scholarly journals Development of an Anti-Idiotypic VHH Antibody and Toxin-Free Enzyme Immunoassay for Ochratoxin A in Cereals

Toxins ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 280 ◽  
Author(s):  
Caixia Zhang ◽  
Qi Zhang ◽  
Xiaoqian Tang ◽  
Wen Zhang ◽  
Peiwu Li
Keyword(s):  
Enzyme Immunoassay ◽  
Ochratoxin A ◽  
High Performance ◽  
Elisa Test ◽  
Hplc Method ◽  
Free Enzyme ◽  
Agricultural Products ◽  
Enzyme Linked Immunosorbent Assay ◽  
Highly Sensitive ◽  
Ic50 Value

Enzyme-linked immunosorbent assay (ELISA) test kits have been widely used for the determination of mycotoxins in agricultural products and foods, however, this test uses toxin standards with high toxicity and carcinogenicity that seriously threaten human health. In this work, the anti-idiotypic nanobody VHH 2-24 was first developed and then, using it as a surrogate standard, a toxin-free enzyme immunoassay for ochratoxin A (OTA) was established. The IC50 value of the VHH 2-24 surrogate standard-based ELISA was 0.097 µg/mL, with a linear range of 0.027–0.653 µg/mL. The average recoveries were tested by spike-and-recovery experiments, and ranged from 81.8% to 105.0%. The accuracy of the developed ELISA for detecting OTA was further verified by using the high performance liquid chromatography (HPLC) method, and an excellent correlation was observed. In summary, the toxin-free ELISA established in this study proves the latent use of the anti-idiotypic VHH as a surrogate calibrator for other mycotoxins and highly toxic small molecule analysis to improve assay properties for highly sensitive analyte determination in agricultural products.

Development of a Flow-Through Enzyme Immunoassay and Application in Screening Green Coffee Samples for Ochratoxin A with Confirmation by High-Performance Liquid Chromatography

2001 ◽  
Vol 64 (10) ◽  
pp. 1597-1602 ◽  
Author(s):  
L. SIBANDA ◽  
S. DE SAEGER ◽  
T. G. M. BAUTERS ◽  
H. J. NELIS ◽  
C. VAN PETEGHEM
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Enzyme Immunoassay ◽  
Ochratoxin A ◽  
High Performance ◽  
Hplc Method ◽  
Coffee Bean ◽  
Green Coffee ◽  
Penicillium Species ◽  
Flow Through

A flow-through enzyme immunoassay has been developed for the screening of green coffee bean samples for ochratoxin A (OA) and was later used in a survey on OA in green coffee from different countries. The test has a sensitivity of 8 ng/g, and calculated recoveries ranged from 70 to 89% and from 86 to 95% for spiked and naturally contaminated samples, respectively. There were no significant differences in within-day and between-day assay performance (P > 0.05). Green coffee samples (15 Arabica and 7 Robusta) received from an international coffee trader were analyzed for intrinsic fungal contamination, screened for OA, and subsequently confirmed by high-performance liquid chromatography (HPLC). All 22 samples were contaminated by fungal species of the genus Aspergillus, while Penicillium species were isolated from a mere 13.6% of the total number of samples. Isolates were tested for their ability to produce OA, and only 3.9% were positive. There was no correlation between occurrence of OA-producing isolates and levels of OA in contaminated samples. Results of the screening procedure showed that 4 of the 22 samples were contaminated with 8 ng/g or higher. The HPLC method confirmed that the OA levels ranged from 27 to 168 ng/g. A fifth sample, which was shown to be negative during screening, had an OA concentration of 4 ng/g. There were no false negatives or positives recorded, and the flow-through enzyme immunoassay results correlated with those obtained by HPLC.

scholarly journals A comparison between enzyme immunoassay and HPLC for ochratoxin A detection in green, roasted and instant coffee

2007 ◽  
Vol 50 (2) ◽  
pp. 349-359 ◽  
Author(s):  
Simone Fujii ◽  
Elisabete Yurie Sataque Ono ◽  
Ricardo Marcelo Reche Ribeiro ◽  
Fernanda Garcia Algarte Assunção ◽  
Cássia Reika Takabayashi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Ochratoxin A ◽  
High Performance ◽  
Screening Tool ◽  
Correlation Coefficients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Assay ◽  
Quality And Safety ◽  
Green Coffee ◽  
Instant Coffee

