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The Journal of Neuroscience : the... Sep 1988The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce the steroid hormone ecdysone thus initiating...
The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce the steroid hormone ecdysone thus initiating molting and metamorphosis. "Big" PTTH, one of several molecular forms of the neurohormone, was isolated from brains of the tobacco hornworm Manduca sexta, and fractionated by high-pressure liquid chromatography (HPLC) for use in antibody production. A murine polyclonal antiserum and a monoclonal antibody (MAb) have been generated using this highly purified preparation of big PTTH. Antisera and hybridoma supernatants were screened with an indirect, brain whole-mount immunocytological assay, and antibody specificity was confirmed by immunocytological, ELISA, and functional criteria. In brain whole-mount preparations, the MAb (A2H5) and antiserum specifically immunostained the lateral protocerebral neurosecretory cells (L-NSC III), the prothoracicotropes, which produce PTTH. This immunostaining was blocked by preadsorbing the antibodies with big PTTH. Analysis of the elution of HPLC-fractionated big PTTH with an in vitro bioassay for the neurohormone and an ELISA employing the A2H5 MAb resulted in peaks of activity that were superimposable. Finally, the antiserum and A2H5 MAb inhibited big PTTH activation of the prothoracic glands to synthesize ecdysone in the in vitro bioassay for the neurohormone. With these specific antibodies, the organization of the PTTH neuroendocrine axis has been defined. It is now evident that both of the peptidergic neurons that comprise the L-NSC III are prothoracicotropes, and that the corpora allata are the neurohemal organs for the release of big PTTH into the hemolymph. This study indicates that these specific antibodies will be useful in investigations of numerous aspects of the biology of this cerebral neuroendocrine axis.
Topics: Animals; Antibodies, Monoclonal; Antibody Formation; Antibody Specificity; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Immune Sera; Immunologic Techniques; Insect Hormones; Lepidoptera; Mice; Neurosecretory Systems
PubMed: 3049956
DOI: 10.1523/JNEUROSCI.08-09-03247.1988 -
Bulletin of the World Health... 1967The determination of the activity of viper antiserum in terms of the effective dose against 1 minimum lethal dose (MLD) of venom often leads to results which are not...
The determination of the activity of viper antiserum in terms of the effective dose against 1 minimum lethal dose (MLD) of venom often leads to results which are not very reproducible. The present study concerns the preparation of a standard Vipera ammodytes antiserum and the choice of a suitable venom test dose. Portions of purified, concentrated antiserum were distributed into vacuum-sealed ampoules for use as a standard, and the amounts of this standard antiserum needed to neutralize various amounts of ammodytes venom were determined. The antivenin unit (AU) was fixed at 1.90 mg; 1 AU is the amount of standard serum required to protect 50% of white mice weighing 18 g against 0.05 mg (5 MLD) of venom. The use of the standard antiserum for titration of crude antisera in terms of this AU is described.
Topics: Animals; Antivenins; Immune Sera; Mice; Snake Bites; Snakes
PubMed: 5300059
DOI: No ID Found -
The Biochemical Journal Dec 1992Angiotensin converting enzyme (ACE; EC 3.4.15.1) was purified from porcine kidney and lung (endothelial isoenzyme) and testis (testicular isoenzyme) by affinity... (Comparative Study)
Comparative Study
A comparison of the zinc contents and substrate specificities of the endothelial and testicular forms of porcine angiotensin converting enzyme and the preparation of isoenzyme-specific antisera.
