scholarly journals Evidence for a Phytoreovirus Associated with Tobacco Exhibiting Leaf Curl Symptoms in South Africa

1999 ◽  
Vol 89 (4) ◽  
pp. 303-307 ◽  
Author(s):  
Marie Emma Christine Rey ◽  
Elvera D'Andrea ◽  
Jennifer Calvert-Evers ◽  
Maria Paximadis ◽  
Guido Boccardo
Keyword(s):  
South Africa ◽  
Inner Core ◽  
Sequence Similarity ◽  
First Record ◽  
Outer Core ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Class I ◽  
Enzyme Linked Immunosorbent Assay ◽  
Leaf Curl

Three forms of tobacco leaf curl (termed classes I, II, and III, based on symptomatology) recently have been described in southern Africa. Numerous attempts to isolate virus particles responsible for a nongeminivirus-induced leaf curl disease (class I) of tobacco in South Africa have been unsuccessful. Recently, 12 dsRNA segments were isolated from tobacco exhibiting class I leaf curl symptoms, suggesting a possible reovirus genome. The objective of our study was to confirm whether the dsRNA segments are associated with a reovirus. Isolation of icosahedral particles with an outer core 60 to 65 nm in diameter and an inner core 40 to 45 nm in diameter was achieved. Twelve distinct nonpolyadenylated dsRNAs were isolated from purified virions, and the total molecular masses of the dsRNAs ranged from 17.86 to 18.40 × 106 Da in polyacrylamide and agarose gels, respectively. Using hybridization analysis, dsRNAs were identified as non-homologous distinct segments. Comparisons with other known reoviruses revealed a unique banding pattern that was most similar to the wound tumor virus (WTV), the type species of the genus Phytoreovirus. Hybridizations of WTV cloned DNA probes (segments S4 and S6 to S9) and dsRNAs from infected tobacco indicated no significant sequence similarity, whereas indirect enzyme-linked immunosorbent assay with a polyclonal antiserum to WTV showed strong positive cross-reactivity to tobacco virions. Our results indicate a virus with features consistent with those of phytoreoviruses. This is the first report of a plant reovirus in tobacco, the first record in Africa, and the second example of a field-isolated dicot phytoreovirus.

An Enzyme-Linked Immunosorbent Assay (ELISA) Used to Study the Cellular Secretion of Endothelial Plasminogen Activator Inhibitor (PAI-1)

1988 ◽  
Vol 59 (01) ◽  
pp. 068-072 ◽  
Author(s):  
I R MacGregor ◽  
N A Booth
Keyword(s):  
Specific Activity ◽  
Umbilical Vein ◽  
Sandwich Elisa ◽  
Secretory Protein ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wide Range ◽  
Pai 1 ◽  
Cellular Secretion

SummaryA two-site sandwich ELISA was developed to measure PAI-1 antigen and utilised a polyclonal antiserum produced against PAI-1 purified from human endothelial cell secretory products. The assay was calibrated against a preparation of pure PAI-1 whose protein concentration had been determined by amino acid analysis and the detection limit was 30 pg PAI-1 ml“3 sample. PAI-1 was detected in primate sera but not in a wide range of nonprimate sera and no cross-reactivity with α2-antiplasmin or antithrombin III was observed.The ELISA was used to study cellular secretion of PAI-1 which was confirmed as a major secretory protein in human umbilical vein endothelial cells (HUVEC). PAI-1 antigen accumulated in the medium in a linear fashion with time and accounted for ≃10% of total secreted protein. Specific activity of intracellular PAI1 was typically 20-fold greater than that of PAI-1 in 24 h conditioned medium and a t½ for inactivation of secreted PAI-1 of 0.53 h was calculated. Purified endotoxin stimulated the secretion of PAI-1 antigen and raised the intracellular levels in HUVEC cultures showing that the anti-fibrinolytic actions of endotoxin are effected by increasing the rate of synthesis and secretion of PAI-1.

scholarly journals Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2622
Author(s):  
Aliah Zannierah Mohsin ◽  
Rashidah Sukor ◽  
Jinap Selamat ◽  
Anis Shobirin Meor Hussin ◽  
Intan Hakimah Ismail ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Analytical Methods ◽  
Polyclonal Antibodies ◽  
Sequence Similarity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Sequence Similarity ◽  
High Affinity