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for ochratoxin A (OTA) detection in green, roasted and instant coffees was developed using anti-OTA monoclonal antibody. Immunological reagents prepared were OTA-BSA (4.76 mg/mL), anti-OTA.7 MAb (2x10³-fold dilution) and HRP-anti IgG (10³-fold dilution). The detection limit was 3.73 ng OTA/g and correlation coefficients (r) between this immunoassay and high performance liquid chromatography were 0.98 for green coffee, 0.98 for roasted and 0.86 for instant. OTA levels detected by ic-ELISA were higher than by HPLC, with ELISA/HPLC ratio of 0.66 - 1.46 (green coffee), 0.96 - 1.11 (roasted) and 0.93 - 1.82 (instant). ELISA recoveries for OTA added to coffee (5 - 70 ng/g) were 81.53 % for green coffee, 46.73 % for roasted and 64.35 % for instant, while recoveries by HPLC were 80.54 %, 45.91 % and 55.15 %, respectively. Matrices interferences were minimized by samples dilution before carrying out the ELISA assay. The results indicate that MAb-based ic-ELISA could be a simple, sensitive and specific screening tool for OTA detection, contributing to quality and safety of coffee products.

scholarly journals Highly Sensitive and Selective Fluorescent Detection of Gossypol Based on BSA-Stabilized Copper Nanoclusters

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 95 ◽  
Author(s):  
Shuangjiao Xu ◽  
Kehai Zhou ◽  
Dan Fang ◽  
Lei Ma
Keyword(s):  
High Performance ◽  
Signal To Noise Ratio ◽  
High Sensitivity ◽  
Hplc Method ◽  
Fluorescent Detection ◽  
Copper Nanoclusters ◽  
Highly Sensitive ◽  
The Us ◽  
Fluorescence Quenching Mechanism ◽  
First Time

In this paper, fluorescent copper nanoclusters (NCs) are used as a novel probe for the sensitive detection of gossypol for the first time. Based on a fluorescence quenching mechanism induced by interactions between bovine serum albumin (BSA) and gossypol, fluorescent BSA-Cu NCs were seen to exhibit a high sensitivity to gossypol in the range of 0.1–100 µM. The detection limit for gossypol is 25 nM at a signal-to-noise ratio of three, which is approximately 35 times lower than the acceptable limit (0.9 µM) defined by the US Food and Drug Administration for cottonseed products. Moreover, the proposed method for gossypol displays excellent selectivity over many common interfering species. We also demonstrate the application of the present method to the measurement of several real samples with satisfactory recoveries, and the results agree well with those obtained using the high-performance liquid chromatography (HPLC) method. The method based on Cu NCs offers the followings advantages: simplicity of design, facile preparation of nanomaterials, and low experimental cost.

Polyclonal Antibody-Based ELISA and HPLC Methods for the Determination of Fumonisins in Corn: A Comparative Study

1996 ◽  
Vol 59 (8) ◽  
pp. 893-897 ◽  
Author(s):  
ERIC W. SYDENHAM ◽  
SONJA STOCKENSTRÖM ◽  
PIETER G. THIEL ◽  
JOHN P. RHEEDER ◽  
M. BRUNO DOKO ◽  
...  
Keyword(s):  
Comparative Study ◽  
Polyclonal Antibody ◽  
High Performance ◽  
Linear Regression Analysis ◽  
Correlation Coefficients ◽  
Hplc Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Regression Slopes ◽  
The Individual ◽  
The Comparative Study