Angiotensin converting enzyme (ACE; EC 3.4.15.1) was purified from porcine kidney and lung (endothelial isoenzyme) and testis (testicular isoenzyme) by affinity chromatography on lisinopril-2.8 nm-Sepharose. Atomic-absorption spectroscopy revealed that ACE purified from kidney and lung contained 2.58 and 2.35 atoms of zinc per molecule of enzyme (M(r) 147,000) respectively. In contrast, ACE purified from testis contained only 1.58 atoms of zinc per molecule of enzyme (M(r) 80,000). Thus it would appear that both putative zinc-binding sites in endothelial ACE contain zinc and may therefore be catalytically active. No differences were observed in the pattern of products generated on hydrolysis of benzoyl (Bz)-Gly-His-Leu, substance P, luteinizing-hormone-releasing hormone (LH-RH) and its analogue, des-Gly10-LH-RH-ethylamide, by kidney and testicular ACE. There was also no difference in the initial rates of hydrolysis of Bz-Gly-His-Leu or substance P by the two isoenzymes, although LH-RH and its analogue were hydrolysed twice as rapidly by kidney ACE. It is therefore unlikely that the N-terminal catalytic site in porcine endothelial ACE is predominantly responsible for the atypical cleavage of LH-RH generating the N-terminal tripeptide. Two polyclonal antisera were raised to the affinity-purified forms of pig kidney and testicular ACE. Isoenzyme-specific antisera were then isolated from these by absorbing out those antibodies recognizing determinants on the other isoenzyme. Immunoelectrophoretic blot analyses and immunofluorescent staining of sections of pig kidney were used to demonstrate the specificity of the antisera. Immunofluorescent staining of sections of pig testis with the antiserum specific to testicular ACE localized testicular ACE solely to the lumen of the seminiferous tubules, whereas the antiserum specific to endothelial ACE revealed the presence of this isoenzyme only in blood vessels. The antiserum to endothelial ACE, which recognizes determinants in the unique N-terminal domain, was investigated as a possible specific inhibitor of the N-terminal catalytic site. Although this antiserum failed to inhibit testicular ACE, the effect on the activity of endothelial ACE appeared to be due to inhibition of both the N- and C-terminal catalytic sites.
Topics: Amino Acid Sequence; Angiotensin-Converting Enzyme Inhibitors; Animals; Antibodies; Antibody Formation; Antibody Specificity; Binding Sites; Cross Reactions; Endothelium; Enzymes, Immobilized; Fluorescent Antibody Technique; Hydrolysis; Immune Sera; Immunohistochemistry; Isoenzymes; Kidney; Kinetics; Lung; Male; Molecular Sequence Data; Peptide Mapping; Peptides; Peptidyl-Dipeptidase A; Rabbits; Substrate Specificity; Swine; Testis; Zinc
PubMed: 1335236
DOI: 10.1042/bj2880875 -
Scientific Reports Jul 2020Snakebite envenomation is a neglected tropical disease of high mortality and morbidity largely due to insufficient supply of effective and affordable antivenoms. Snake...
Snakebite envenomation is a neglected tropical disease of high mortality and morbidity largely due to insufficient supply of effective and affordable antivenoms. Snake antivenoms are mostly effective against the venoms used in their production. It is thus crucial that effective and affordable antivenom(s) with wide para-specificity, capable of neutralizing the venoms of a large number of snakes, be produced. Here we studied the pan-specific antiserum prepared previously by a novel immunization strategy involving the exposure of horses to a 'diverse toxin repertoire' consisting of 12 neurotoxic Asian snake toxin fractions/ venoms from six species. This antiserum was previously shown to exhibit wide para-specificity by neutralizing 11 homologous and 16 heterologous venoms from Asia and Africa. We now show that the antiserum can neutralize 9 out of 10 additional neurotoxic venoms. Altogether, 36 snake venoms belonging to 10 genera from 4 continents were neutralized by the antiserum. Toxin profiles previously generated using proteomic techniques of these 36 venoms identified α-neurotoxins, β-neurotoxins, and cytotoxins as predominant toxins presumably neutralized by the antiserum. The bases for the wide para-specificity of the antiserum are discussed. These findings indicate that it is feasible to generate antivenoms of wide para-specificity against elapid neurotoxic venoms from different regions in the world and raises the possibility of a universal neurotoxic antivenom. This should reduce the mortality resulting from neurotoxic snakebite envenomation.
