scholarly journals Nanobody-Alkaline Phosphatase Fusion Protein-Based Enzyme-Linked Immunosorbent Assay for One-Step Detection of Ochratoxin A in Rice

Sensors ◽  
2018 ◽  
Vol 18 (11) ◽  
pp. 4044 ◽  
Author(s):  
Zhichang Sun ◽  
Xuerou Wang ◽  
Qi Chen ◽  
Yonghuan Yun ◽  
Zongwen Tang ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
Solvent Tolerance ◽  
Enzyme Linked Immunosorbent Assay ◽  
One Step

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL−1 and a limit of detection of 0.059 ng mL−1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.

Production and characterization of a single-chain variable fragment-alkaline phosphatase fusion protein for glycocholic acid detection in a one-step enzyme-linked immunosorbent assay

2018 ◽  
Vol 10 (22) ◽  
pp. 2629-2635 ◽  
Author(s):  
Xiping Cui ◽  
Qiyi He ◽  
Ding Shen ◽  
Zhengyun Jiang ◽  
Yingshan Chen ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Glycocholic Acid ◽  
One Step

One-step enzyme-linked immunosorbent assay for glycocholic acid based on single-chain variable fragment-alkaline phosphatase fusion protein.

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.

scholarly journals Single-Laboratory Validation of the Biosense Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA) for Determination of Domoic Acid Toxins in Shellfish

2007 ◽  
Vol 90 (4) ◽  
pp. 1000-1010 ◽  
Author(s):  
Hans Kleivdal ◽  
Sven-Inge Kristiansen ◽  
Mona V Nilsen ◽  
Lyn Briggs
Keyword(s):  
Immunosorbent Assay ◽  
Domoic Acid ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Method Comparison ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Shellfish Poisoning ◽  
Relative Standard

Abstract Method validation was conducted for an enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins, known to give amnesic shellfish poisoning (ASP) symptoms, in shellfish. The calibration curve range of the assay is approximately 10260 pg/mL, with a dynamic working range for DA toxins in shellfish from 0.01 to at least 250 mg/kg. The ASP ELISA showed no significant cross-reactivity to structural analogs, and proved to be robust to deliberate alterations of the optimal running conditions. The shellfish matrix effects observed with mussels, oysters, and scallops were eliminated by diluting shellfish extracts 1:200 prior to analysis, leading to a limit of detection at 0.003 mg/kg. Thirteen blank shellfish homogenates were spiked with certified mussel material containing DA to levels in the range of 0.125 mg DA/kg, and analyzed in quadruplicate on 3 different days. The relative standard deviation (RSD) under intra-assay repeatability conditions ranged from 6.5 to 13.1%, and under interassay repeatability conditions the RSD ranged from 5.7 to 13.4%, with a mean value of 9.3%. The recoveries ranged from 85.5 to 106.6%, with a mean recovery of 102.2%. A method comparison was conducted with liquid chromatography with ultraviolet detection, using naturally contaminated scallop samples (n = 27) with DA levels at 0244 mg/kg. The overall correlation coefficient was 0.960 and the slope of the regression was 1.218, indicating a good agreement between the methods.

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay Based on the Multiepitope Peptide for the Synchronous Detection of Staphylococcal Enterotoxin A and G Proteins in Milk

2015 ◽  
Vol 78 (2) ◽  
pp. 362-369 ◽  
Author(s):  
MINGYAN LIANG ◽  
TINGTING ZHANG ◽  
XUELAN LIU ◽  
YANAN FAN ◽  
SHENGLIN XIA ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Signal To Noise Ratio ◽  
Food Poisoning ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Synchronous Detection ◽  
Standard Curve ◽  
Stability And Accuracy

