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.

scholarly journals New Resources for the Specific and Sensitive Detection of the Emerging Tomato Brown Rugose Fruit Virus

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1680
Author(s):  
Joan Miquel Bernabé-Orts ◽  
Covadonga Torre ◽  
Eduardo Méndez-López ◽  
Yolanda Hernando ◽  
Miguel A. Aranda
Keyword(s):  
Internal Control ◽  
Plant Protection ◽  
Genetic Resistance ◽  
Diagnostic Techniques ◽  
Plant Viruses ◽  
Elisa Test ◽  
Cross Reactivity ◽  
Sensitive Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Effective Prevention

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

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 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.

scholarly journals Inter-relatedness of some isoenzymes of cytochrome P-450 from rat, rabbit and human, determined with monoclonal antibodies

1986 ◽  
Vol 236 (2) ◽  
pp. 569-577 ◽  
Author(s):  
D Sesardic ◽  
A R Boobis ◽  
J McQuade ◽  
S Baker ◽  
E A Lock ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Epitope Mapping ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Fragments ◽  
Structural Relationships ◽  
Homologous Antigen ◽  
Complex Structural ◽  
Cytochrome P 450

Monoclonal antibodies have been raised to rat liver cytochromes P-450 b and c, and rabbit liver cytochrome P-450 form 4. A total of six antibodies have been studied. Each antibody reacted strongly both with its homologous antigen and with microsomal fractions selectively enriched with that antigen by treatment of animals with inducing compounds. However, several of the antibodies showed cross-reactivity, either within or between species. A combination of enzyme-linked immunosorbent assay, immunoadsorption, Western blotting and competitive radioimmunoassay revealed that each of the antibodies reacted with a different epitope. Proteolytic digestion of antigen followed by Western blotting of the peptide fragments enabled antibodies, otherwise identical in their reactivity, to be distinguished. It is concluded that complex structural relationships exist amongst the different isoenzymes of cytochrome P-450 and that epitope mapping will help in characterizing both animal and human cytochromes P-450.

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 <1.5 mg of protein per kg would represent an amount of mustard seed protein of <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.

scholarly journals An Immunity (QL3) Breaking Strain of Sorghum Mosaic Potyvirus in India

Plant Disease ◽  
1999 ◽  
Vol 83 (9) ◽  
pp. 877-877 ◽  
Author(s):  
V. R. Mali
Keyword(s):  
Electron Microscopy ◽  
Saccharum Officinarum ◽  
Plant Viruses ◽  
Polyclonal Antiserum ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Plant ◽  
Breaking Strain ◽  
Symptomless Infection ◽  
Sorghum Grain

Sorghum (Sorghum bicolor) line Tx2786 is immune to strains or isolates of sorghum mosaic potyvirus (SrMV strains SCH and SCH) but susceptible to strains of maize dwarf mosaic (MDMV) and sugarcane mosaic (SCMV) potyviruses (1,2). When grown in proximity to sugarcane, Tx2786 was infected with a virus that caused mosaic and necrotic symptoms during the postrainy season of 1994 (14 to 30°C) at Parbhani (Maharashtra, India). Leaf-dip electron microscopy performed on virus-infected sorghum and sugarcane tissues revealed particles, 720 × 12 nm, typical of a potyvirus. A potyvirus isolate (designated IBS) was readily transmitted mechanically from field-infected sorghum (Tx2786) and sugarcane (Saccharum officinarum cv. Co.740) to 18 glasshouse-grown sorghum inbred lines. All sorghum lines (QL-3 from India and Tx2786), including those resistant to SrMV, SCMV, MDMV, and johnsongrass mosaic potyvirus (JGMV), were infected. IBS caused necrosis in Cargill-40, Hegari, PI-35038, RTx09, Tx2786, QL-3 from India, and Redlan lines and mosaic symptoms in RTx430, SA-394, Atlas, Martin, BTx3048, Caprock, NM-31, QL-11, SCO-175-14E, Rio, and BTx623 lines. Sorghum reactions to sorghum and sugarcane virus isolates were similar. IBS also infected maize (Zea mays cv. African Tall), johnsongrass (S. halepense cv. 81, symptomless infection), and sorghum (S. bicolor cv. CS-3541) but not prosomillet (Panicum miliaceum), fingermillet (Eleusine coracana cv. IE-2540), pearl millet (Pennisetum typhoides cv. NHB-3), or oat (Avena sativa cv. Kent) when sap transmitted. IBS also was transmitted by the corn leaf aphid (Rhopalosiphum maidis) in a nonpersistent manner from sorghum and sugarcane to all 18 sorghum lines in a glasshouse. Based on direct antigen-coating enzyme-linked immunosorbent assay (DAC-ELISA), serologically specific electron microscopy, and Western blot tests, IBS was serologically related to SrMV strains SCH and SCI (2–4) but not to MDMV strains A, D, and E; SCMV strains A, B, D, SC, and MDB; or JGMV type strain from Australia and strain O from Texas (2). IBS induced formation of cytoplasmic cylindrical inclusions consisting of pinwheels associated with long, straight, laminated aggregates (subdivision type II) in sorghum and sugarcane host cells. Purified IBS contained one major polypeptide of 40 kDa and a ribonucleic acid of 3.0 × 106 Da. Polyclonal antiserum to IBS was produced in rabbits and used in DAC-ELISA to confirm the identity of IBS in sugarcane, sorghum lines, and other test plant species. On the basis of biological and serological properties, IBS isolated from field-infected sorghum grain Tx2786 and sugarcane cv. Co.740 is identified as an immunity (QL3) breaking strain of SrMV (SrMV-IBS). References: (1) L. M. Giorda et al. Plant Dis. 70:624, 1986. (2) D. D. Shukla et al. Phytopathology 79: 223, 1989. (3) D. D. Shukla et al. 1998. AAB Description of Plant Viruses No. 359. AAB, Kew, Surrey, England. (4) Z. N. Yang and T. E. Mirkov. Phytopathology 87:932, 1997.