Topics: Animals; Antivenins; Elapid Venoms; Elapidae; Immune Sera; Immunization; Neurotoxins; Proteomics; Snake Venoms; Snakes; Vaccination
PubMed: 32647261
DOI: 10.1038/s41598-020-66657-8 -
Free Radical Biology & Medicine Sep 2012Methionine residues in protein can be oxidized by reactive oxygen or nitrogen species to generate methionine sulfoxide. This covalent modification has been implicated in...
Methionine residues in protein can be oxidized by reactive oxygen or nitrogen species to generate methionine sulfoxide. This covalent modification has been implicated in processes ranging from normal cell signaling to neurodegenerative diseases. A general method for detecting methionine sulfoxide in proteins would be of great value in studying these processes, but development of a chemical or immunochemical technique has been elusive. Recently, an antiserum raised against an oxidized corn protein, DZS18, was reported to be specific for methionine sulfoxide in proteins (Arch. Biochem. Biophys. 485:35-40; 2009). However, data included in that report indicate that the antiserum is not specific. Utilizing well-characterized native and methionine-oxidized glutamine synthetase and aprotinin, we confirm that the antiserum does not possess specificity for methionine sulfoxide.
Topics: Animals; Antibodies; Antibody Specificity; Aprotinin; Blotting, Western; Escherichia coli Proteins; Glutamate-Ammonia Ligase; Immune Sera; Methionine; Protein Processing, Post-Translational; Rabbits; Reference Standards
PubMed: 22771451
DOI: 10.1016/j.freeradbiomed.2012.06.036 -
The Journal of Clinical Investigation Apr 1970Identifying posterior pituitary hormones in body fluids or neurohypophysial extracts was heretofore partially achieved by using pharmacologic potency ratios or...
Identifying posterior pituitary hormones in body fluids or neurohypophysial extracts was heretofore partially achieved by using pharmacologic potency ratios or semispecific inactivation by thioglycolate or enzymes. Production of antisera against oxytocin and lysine-vasopressin has prompted us to test their specificity against lysine-vasopressin, arginine-vasopressin, arginine-vasotocin, and oxytocin. In ethanol anesthetized rats, antidiuretic and milk-ejection activities were assayed for each peptide-antiserum combination after 0, 30, 60, and 90 min of incubation. Results indicate that (a) oxytocin antiserum inactivates oxytocin, but not arginine-vasopressin, lysine-vasopressin, or arginine-vasotocin; vasopressin antiserum inactivates arginine-vasopressin and lysine-vasopressin, but neither oxytocin nor arginine-vasotocin; (b) an identifiable antigenic site exists for each hormone; (c) relatively specific identifications of natural neurohypophysial peptides are possible using antisera and bioassays; (d) this method is promising for identifying neurohypophysial peptides in body fluids and pituitary extracts; and (e) active and passive immunization against oxytocin and vasopressin may increase our understanding of their physiologic functions.
Topics: Animals; Antibody Formation; Arginine; Diuresis; Female; Immune Sera; Lactation; Lysine; Oxytocin; Pituitary Gland, Posterior; Pituitary Hormones, Posterior; Pregnancy; Rats; Thioglycolates; Tissue Extracts; Vasopressins; Vasotocin
PubMed: 5443183
DOI: 10.1172/JCI106296 -
Genetics and Molecular Research : GMR Nov 2014The aim of this study was to separate, purify, and identify Salmonella paratyphi A flagellin, and to prepare its antisera. Primary flagellin was isolated from S....