Staphylococcal food poisoning (SFP), one of the most common foodborne diseases, results from ingestion of staphylococcal enterotoxins (SEs) in foods. In our previous studies, we found that SEA and SEG were two predominant SE proteins produced by milk-acquired S. aureus isolates. Here, a tandemly arranged multiepitope peptide (named SEAGepis) was designed with six linear B-cell epitopes derived from SEA or SEG and was heterologously expressed. The SEAGepis-specific antibody was prepared by immunizing rabbit with rSEAGepis. Then, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on rSEAGepis and the corresponding antibody was developed to simultaneously detect SEA and SEG. Under the optimized conditions, the ic-ELISA standard curve for rSEAGepis was constructed in the concentration range of 0.5 to 512 ng/ml, and the average coefficients of variation of intra-and interassay were 4.28 and 5.61% during six standard concentrations. The average half-maximal inhibitory concentration was 5.07 ng/ml, and the limit of detection at a signal-to-noise ratio of 3 was 0.52 ng/ml. The anti-rSEAGepis antibody displayed over 90% cross-reactivity with SEA and SEG but less than 0.5% cross-reactivity with other enterotoxins. Artificially contaminated milk with different concentrations of rSEAGepis, SEA, and SEG was detected by the established ic-ELISA; the recoveries of rSEAGepis, SEA, and SEG were 91.1 to 157.5%, 90.3 to 134.5%, and 89.1 to 117.5%, respectively, with a coefficient of variation below 12%. These results demonstrated that the newly established ic-ELISA possessed high sensitivity, specificity, stability, and accuracy and could potentially be a useful analytical method for synchronous detection of SEA and SEG in milk.

Sensitive Measurement of Thrombopoietin by a Monoclonal Antibody Based Sandwich Enzyme-linked Immunosorbent Assay

1997 ◽  
Vol 78 (04) ◽  
pp. 1262-1267 ◽  
Author(s):  
Claudia C Folman ◽  
Albert E G K von dem Borne ◽  
Irma H J A M Rensink ◽  
Winald Gerritsen ◽  
C Ellen van der Schoot ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Platelet Transfusion ◽  
Large Scale ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reliable Tool

SummaryIn this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs).The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 ± 0.8 A.U./ml, mean ± sd) was lower than the concentration found in controls (11 ± 8 A.U./ml, mean ± sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma.We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels.In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

scholarly journals Quantitation of Indian Krait (Bungarus caeruleus) Venom in Human Specimens of Forensic Origin by Indirect Competitive Inhibition Enzyme-Linked Immunosorbent Assay

2006 ◽  
Vol 89 (5) ◽  
pp. 1360-1366 ◽  
Author(s):  
Ganneru Brunda ◽  
Beedu Sashidhar Rao ◽  
Rajendra Kumar Sarin
Keyword(s):  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Limit Of Detection ◽  
Linear Regression Analysis ◽  
Cross Reactivity ◽  
Assay System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Calibration Plot ◽  
Rat Tissues ◽  
Capture Assay

Abstract An indirect competitive inhibition enzyme-linked immunosorbent assay was reported to detect krait venom in human specimens of forensic origin. Polyclonal anti-krait venom antibodies were characterized by indirect antibody capture assay. The calibration plot was constructed based on linear regression analysis (y = 72.85 12.29x, r2 = 0.98) with concentration ranges from 0.013 to 1000 ng/well of krait venom with a limit of detection of 0.2 ng/mL in the assay system. The IC50 (inhibitory concentration at 50% displacement) value of krait venom was observed to be 70 ng. Spiking studies indicated recoveries of 95100% and 94100% when various concentrations of krait venom were spiked to rat tissues (skin, liver, and kidneys) and pooled human serum, respectively. Polyclonal anti-krait venom antibodies showed no cross-reactivity with cobra and viper venom when tested in the assay system. The coefficient of variation of various concentrations of working range in intra-assay (n = 6) was &lt;5%, whereas in interassay (n = 6) it was observed to be 7%. Further, the method was used to quantitate krait venom in human autopsy and biopsy specimens of forensic origin. Concentration of krait venom was found to be in the range of 4172 ng/100 mg skin or skin scrapings and 64378 ng/mL blood or serum. The methodology may find application in forensic laboratories to assess the cause of death in the cases of krait-bite victims.

