonal dog antiserum. This antiserum treatment is expensive and effective only in the early stages of paralysis. The aim of current research is to develop a recombinant veterinary vaccine based on the tick neurotoxin peptide sequence. A successful vaccine would provide cost-effective, long-term protective immunity against tick-induced paralysis.     

Characterization of a stable L-form of the fish pathogen Ameromonas salmonicida

A stable L-form of Aeromonas salmonicida , which resulted form induction with benzylpenicillin, contained more of an outer membrane protein, with an estimated molecular wight of 40 kDza but less of 47.9 and 38 kDa proteins, than did parental walled cells. In addition from Western blots, two protein bands reacted strongly with a polyclonal antiserum. The antiserum did not react demonstrably with teh band detect in the L-forms on the gels.