The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10−3 μM compared to 9.046 × 10−3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.

scholarly journals Serological Methods for Detection of Polymyxa graminis, an Obligate Root Parasite and Vector of Plant Viruses

2000 ◽  
Vol 90 (5) ◽  
pp. 537-545 ◽  
Author(s):  
P. Delfosse ◽  
A. S. Reddy ◽  
A. Legréve ◽  
K. Thirumala Devi ◽  
M. D. Abdurahman ◽  
...  
Keyword(s):  
Simple Procedure ◽  
Plant Viruses ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Northern India ◽  
Homologous Antigen ◽  
Root Parasite ◽  
Controlled Environments ◽  
Olpidium Brassicae

A purification procedure was developed to separate Polymyxa graminisresting spores from sorghum root materials. The spores were used as im-munogen to produce a polyclonal antiserum. In a direct antigen coating enzyme-linked immunosorbent assay (DAC ELISA), the antiserum could detect one sporosorus per well of the ELISA plate. In spiked root samples, the procedure detected one sporosorus per mg of dried sorghum roots. The majority of isolates of P. graminis from Europe, North America, and India reacted strongly with the antiserum. Interestingly, P. graminis isolates from the state of Rajasthan (northern India), from Pakistan, and an isolate from Senegal (West Africa) reacted weakly with the antiserum. The cross-reactivity of the serum with P. betae isolates from Belgium and Turkey was about 40% of that observed for the homologous isolate. There was no reaction with common fungi infecting roots or with the obligate parasite Olpidium brassicae. However, two isolates of Spongospora sub-terranea gave an absorbance similar to that observed with the homologous antigen. The DAC ELISA procedure was successfully used to detect various stages in the life cycle of P. graminis and to detect infection that occurred under natural and controlled environments. A simple procedure to conjugate antibodies to fluorescein 5-isothiocyanate (FITC) is described. Resting spores could be detected in root sections by using FITC-labeled antibodies. The potential for application of the two serological techniques for studying the epidemiology of peanut clump disease and for the characterization of Polymyxa isolates from various geographical origins is discussed.

Development of an Enzyme-Linked Immunosorbent Assay Method To Detect Mustard Protein in Mustard Seed Oil

2007 ◽  
Vol 70 (1) ◽  
pp. 179-183 ◽  
Author(s):  
STEF J. KOPPELMAN ◽  
RIEK VLOOSWIJK ◽  
GINA BOTTGER ◽  
GERT van DUIJN ◽  
PETER van der SCHAFT ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Seed Oil ◽  
Food Product ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mustard Seed ◽  
Calculated Level ◽  
Mustard Protein

An enzyme-linked immunosorbent assay for the detection of mustard protein was developed. The assay is based on a polyclonal antiserum directed against a mixture of mustard proteins raised in rabbits. The assay has a detection limit of 1.5 ppm (milligrams per kilogram) and is suitable for the detection of traces of mustard protein in mustard seed–derived flavoring ingredients. Limited cross-reactivity testing showed that no other plant proteins reacted significantly. From the animal proteins tested, only milk showed some cross-reactivity. With this sensitive assay, it was shown that refined mustard seed oil produced by steam distillation does not contain detectable amounts of mustard protein. Mustard seed oil is used as a flavoring in very low quantities, typically between 40 and 200 mg/kg. Thus, 100 g of a food product flavored with 200 mg of mustard seed oil per kg containing &lt;1.5 mg of protein per kg would represent an amount of mustard seed protein of &lt;30 ng. Taking into account the published literature on allergic reactions to the unintended ingestion of mustard, this conservatively low calculated level indicates that it is unlikely that food products containing mustard seed oil as a flavoring ingredient will elicit an allergic reaction in mustard-allergic individuals.