Immunoassay of Ochratoxin A, Using Antibodies Developed Against a New Ochratoxin-Albumin Conjugate

1992 ◽  
Vol 75 (5) ◽  
pp. 824-828 ◽  
Author(s):  
Anna Breitholtz-Emanuelsson ◽  
Gunnel Dalhammar ◽  
Karl Hult
Keyword(s):  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Preparation Method ◽  
Detection Limits ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Sample Preparation Method ◽  
Ochratoxin Α ◽  
Incubation Buffer

Abstract A derivative of ochratoxin A was linked to bovine serum albumin in such a way that the carboxylic group of ochratoxin A was left unmodified. Lysine was substituted for phenylalanine in ochratoxin A, and the ε-amino group was linked to the protein. The conjugate was injected into 2 rabbits; antibodies against ochratoxin A were developed and used to develop an indirect competitive enzyme-linked immunosorbent assay. The detection limits for ochratoxin A in incubation buffer were 0.07 and 0.02 ng ochratoxin A/mL with the 2 antisera, respectively. Three hundred human plasma samples were purified by a novel sample preparation method and were analyzed by the immunosorbent assay. The detection limits for ochratoxin A in plasma samples were 0.2 and 0.1 ng ochratoxin A/mL with the 2 antisera, respectively. The cross-reactivity of the 2 antiochratoxin A sera was high for the ochratoxin A methyl ester, about 20% for ochratoxin C, and low for (4R)-4-hydroxyochratoxin A, ochratoxin α, ochratoxin B, and 4-hydroxyochratoxin B. No cross-reactivity was seen for phenylalanine and lysine.

scholarly journals Co-occurrence of Aflatoxins, Ochratoxin A, Fumonisins, and Zearalenone in Cereals and Feed, Determined by Competitive Direct Enzyme-Linked Immunosorbent Assay and Thin-Layer Chromatography

2009 ◽  
Vol 60 (4) ◽  
pp. 427-434 ◽  
Author(s):  
Maja Klarić ◽  
Zdenka Cvetnić ◽  
Stjepan Pepeljnjak ◽  
Ivan Kosalec
Keyword(s):  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Permissible Limit ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wide Range ◽  
Food And Feed ◽  
Layer Chromatography

Co-occurrence of Aflatoxins, Ochratoxin A, Fumonisins, and Zearalenone in Cereals and Feed, Determined by Competitive Direct Enzyme-Linked Immunosorbent Assay and Thin-Layer ChromatographyAspergillus, Penicillium, andFusariumspecies frequently contaminate crops. For this reason mycotoxins such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), and zearalenone (ZEA) are found in food and feed in a wide range of concentrations, depending on environmental and storage conditions. Consumption of mycotoxin-contaminated food and feed has been associated with acute and chronic poisoning and carcinoma. The aim of this study was to determine the incidence and co-occurrence of AFs (B1+B2+G1+G2), OTA, FBs (B1+B2+B3), and ZEA in 37 samples of cereals and feed randomly collected in 2007 from households of an endemic nephropathy (EN) area in Croatia. The mycotoxins were determined using the competitive direct ELISA test (CD-ELISA) in combination with thin-layer chromatography (TLC). The most frequent mycotoxin was ZEA (92%, mean 318.3 μg kg-1), followed by FBs (27%, 3690 μg kg-1), AFs (24.3%, 4.6 μg kg-1), and OTA (16.2%, 9.8 μg kg-1). Levels of AFs, ZEA, and FBs detected by CD-ELISA significantly correlated with the TLC results. However, only one OTA-positive sample was confirmed by TLC due to its high limit of detection. The levels of these mycotoxins were below the permissible limit for animal feed. Twenty-nine percent of cereals were contaminated with FBs, OTA, or ZEA in mass fractions above the permissible limit for humans. Co-occurrence of two toxins varied between 4.2% and 54% and of three between 4.2% and 7.6%. Prolonged co-exposure to AFs, OTA, FBs, and ZEA might increase the risk of various chronic diseases.

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