scholarly journals Development of an antigen-capture enzyme-linked immunosorbent assay for Clostridium perfringens beta2-toxin in porcine feces and the neonatal piglet intestine

2012 ◽  
Vol 24 (5) ◽  
pp. 895-902 ◽  
Author(s):  
Jasmina Kircanski ◽  
Douglas Hodgins ◽  
Glenn Soltes ◽  
Yanlong Pei ◽  
Valeria R. Parreira ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Protease Activity ◽  
Limit Of Detection ◽  
Intestinal Content ◽  
Enzyme Linked Immunosorbent Assay ◽  
Neonatal Piglet ◽  
Antigen Capture ◽  
Field Samples ◽  
Intestinal Contents ◽  
Beta2 Toxin

An enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantitation of beta2-toxin in neonatal piglet intestinal contents. Polystyrene plates were coated with polyclonal capture antibodies prepared against consensus recombinant beta2-toxin. The ELISA was developed using consensus recombinant beta2-toxin, atypical recombinant beta2-toxin, purified consensus native beta2-toxin, and field samples of neonatal porcine intestinal contents. Captured antigen was detected using a horseradish peroxidase–labeled monoclonal antibody against consensus recombinant beta2-toxin. The limit of detection of the ELISA for consensus beta2-toxin was between 2.0 and 3.5 ng/ml. The ELISA detected atypical recombinant beta2-toxin only weakly. Optical density was protein concentration dependent. The test confirmed differences between consensus and atypical recombinant beta2-toxin, but similar results obtained when testing pure consensus recombinant beta2-toxin and native beta2-toxin. Results obtained from intestinal content samples, particularly from the small intestine, were highly inconsistent and suggested variable protease activity. Addition of protease inhibitors partially prevented degradation of the toxin; however, sample processing at low temperature, at a lower pH (citrate buffer with 5% of bovine serum albumin, pH 6.1), and “cold incubation” of applied antigens abolished protease activity. The recombinant toxin was preserved in spiked intestinal samples by freezing at −70°C, suggesting that necropsy samples can be stored frozen for periodic testing. With appropriate sample preparation, antigen-capture ELISA can detect beta2-toxin in the intestinal content and feces of neonatal piglets.

scholarly journals Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Saengsoon Charoenvilaisiri ◽  
Channarong Seepiban ◽  
Mallika Kumpoosiri ◽  
Sombat Rukpratanporn ◽  
Nuchnard Warin ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Immunosorbent Assay ◽  
Mosaic Disease ◽  
Cassava Mosaic Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sri Lankan ◽  
Stem Cuttings ◽  
Field Samples ◽  
Cassava Stem ◽  
Cassava Mosaic Virus

Abstract Background Cassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA). Methods Monoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard. Results A TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA. Conclusions Our findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

Simulation model for predicting limit of detection and range of quantitation of competitive enzyme-linked immunosorbent assay

2007 ◽  
Vol 103 (5) ◽  
pp. 427-431 ◽  
Author(s):  
Dong Hwan Choi ◽  
Yoshio Katakura ◽  
Rieko Matsuda ◽  
Yuzuru Hayashi ◽  
Kazuaki Ninomiya ◽  
...  
Keyword(s):  
Simulation Model ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay

scholarly journals Aflatoxigenic and ochratoxigenic moulds and their toxins in tree nuts on Serbian market

2020 ◽  
pp. 9-18
Author(s):  
Irena Rakic ◽  
Gordana Dimic ◽  
Marija Skrinjar ◽  
Suncica Kocic-Tanackov
Keyword(s):  
Aspergillus Niger ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Alternaria Alternata ◽  
Rhizopus Oryzae ◽  
Limit Of Detection ◽  
Average Concentration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mean Values ◽  
Tree Nuts

In this study, moulds and mycotoxins presence in different tree nuts were investigated. The results showed that all of the 25 samples were contaminated with moulds. Mean values of total mould count varied from 1-4.9 cfu per grain. The most frequent species in hazelnut samples were Rhizopus oryzae (32.2%) and Aspergillus niger (28.9%). In walnuts A. niger (75.6%), in cashews also A. niger (42.4%) while in pistachio samples Alternaria alternata (20.7%), and Cladosporium cladosporioides (20.7%) were the most dominant. Rhizopus oligosporus was the only identified species in all almond samples (100%). Using Enzyme Linked Immunosorbent Assay (ELISA), the presence of total aflatoxins and ochratoxin A was examinated. In all analyzed samples, levels of ochratoxin A were below the limit of detection. Total aflatoxins were detected only in walnut samples with average concentration of 7.1 ?g/kg.