Enzyme-linked Immunosorbent Assay of Versicolorin A and Related Aflatoxin Biosynthetic Precursors

1991 ◽  
Vol 54 (2) ◽  
pp. 105-108 ◽  
Author(s):  
GWEN REYNOLDS ◽  
JAMES J. PESTKA
Keyword(s):  
High Performance ◽  
Cross Reactivity ◽  
Competitive Elisa ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enhanced Production ◽  
Rabbit Polyclonal Antiserum ◽  
Norsolorinic Acid ◽  
Versicolorin A ◽  
Culture Procedure

An immunochemical approach is described for the detection of versicolorin (VA) and other aflatoxin precursors in Aspergillus parasiticus cultures. VA was purified from A. parasiticus ATCC 36537 cultures by semipreparative high performance liquid chromatography and confirmed by mass spectrometry and ultraviolet (UV) absorption. To be rendered immunogenic, VA was converted to a hemiacetal and conjugated to bovine serum albumin (BSA) by reductive alkylation. Rabbit polyclonal antiserum prepared against the VA hemiacetal-BSA conjugate was employed in a competitive ELISA using VA hemiacetal-horseradish peroxidase conjugate as the marker ligand. Based on the amount of VA analogue required to inhibit binding by 50% in competitive ELISA, cross-reactivity relative to VA for VA hemiacetal, averufanin, averantin, norsolorinic acid, averufin, sterigmatocystin, and aflatoxin B1 were 106, 85, 7, 6, 2, &lt;1, and &lt;1%, respectively. The ELISA was used to monitor enhanced production of VA equivalents by A. parasiticus ATCC 36537 in a modified culture procedure. The VA antibody should be extremely useful in the biochemical and genetic investigation of aflatoxin biosynthesis.

The Major Proteins of Wheat Endosperm Starch Granules

1995 ◽  
Vol 22 (5) ◽  
pp. 793 ◽  
Author(s):  
S Rahman ◽  
B Kosar-Hashemi ◽  
MS Samuel ◽  
A Hill ◽  
DC Abbott ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Soluble Starch ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Starch Synthase ◽  
Surface Proteins ◽  
Wheat Starch ◽  
Proteinase K ◽  
Starch Granules ◽  
Terminal Sequence

Wheat starch contains two classes of associated proteins: proteins which are embedded within the granule and loosely associated surface proteins. The characterisation of the major proteins that are embedded in the granule are described. Gel electrophoresis on the basis of size resolved these proteins into five bands of molecular weights 60, 75, 85, 100 and 105 kDa. These polypeptides were demonstrated to be within the granule by their resistance to proteinase K digestion when granules were ungelatinised. The N-terminal sequences of these polypeptides are reported. The most prominent polypeptide is the 60 kDa granule-bound starch synthase. The N-terminal sequence obtained from the 75 kDa polypeptide shows homology to rice soluble starch synthase. The 85 kDa band was resolved into at least two types of polypeptides, one of which reacted with polyclonal antiserum to the maize branching enzyme IIb. The 100 and 105 kDa polypeptides were located only in the granule and are related, on the basis of N-terminal sequence similarity and cross-reactivity to monoclonal antibodies. SDS-PAGE and monoclonal antibody cross-reactivity experiments suggest that the 100 and 105 kDa polypeptides are absent from starch granules from all other species examined, including other cereals. It is speculated that all the major granule proteins are involved in starch biosynthesis.

scholarly journals Characterization of Four Viral Species Belonging to the Family Potyviridae Isolated from Ranunculus asiaticus

2006 ◽  
Vol 96 (6) ◽  
pp. 560-566 ◽  
Author(s):  
M. Turina ◽  
M. Ciuffo ◽  
R. Lenzi ◽  
L. Rostagno ◽  
L. Mela ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Aphid Transmission ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coated Plate ◽  
Ranunculus Asiaticus ◽  
Das Elisa

Four different viral species were isolated from diseased Ranunculus asiaticus plants growing in Imperia Province (Italian Riviera-Liguria Region). Infected plants exhibited mosaic symptoms and growth abnormalities. The viruses were mechanically inoculated to a range of herbaceous hosts and differentiated biologically. Long flexuous particles were present in leaf dip extracts observed by electron microscopy. A general protocol for the amplification of potyvirus genome fragments through reverse transcription-polymerase chain reaction generated products that were cloned and sequenced. Sequence and phylogenetic analysis suggested that three of these isolates could be considered new viral species belonging to the genus Potyvirus. The fourth isolate is a new member of the genus Macluravirus. Purified virus was used as antigen to produce a specific polyclonal antiserum in rabbit; serological features were established through double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), antigen coated plate (ACP)-ELISA, and western blot analysis. DAS-ELISA was highly specific for each virus isolate, whereas some cross-reactivity was shown in ACP-ELISA and western blot analysis. Aphid transmission by Myzus persicae was demonstrated in a controlled environment for each of the four viral isolates, whereas no transmission through seed was observed.