The aim of this study was to separate, purify, and identify Salmonella paratyphi A flagellin, and to prepare its antisera. Primary flagellin was isolated from S. paratyphi A using the acid lysis method. The flagellin was purified with weak anion exchange chromatography and the protein was identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and negative staining with phosphotungstic acid with scanning electron microscopy (SEM). The production of the obtained flagellin was then quantified. New Zealand white rabbits were then immunized with the isolated flagellin, the presence of serum anti-flagellin antibodies was assessed with the immunoblot test, and its potency was determined with the double immunodiffusion test. The results of SDS-PAGE showed that the molecular weight (m.w.) of the purified flagellin was 52 x 10(3). The immunoblot test also showed a band at 52 x 10(3) m.w. The SEM results showed that the flagellin was filamentous. These three results showed that the protein was homogeneous. The protein quantification analysis found that 4.8 ± 0.5 mg flagellin could be extracted per 1 g wet weight bacteria. The titer of the anti-flagellin antiserum was 1:64. Through this method, we obtained high productions of flagellin, which could be easily purified, identified, and prepared into high titer antiserum.
Topics: Animals; Blotting, Western; Chromatography, Ion Exchange; Complex Mixtures; Electrophoresis, Polyacrylamide Gel; Flagellin; Immune Sera; Microscopy, Electron, Scanning; Rabbits; Salmonella paratyphi A
PubMed: 25501138
DOI: 10.4238/2014.November.7.3 -
The American Journal of Pathology Jul 1981The molecular nature of neurofibrillary tangles of senile dementia of the Alzheimer type (SDAT) was studied by immunoperoxidase and immunofluorescence techniques. Five...
The molecular nature of neurofibrillary tangles of senile dementia of the Alzheimer type (SDAT) was studied by immunoperoxidase and immunofluorescence techniques. Five antiserums, including anti-humanbrain-2-cycle-purified-microtubule-fractions (2 x MT), anti-calf-brain-2 x MT, anti-sea-urchin-egg-tubulin, antibeef-brain-tubulin, and anti-human-brain-neurofilament(NF)-210-kilodalton(kd)-protein were tested for their binding to neurofibrillary tangles. The antihuman-2 x MT serum stained structures resembling neurofibrillary tangles, neurites of neuritic plaques, and microglialike cells in SDAT brains, but no such staining pattern was detected in normal brain sections. In neurons isolated from SDAT brains, about 40% of the tangles were labeled by the anti-human-2xMT serum with an identical pattern. Other antiserums tested did not preferentially bind tanglelike structures in tissue sections and bound to less than 5% of the tangles in isolated neurons. These results suggest that the antigenic sites of tubulin and NF proteins are not shared by neurofibrillary tangles. Different from the calf preparation, the human-2 x MT fractions contained a prominent protein band that was identical to ferritin in molecular weight and cross-reacted with anti-human-2 x MT and anti-human-ferritin serums. However, antiserums to this ferritinlike protein, or anti-ferritin, did not stain neurofibrillary tangles. Although neither the calf 2 x MT nor two other human MT fractions failed to elicit an antiserum that stained tangles, these fractions were able to remove the antihuman-2 x MT serum activity that binds to tangles. The data suggest that the protein (or proteins) that makes up neurofibrillary tangles of SDAT is present in various quantities in microtubule fractions of normal brain.
Topics: Alzheimer Disease; Animals; Brain Chemistry; Cattle; Cytoskeleton; Dementia; Electrophoresis, Polyacrylamide Gel; Ferritins; Fluorescent Antibody Technique; Goats; Humans; Immune Sera; Neurons; Rabbits; Tubulin
PubMed: 7020426
DOI: No ID Found -
The Journal of Cell Biology Feb 1979An antiserum prepared against purified surface membranes of transformed BHK21/C13 cells (C13/B4) reversibly rounded and detached hamster cells from plastic tissue...
Studies on the function of cell surface glycoproteins. I. Use of antisera to surface membranes in the identification of membrane components relevant to cell-substrate adhesion.