The performance of an experimental polyclonal antibody (PAb)-based competitive direct enzyme-linked immunosorbent assay (CD-ELISA) developed for the analysis of fumonisins in corn was assessed by comparison with an established high-performance liquid chromatography (HPLC) method. The comparative study was conducted using a series of 20 corn samples naturally contaminated with combined fumonisin levels ranging from <0.05 to >5 μg/g (ppm). Linear regression analysis between the results generated by HPLC and CD-ELISA provided correlation coefficients (r) and regression slopes (b) of r = 0.960, b = 1.493 (P < 0.001); r = 0.865, b = 3.903 (P < 0.001); and r = 0.832, b = 0.107 (P < 0.001) for the individual fumonisins B1 (FB1), B2 (FB2) and B3 (FB3), respectively, while corresponding values of r = 0.967, b = 1.059 (P < 0.001) were obtained for the combined FB1, FB2, and FB3 concentrations. In 3 of 18 fumonisin-positive corn samples, combined fumonisin levels determined by CD-ELISA were between 85 and 100% higher than those determined in the same extracts by HPLC, while in 13 other samples, CD-ELISA results were between 1.8 and 53% higher than those determined by HPLC. Conversely, in 2 of 18 samples, CD-ELISA results were lower than those determined by HPLC. The differences recorded between HPLC and the experimental PAb-based CD-ELISA were far less than those previously recorded for other mono- and polyclonal antibody-based CD-ELISA systems. The results indicate that the experimental PAb-based CD-ELISA may be effectively applied for the initial screening for fumonisins in corn.

scholarly journals Highly Sensitive and Specific SARS-CoV-2 Serological Assay Using a Magnetic Modulation Biosensing System

Biosensors ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 7
Author(s):  
Shira Avivi-Mintz ◽  
Yaniv Lustig ◽  
Victoria Indenbaum ◽  
Eli Schwartz ◽  
Amos Danielli
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
Turnaround Time ◽  
Elisa Test ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Serological Assay ◽  
Clinical Sensitivity ◽  
Highly Sensitive

Sensitive serological assays are needed to provide valuable information about acute and past viral infections. For example, detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies could serve as the basis for an “immunity passport” that would enable individuals to travel internationally. Here, utilizing a novel Magnetic Modulation Biosensing (MMB) system and the receptor-binding domain of the SARS-CoV-2 spike protein, we demonstrate a highly sensitive and specific anti-SARS-CoV-2 IgG serological assay. Using anti-SARS-CoV-2 IgG antibodies, RT-qPCR SARS-CoV-2-positive and healthy patients’ samples, and vaccinees’ samples, we compare the MMB-based SARS-CoV-2 IgG assay’s analytical and clinical sensitivities to those of the enzyme-linked immunosorbent assay (ELISA). Compared with ELISA, the MMB-based assay has an ~6-fold lower limit of detection (129 ng/L vs. 817 ng/L), and it detects an increase in the IgG concentration much earlier after vaccination. Using 85 RT-qPCR SARS-CoV-2-positive samples and 79 -negative samples, the MMB-based assay demonstrated similar clinical specificity (98% vs. 99%) and sensitivity (93% vs. 92%) to the ELISA test, but with a much faster turnaround time (45 min vs. 245 min). The high analytical and clinical sensitivity, short turnaround time, and simplicity of the MMB-based assay makes it a preferred method for antibody detection.

scholarly journals Implementation of Isavuconazole in a Fluorescence-Based High-Performance Liquid Chromatography Kit Allowing Simultaneous Detection of All Four Currently Licensed Mold-Active Triazoles

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
René Jørgensen ◽  
Siri Rytcher Andersen ◽  
Karen Marie Thyssen Astvad ◽  
Maiken Cavling Arendrup
Keyword(s):  
Invasive Aspergillosis ◽  
Human Plasma ◽  
Broad Spectrum ◽  
High Performance ◽  
Simultaneous Detection ◽  
Hplc Method ◽  
Therapeutic Window ◽  
Serum Samples ◽  
Accuracy And Precision ◽  
Highly Sensitive