Immunoassay for human serum hepcidin

Blood ◽  
2008 ◽  
Vol 112 (10) ◽  
pp. 4292-4297 ◽  
Author(s):  
Tomas Ganz ◽  
Gordana Olbina ◽  
Domenico Girelli ◽  
Elizabeta Nemeth ◽  
Mark Westerman
Keyword(s):  
Healthy Volunteers ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Deficiency Anemia ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reactive Protein ◽  
Transient Rise ◽  
Juvenile Hemochromatosis ◽  
Serum Hepcidin ◽  
Clinical Conditions

Abstract We developed and validated the first serum enzyme-linked immunosorbent assay for hepcidin, the principal iron-regulatory hormone that has been very difficult to measure. In healthy volunteers, the 5% to 95% range of hepcidin concentrations was 29 to 254 ng/mL in men (n = 65) and 17 to 286 ng/mL in women (n = 49), with median concentrations 112 versus 65 (P < .001). The lower limit of detection was 5 ng/mL. Serum hepcidin concentrations in 24 healthy subjects correlated well with their urinary hepcidin (r = 0.82). Serum hepcidin appropriately correlated with serum ferritin (r = 0.63), reflecting the regulation of both proteins by iron stores. Healthy volunteers showed a diurnal increase of serum hepcidin at noon and 8 pm compared with 8 am, and a transient rise of serum hepcidin in response to iron ingestion. Expected alterations in hepcidin levels were observed in a variety of clinical conditions associated with iron disturbances. Serum hepcidin concentrations were undetectable or low in patients with iron deficiency anemia (ferritin < 10 ng/mL), iron-depleted HFE hemochromatosis, and juvenile hemochromatosis. Serum hepcidin concentrations were high in patients with inflammation (C-reactive protein > 10 mg/dL), multiple myeloma, or chronic kidney disease. The new serum hepcidin enzyme-linked immunosorbent assay yields accurate and reproducible measurements that appropriately reflect physiologic, pathologic, and genetic influences, and is informative about the etiology of iron disorders.

A Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for Determination of Homoharringtonine

Planta Medica ◽  
2018 ◽  
Vol 84 (14) ◽  
pp. 1038-1044 ◽  
Author(s):  
Benyakan Pongkitwitoon ◽  
Seiichi Sakamoto ◽  
Rika Nagamitsu ◽  
Waraporn Putalun ◽  
Hiroyuki Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Myeloid Leukemia ◽  
High Performance ◽  
Sodium Periodate ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Study Support ◽  
Mediated Oxidation

AbstractHomoharringtonine (HHT), also known as omacetaxine, is a natural compound found in the genus Cephalotaxus and is a promising pharmaceutical drug used for the treatment of chronic or accelerated phase chronic myeloid leukemia. As a tool for the quantitative determination of HHT, a specific monoclonal antibody against HHT (MAb 6A1) was generated by conjugates prepared via sodium periodate-mediated oxidation. The developed indirect competitive enzyme-linked immunosorbent assay (icELISA) using MAb 6A1 was found to be highly specific and sensitive with a limit of detection for HHT of 48.8 ng/mL. Validation assays to evaluate precision and accuracy of the method were conducted by the use of intra- and inter-assay analysis, recovery test, and comparison analysis between the amounts of HHT determined by ELISA and high-performance liquid chromatography. These results revealed that the established icELISA using MAb 6A1 is specific, sensitive, and reliable enough to be applied to the qualitative analysis for HHT. Furthermore, the results of this study support the usefulness of sodium periodate as a reagent for the conjugation between Cephalotaxus alkaloids and proteins for producing specific antibodies.

scholarly journals Validation of an Enzyme-Linked Immunosorbent Assay That Detects Histoplasma capsulatum Antigenuria in Colombian Patients with AIDS for Diagnosis and Follow-Up during Therapy

2014 ◽  
Vol 21 (9) ◽  
pp. 1364-1368 ◽  
Author(s):  
Diego H. Caceres ◽  
Christina M. Scheel ◽  
Ángela M. Tobón ◽  
Angela Ahlquist Cleveland ◽  
Ángela Restrepo ◽  
...  
Keyword(s):  
Clinical Improvement ◽  
Immunosorbent Assay ◽  
Histoplasma Capsulatum ◽  
Response To Therapy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Test ◽  
Content Type ◽  
Antigen Capture

ABSTRACTWe validated an antigen capture enzyme-linked immunosorbent assay (ELISA) in Colombian persons with AIDS and proven histoplasmosis and evaluated the correlation between antigenuria and clinical improvement during follow-up. The sensitivity of theHistoplasma capsulatumELISA was 86%, and the overall specificity was 94%. The antigen test successfully monitored the response to therapy.

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