scholarly journals Antigenic Diversity of Haemophilus somnus Lipooligosaccharide: Phase-Variable Accessibility of the Phosphorylcholine Epitope

2000 ◽  
Vol 38 (12) ◽  
pp. 4412-4419 ◽  
Author(s):  
Michael D. Howard ◽  
Andrew D. Cox ◽  
Jeffrey N. Weiser ◽  
Gerhardt G. Schurig ◽  
Thomas J. Inzana
Keyword(s):  
Inner Core ◽  
Random Phase ◽  
Phase Variation ◽  
Outer Core ◽  
Enzyme Linked Immunosorbent Assay ◽  
Phase Variable ◽  
Core Oligosaccharide ◽  
Antigenic Diversity ◽  
Haemophilus Somnus ◽  
Antigenic Heterogeneity

The lipooligosaccharide (LOS) of Haemophilus somnusundergoes antigenic phase variation, which may facilitate evasion from the bovine host immune response and/or colonization and dissemination. However, LOS antigenic diversity in H. somnus has not been adequately investigated. In this study, monoclonal antibodies (MAbs) specific to various LOS epitopes were used to investigate antigenic variation and stability in LOS from H. somnus strains and phase variants. Clinical isolates of H. somnus exhibited intrastrain, as well as interstrain, antigenic heterogeneity in LOS when probed with MAbs to outer core oligosaccharide epitopes in an enzyme-linked immunosorbent assay (ELISA). However, epitopes reactive with MAbs directed predominately to the inner core heptose region were highly conserved. At least one epitope, which was expressed in few strains, was identified. One LOS component affected by phase variation was identified as phosphorylcholine (PCho), which is linked to the primary glucose residue. Inhibition ELISA, immunoblotting, and electrospray-mass spectrometry were used to confirm that MAb 5F5.9 recognized PCho. LOS reactivity with MAb 5F5.9 was associated with loss of most of the outer core oligosaccharide, indicating that reactivity with PCho was affected by phase variation of the glycose residues in this region. Our results indicate that outer core epitopes of H. somnus LOS exhibit a high degree of random, phase-variable antigenic heterogeneity and that such heterogeneity must be considered in the design of vaccines and diagnostic tests.

scholarly journals Osmotic stress and vesiculation as key mechanisms controlling bacterial sensitivity and resistance to TiO2 nanoparticles

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Christophe Pagnout ◽  
Angelina Razafitianamaharavo ◽  
Bénédicte Sohm ◽  
Céline Caillet ◽  
Audrey Beaussart ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Osmotic Stress ◽  
Inner Core ◽  
Metal Oxide Nanoparticles ◽  
Membrane Vesicles ◽  
Outer Core ◽  
Membrane Composition ◽  
Cell Turgor ◽  
Bacterial Sensitivity ◽  
Membrane Components

AbstractToxicity mechanisms of metal oxide nanoparticles towards bacteria and underlying roles of membrane composition are still debated. Herein, the response of lipopolysaccharide-truncated Escherichia coli K12 mutants to TiO2 nanoparticles (TiO2NPs, exposure in dark) is addressed at the molecular, single cell, and population levels by transcriptomics, fluorescence assays, cell nanomechanics and electrohydrodynamics. We show that outer core-free lipopolysaccharides featuring intact inner core increase cell sensitivity to TiO2NPs. TiO2NPs operate as membrane strippers, which induce osmotic stress, inactivate cell osmoregulation and initiate lipid peroxidation, which ultimately leads to genesis of membrane vesicles. In itself, truncation of lipopolysaccharide inner core triggers membrane permeabilization/depolarization, lipid peroxidation and hypervesiculation. In turn, it favors the regulation of TiO2NP-mediated changes in cell Turgor stress and leads to efficient vesicle-facilitated release of damaged membrane components. Remarkably, vesicles further act as electrostatic baits for TiO2NPs, thereby mitigating TiO2NPs toxicity. Altogether, we highlight antagonistic lipopolysaccharide-dependent bacterial responses to nanoparticles and we show that the destabilized membrane can generate unexpected resistance phenotype.

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