ABSTRACT Isavuconazole is a new broad-spectrum triazole agent recently approved for the treatment of both invasive aspergillosis and mucormycosis. Currently, there is no consensus regarding the potential need for TDM of isavuconazole, and no therapeutic window has been defined. However, at the ECIL-6 meeting in 2015, it was advised that TDM is indicated in a number of different settings. In this study, we describe a rapid and validated isocratic HPLC method for fluorescence-based detection and quantification of isavuconazole in human plasma/serum samples. The method is simple and efficient with good accuracy and precision and importantly only requires a small volume of patient plasma/serum. Furthermore, this method is highly sensitive and selective and can be detected simultaneously with the three other triazoles, itraconazole, voriconazole, and posaconazole, without the need for expensive mass spectrometry equipment. Isavuconazole (ISZ) is a newly available broad-spectrum triazole agent recently approved for the treatment of both invasive aspergillosis and mucormycosis. The aim of this study was to develop a simple and reliable method for therapeutic drug monitoring (TDM) of ISZ in human plasma samples. The method involves using a kit from ChromSystems intended for TDM of itraconazole (ITZ), posaconazole (PSZ), and voriconazole (VRZ) in serum/plasma for sample preparation and high-performance liquid chromatography, using fluorescence detection with emission and excitation wavelengths set to 261 and 366 nm, respectively. The assay was linear over the ISZ concentration range of 0.2 to 20.0 mg/liter, using a 0.1-ml sample volume. The inter- and intraday coefficients of variation were all below 3.7%, whereas the accuracies ranged from 95.0 to 106.2% and the mean extraction recovery was 91.9%. In addition, the method worked well using four different Vacutainer types, with six different healthy volunteers and under a number of relevant storage conditions. Finally, the ISZ detection could be seamlessly implemented in the TDM kit for VRZ, PSZ, and ITZ, enabling simultaneous detection of all four triazoles. This method proved to be simple, accurate, precise, and well suited for routine analysis work. It has been implemented in our laboratory for the simultaneous quantitative analysis of ISZ, VRZ, PSZ, and ITZ for TDM and pharmacokinetic research. IMPORTANCE Isavuconazole is a new broad-spectrum triazole agent recently approved for the treatment of both invasive aspergillosis and mucormycosis. Currently, there is no consensus regarding the potential need for TDM of isavuconazole, and no therapeutic window has been defined. However, at the ECIL-6 meeting in 2015, it was advised that TDM is indicated in a number of different settings. In this study, we describe a rapid and validated isocratic HPLC method for fluorescence-based detection and quantification of isavuconazole in human plasma/serum samples. The method is simple and efficient with good accuracy and precision and importantly only requires a small volume of patient plasma/serum. Furthermore, this method is highly sensitive and selective and can be detected simultaneously with the three other triazoles, itraconazole, voriconazole, and posaconazole, without the need for expensive mass spectrometry equipment.

scholarly journals Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 313
Author(s):  
Salvatore Giovanni De-Simone ◽  
Larissa Rodrigues Gomes ◽  
Paloma Napoleão-Pêgo ◽  
Guilherme Curty Lechuga ◽  
Jorge Soares de Pina ◽  
...  
Keyword(s):  
B Cell ◽  
Diphtheria Toxin ◽  
High Performance ◽  
Catalytic Domain ◽  
Elisa Test ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spot Synthesis ◽  
Humoral Immune

Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. Methods: One-hundred and twelve 15 mer peptides covering the entire sequence of diphtheria toxin (DTx) protein were prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Results: Twenty epitopes were identified, with two being in the signal peptide, five in the catalytic domain (CD), seven in the HBFT domain, and five in the receptor-binding domain (RBD). Two 17 mer (CB/Tx-2/12 and CB/DTx-4–13) derived biepitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) for huVS and miVS and, when compared with a commercial ELISA test, demonstrated a high performance. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays.

Determination of Aflatoxins in Foods Using HPLC and a Commercial ELISA System

1991 ◽  
Vol 54 (4) ◽  
pp. 291-294 ◽  
Author(s):  
MOUNIR AZER ◽  
CHONG COOPER
Keyword(s):  
High Performance ◽  
Peanut Butter ◽  
Peanut Meal ◽  
Hplc Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Poor Correlation ◽  
Elisa System ◽  
Three Samples ◽  
Peanut Sample ◽  
High Degree

A commercially available enzyme linked immunosorbent assay (ELISA) system and a high performance liquid chromatography (HPLC) method were used simultaneously to analyze 178 samples of foodstuffs for total aflatoxins. High correlation coefficient values between results of the two methodologies were obtained (>0.96) with nuts, nut products, peanuts, and peanut butter. However, poor correlation was obtained from results of cereals and grain samples. The ELISA system demonstrated a high degree of reproducibility between wells (p<0.01). Of the 35 samples of nut and nut products examined, aflatoxins were detected in five samples at levels ranging from 40 to 276 μg/kg. However, of 73 peanut and peanut butter samples only one peanut sample contained 61 μg/kg, and of 70 samples of processed cereal and grain, three samples contained 6–10 μg/kg total aflatoxins. Both methods were also applied to six peanut meal samples, with known aflatoxin concentrations, provided in an interlaboratory collaborative trial. The results obtained by the HPLC and ELISA methods were in close agreement with the mean results obtained by the participating laboratories.