An antiserum prepared against purified surface membranes of transformed BHK21/C13 cells (C13/B4) reversibly rounded and detached hamster cells from plastic tissue culture plates but did not affect cells of other species. Antiserum treatment did not alter the growth rate of C13/B4 or BHK21/C13 cells; however, NIL-8 cells exposed to the antiserum detached from the substrate and stopped growing, but remained viable for up to 72 h in the presence of the antiserum. Rounding and detachment were not inhibited by DNP or cycloheximide. Antiserum-detached cells did not reattach in the presence of these inhibitors. F(ab)' fragments also induced rounding, thus ruling out the involvement of complement and ligand-induced rearrangement of surface antigens in rounding and detachment. Three different surface-reactive immunoglobulin preparations were used in indirect immunoprecipitation studies in an attempt to identify cell surface antigens involved in regulating adhesion and morphology. Antiserum against surface membranes (anti-M) and against material shed by the cells into serum-free medium (anti-SFM) caused rounding and detachment, but a third antiserum (anti-LIS) prepared against a partially purified glycoprotein did not. All three immunoglobulin preparations precipitated glycoproteins with an apparent mol wt of 120,000 daltons from a crude membrane preparation solubilized by Nonidet NP-40. The two immunoglobulin preparations that caused rounding precipitated an additional glycoprotein peak of 140,000 daltons. Extensive preabsorption of the extract with anti-LIS immunoglobulin enriched the anti-membrane and antiserum-free medium precipitates for the 140,000-dalton peak. Anti-M immunoglobulin eluted from intact cells and subsequently used to precipitate NP-40 solubilized membrane constituents also reacted with a group of glycoproteins of approximately 140,000 mol wt. Therefore, this group of glycoproteins was considered most likely to be the glycoproteins involved in substrate adhesion and maintenance of cellular morphology.
Topics: Animals; Antigens, Surface; Cell Adhesion; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cricetinae; Fibroblasts; Glycoproteins; Immune Sera; Kidney; Membrane Proteins; Neoplasm Proteins; Species Specificity
PubMed: 457748
DOI: 10.1083/jcb.80.2.385 -
Journal of Dairy Science Dec 2000On the background of positive survival data from farms in Mississippi, treating calves with antiserum injection in addition to normal colostrum administration, the... (Clinical Trial)
Clinical Trial Randomized Controlled Trial
On the background of positive survival data from farms in Mississippi, treating calves with antiserum injection in addition to normal colostrum administration, the objective of the present study was to evaluate the influence of a single subcutaneously administered bovine antiserum injection (0.031 g of IgG/kg of body weight) and pooled colostrum administration on efficiency of Ig absorption and on 24-h plasma IgG concentration in neonatal bull calves. Twenty-nine male dairy calves (21 Holsteins and 8 Jerseys) were assigned randomly at parturition to receive one of four treatments: 1) colostrum (n = 9), 2) colostrum and bovine antiserum injection (n = 7), 3) milk replacer (n = 5), or 4) milk replacer and bovine antiserum injection (n = 8). At birth, calves either did or did not receive an injection of bovine antiserum and were fed pooled colostrum or milk replacer (Holsteins, 3.8 L; Jerseys, 1.9 L) via an esophageal feeder. Blood was collected immediately before administration of the colostrum or milk replacer, then again at 24 and 48 h postpartum. Immunoglobulin G concentrations of colostrum, milk replacer, antiserum, and plasma were monitored by single radial immunodiffusion. Colostrum administration and injection of bovine antiserum each increased plasma Ig concentration at 24 h posttreatment. In addition, antiserum injection increased the apparent efficiency of absorption of colostral Ig by 42% over that for calves fed colostrum alone. The increase in plasma IgG for antiserum-treated calves exceeded the total amount of IgG administered in the antiserum injection; hence, this increase appeared to be the result of an increase in total absorption of colostral IgG, or possibly antiserum injection somehow triggered active synthesis of IgG. Injection of antiserum might possibly serve as a beneficial adjunct to a colostrum management program by enhancing the acquisition of passive immunity from colostral sources.
Topics: Animals; Animals, Newborn; Cattle; Colostrum; Immune Sera; Immunity, Maternally-Acquired; Immunoglobulin G; Injections, Subcutaneous; Intestinal Absorption; Kinetics; Male; Survival; Weight Gain
PubMed: 11132854
DOI: 10.3168/jds.S0022-0302(00)75182-4