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, <1, and <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.

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 753. Performance Characteristics of Diagnostic Assays in Blastomycosis

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S424-S424
Author(s):  
Timothy O’Dowd ◽  
Jack McHugh ◽  
Nancy Wengenack ◽  
Elitza Theel ◽  
Paschalis Vergidis
Keyword(s):  
Granulomatous Inflammation ◽  
Cross Reactivity ◽  
Performance Characteristics ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fungal Culture ◽  
Noninvasive Test ◽  
Serum Antigen ◽  
Urine Antigen ◽  
Antigen Tests ◽  
Urine And Serum

Abstract Background Blastomycosis has historically been a difficult diagnosis to establish, often initially misdiagnosed as bacterial pneumonia. Serologic assays and polymerase chain reaction (PCR) tests are available, but their performance is not well defined. The objective of this study was to characterize their performance. Methods Subjects were identified via chart review of patients diagnosed with blastomycosis from 2005 to 2020. A definitive diagnosis was based on fungal culture, histopathology, or cytology. Performance characteristics of the Blastomyces antibody enzyme linked immunosorbent assay (ELISA), immunodiffusion (ID), complement fixation (CF), urine and serum antigen ELISAs, and PCR were evaluated in patients with confirmed blastomycosis. Data on patient demographics, location of disease, and mortality was also collected. Results We identified 193 patients with blastomycosis. The mean age was 51.8 years (range, 11-84) and 73.6% of patients were male. 42.5% resided in Minnesota, 18.1% in Wisconsin, and 12.9% in Iowa. Diagnosis was based on culture in 142 (73.2%) or histopathology/cytology in 67 (34.7%) patients. Granulomatous inflammation was present in 73.1% (38/52) while 21.2% (41/193) had evidence of extrapulmonary dissemination. The antibody, ID, and CF assays were positive in 43.5% (37/85), 35.1% (33/94) and 20.5% (8/39) of patients, respectively. Sensitivity of Blastomyces PCR was 40% (4/10) in sputum and 75% (21/28) in bronchoalveolar lavage (BAL) fluid. Blastomyces urine and serum antigen tests were positive in 68% (34/50) and 50% (9/18) of cases, respectively, while the urine antigen was positive in 63.6% (7/11) of disseminated cases. Patients had a positive Histoplasma urine antigen test in 54.1% (20/37) and Aspergillus galactomannan in BAL in 34.8% (8/23) of cases. Serum beta-D-glucan test was positive in 16.7% (2/12). 90-day mortality was 21/193 (10.9%) and median time from diagnosis to death was 18 days. Conclusion In this cohort, Blastomyces urine antigen was the most sensitive noninvasive test, with similar performance in pulmonary and disseminated disease. However, its utility is limited by poor specificity due to cross-reactivity. Blastomyces PCR from BAL fluid demonstrated the highest sensitivity. Blastomyces antibody, ID, and CF had poor sensitivity. Disclosures All Authors: No reported disclosures

scholarly journals Characterization of the Diagnostic Performance of a Novel COVID-19 PETIA in Comparison to Four Routine N-, S- and RBD-Antigen Based Immunoassays

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1332
Author(s):  
Alexander Spaeth ◽  
Thomas Masetto ◽  
Jessica Brehm ◽  
Leoni Wey ◽  
Christian Kochem ◽  
...  
Keyword(s):  
Immune Response ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chemiluminescence Immunoassay ◽  
Face To Face ◽  
Comprehensive Comparison ◽  
Broad Variety ◽  
Viral Responses ◽  
Novel Coronavirus ◽  
High Consistency

In 2019, a novel coronavirus emerged in Wuhan in the province of Hubei, China. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) quickly spread across the globe, causing the neoteric COVID-19 pandemic. SARS-CoV-2 is commonly transmitted by droplet infection and aerosols when coughing or sneezing, as well as high-risk exposures to infected individuals by face-to-face contact without protective gear. To date, a broad variety of techniques have emerged to assess and quantify the specific antibody response of a patient towards a SARS-CoV-2 infection. Here, we report the first comprehensive comparison of five different assay systems: Enzyme-Linked Immunosorbent Assay (ELISA), Chemiluminescence Immunoassay (CLIA), Electro-Chemiluminescence Immunoassay (ECLIA), and a new Particle-Enhanced Turbidimetric Immunoassay (PETIA) for SARS-CoV-2. Furthermore, we also evaluated the suitability of N-, S1- and RBD-antigens for quantifying the SARS-CoV-2 specific immune response. Linearity and precision, overall sensitivity and specificity of the assays, stability of samples, and cross-reactivity of general viral responses, as well as common coronaviruses, were assessed. Moreover, the reactivity of all tests to seroconversion and different sample matrices was quantified. All five assays showed good overall agreement, with 76% and 87% similarity for negative and positive samples, respectively. In conclusion, all evaluated methods showed a high consistency of results and suitability for the robust quantification of the SARS-CoV-2-derived immune response.

Sign in / Sign up

Export Citation Format

Share Document