scholarly journals Development of a Direct Competitive ELISA Kit for Detecting Deoxynivalenol Contamination in Wheat

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 50
Author(s):  
Li Han ◽  
Yue-Tao Li ◽  
Jin-Qing Jiang ◽  
Ren-Feng Li ◽  
Guo-Ying Fan ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
High Performance ◽  
Limit Of Detection ◽  
High Specificity ◽  
Hplc Method ◽  
Rapid Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
Detection Range ◽  
Technical Requirements ◽  
Relative Standard

This study was conducted to develop a self-assembled direct competitive enzyme-linked immunosorbent assay (dcELISA) kit for the detection of deoxynivalenol (DON) in food and feed grains. Based on the preparation of anti-DON monoclonal antibodies, we established a standard curve with dcELISA and optimized the detection conditions. The performance of the kit was evaluated by comparison with high-performance liquid chromatography (HPLC). The minimum detection limit of DON with the kit was 0.62 ng/mL, the linear range was from 1.0 to 113.24 ng/mL and the half-maximal inhibition concentration (IC50) was 6.61 ng/mL in the working buffer; there was a limit of detection (LOD) of 62 ng/g, and the detection range was from 100 to 11324 ng/g in authentic agricultural samples. We examined four samples of wheat bran, wheat flour, corn flour and corn for DON recovery. The average recovery was in the range of 77.1% to 107.0%, and the relative standard deviation (RSD) ranged from 4.2% to 11.9%. In addition, the kit has the advantages of high specificity, good stability, a long effective life and negligible sample matrix interference. Finally, wheat samples from farms in the six provinces of Henan, Anhui, Hebei, Shandong, Jiangsu and Gansu in China were analyzed by the kit. A total of 30 samples were randomly checked (five samples in each province), and the results were in good agreement with the standardized HPLC method. These tests showed that the dcELISA kit had good performance and met relevant technical requirements, and it had the characteristics of accuracy, reliability, convenience and high-throughput screening for DON detection. Therefore, the developed kit is suitable for rapid screening of DON in marketed products.

scholarly journals Heterologous immunoassay strategy for enhancing detection sensitivity of banned dye rhodamine B in fraudulent food

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jin Wang ◽  
Xing Shen ◽  
Peng Zhong ◽  
Zhaodong Li ◽  
Qiushi Tang ◽  
...  
Keyword(s):  
Rhodamine B ◽  
High Performance ◽  
High Specificity ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Exposure Level ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Chinese Prickly Ash

Abstract Background The high quality of antibody (Ab) is critical for an immunoassay; usually, an Ab with low affinity is often regarded as a “bad” one in the immunoassay development. How to use a “bad” Ab to develop a highly sensitive immunoassay is still a huge challenge. Methods In this study, a heterologous immunoassay strategy was designed to enhance the sensitivity for the detection of banned dye, rhodamine B (RB), in fraudulent food. The RB Ab could not recognize RB by pairing with homologous coating antigen (Ag). However, the Ab showed unexpected high specificity and sensitivity recognition after being replaced by heterologous coating Ag. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed based on the heterologous strategy. Results The detection limit of icELISA for chilli powder, Chinese prickly ash, hot-pot seasoning, and chilli sauce was 0.002 μg/kg, and the recoveries of the four samples ranged from 76.0 to 102.0%, with the coefficient of variation between 3.9 and 18.8%. Parallel experiment for 20 market samples with high-performance liquid chromatography (HPLC) was performed on to confirm the performance of the practical application of the developed icELISA, and the results of the two methods had good correlation. Molecular modeling inferred that the carboxyl group of hapten and its exposure level played an important role in the hapten-Ab recognition. Conclusions The proposed icELISA can be used for the surveillance screening of RB in a range of seasoning foods, and the heterologous strategy is an effective approach to enhance the sensitivity in an immunoassay.

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