Development of an Enzyme-Linked Immunosorbent Assay and Gold-Labelled Immunochromatographic Strip Assay for the Detection of Ancient Wool

The identification of ancient wool is of great importance in archaeology. Despite lots of meaningful information can be achieved by conventional detection methods, that is, light and electron microscopy, spectroscopy, and chromatography, the efficacy is likely to be limited in the detection of ancient samples with contamination or severe degradation. In this work, an immunoassay was proposed and performed for the identification of ancient wool. First, a specific antibody, which has the benefits of low cost, easy operation, and extensive applicability, was developed directly through immunizing rabbits with complete antigen (keratin). Then, an enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-labelled immunochromatographic strip (ICS) were developed to qualitatively identify the corresponding protein in ancient wool samples unearthed from Kazakhstan and China. The anti-keratin antibody exhibited high sensitivity and specificity for the identification of modern and ancient wool. The limit of detection (LOD) of the ELISA method was 10 ng/mL, and no cross-reactions with other interfering antigens have been noted. It is concluded that the immunoassays are reliable methods for the identification of ancient wool.

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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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-323 ◽

2015 ◽

Vol 78 (2) ◽

pp. 362-369 ◽

Cited By ~ 9

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.

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Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

Journal of Biomedicine and Biotechnology ◽

10.1155/2009/579175 ◽

2009 ◽

Vol 2009 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Yan Zhang ◽

FengXia Wang ◽

Li Fang ◽

Shuo Wang ◽

GuoZhen Fang

Keyword(s):

Immunosorbent Assay ◽

Matrix Effects ◽

Limit Of Detection ◽

Rapid Determination ◽

High Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Liver Sample ◽

Pig Liver ◽

Animal Products ◽

The Matrix

To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver), we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with anIC50of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride) ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2>0.95), which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

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Rapid, Quantitative, and High-Sensitivity Detection of Anti-Phospholipase A2 Receptor Antibodies Using Novel Cdse/Zns-Based Fluorescence Immunosorbent Assay

10.21203/rs.3.rs-134511/v1 ◽

2020 ◽

Author(s):

Chenxi Li ◽

Manyun Qian ◽

Qiaozhen Hong ◽

Xiaohong Xin ◽

Zichun Sun ◽

...

Keyword(s):

Phospholipase A2 ◽

Immunosorbent Assay ◽

Limit Of Detection ◽

High Sensitivity ◽

Detection Sensitivity ◽

Response To Treatment ◽

Enzyme Linked Immunosorbent Assay ◽

The Novel ◽

Serum Samples ◽

Phospholipase A2 Receptor

Abstract Autoantibodies against M-type phospholipase A2 receptor (PLA2R) are specific biomarkers for idiopathic membranous nephropathy (IMN) and their quantification has been helpful to monitor disease activity. In this study, we describe a highly sensitive and rapid quantum dots-based immunochromatography assay (QD-ICA) for quantifying PLA2R autoantibodies. Serum samples from 135 biopsy-confirmed patients with nephrotic syndrome were analyzed for PLA2R autoantibodies using the novel QD-ICA as well as enzyme-linked immunosorbent assay (ELISA). The detection sensitivity and specificity of QD-ICA (80.9 and 100%, respectively) exceeded those of ELISA (72.1 and 98.5%, respectively). The optimum cut-off value of QD-ICA was 18.18 RU/mL and limit of detection was 2.86 relative units/mL. The novel QD-ICA outperforms ELISA in detecting PLA2R autoantibodies, with shorter detection time, fewer steps, smaller equipment size, and broader testing application, suggesting its capability to improve IMN diagnosis and monitor patient response to treatment.

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Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for detecting aflatoxin M1 in milk

Food Control ◽

10.1016/j.foodcont.2016.01.036 ◽

2016 ◽

Vol 66 ◽

pp. 1-7 ◽

Cited By ~ 19

Author(s):

Biing-Hui Liu ◽

Kuang–Chun Chu ◽

Feng-Yih Yu

Keyword(s):

Monoclonal Antibody ◽

Immunosorbent Assay ◽

Aflatoxin M1 ◽

Enzyme Linked Immunosorbent Assay ◽

Immunochromatographic Strip

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Rapid and Sensitive Detection of miRNA Based on AC Electrokinetic Capacitive Sensing for Point-of-Care Applications

Sensors ◽

10.3390/s21123985 ◽

2021 ◽

Vol 21 (12) ◽

pp. 3985

Author(s):

Nan Wan ◽

Yu Jiang ◽

Jiamei Huang ◽

Rania Oueslati ◽

Shigetoshi Eda ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Clinical Diagnostics ◽

Detection Methods ◽

Capacitive Sensing ◽

Application Potential ◽

Mirna Detection ◽

Mirna 25 ◽

Low Sensitivity

A sensitive and efficient method for microRNAs (miRNAs) detection is strongly desired by clinicians and, in recent years, the search for such a method has drawn much attention. There has been significant interest in using miRNA as biomarkers for multiple diseases and conditions in clinical diagnostics. Presently, most miRNA detection methods suffer from drawbacks, e.g., low sensitivity, long assay time, expensive equipment, trained personnel, or unsuitability for point-of-care. New methodologies are needed to overcome these limitations to allow rapid, sensitive, low-cost, easy-to-use, and portable methods for miRNA detection at the point of care. In this work, to overcome these shortcomings, we integrated capacitive sensing and alternating current electrokinetic effects to detect specific miRNA-16b molecules, as a model, with the limit of detection reaching 1.0 femto molar (fM) levels. The specificity of the sensor was verified by testing miRNA-25, which has the same length as miRNA-16b. The sensor we developed demonstrated significant improvements in sensitivity, response time and cost over other miRNA detection methods, and has application potential at point-of-care.

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Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin

Toxins ◽

10.3390/toxins10110458 ◽

2018 ◽

Vol 10 (11) ◽

pp. 458 ◽

Cited By ~ 1

Author(s):

Hisaya Ono ◽

Nobuaki Hachiya ◽

Yasunori Suzuki ◽

Ikunori Naito ◽

Shouhei Hirose ◽

...

Keyword(s):

Limit Of Detection ◽

Expression System ◽

Sandwich Elisa ◽

Food Poisoning ◽

High Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

E Coli ◽

Detection Systems ◽

C Terminus ◽

Culture Supernatants

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

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Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

Toxins ◽

10.3390/toxins13110781 ◽

2021 ◽

Vol 13 (11) ◽

pp. 781

Author(s):

Zhuolin Song ◽

Lin Feng ◽

Yuankui Leng ◽

Mingzhu Huang ◽

Hao Fang ◽

...

Keyword(s):

Ochratoxin A ◽

Limit Of Detection ◽

High Sensitivity ◽

Cross Reaction ◽

Molar Ratio ◽

Quantitative Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Enzyme Loading ◽

M13 Bacteriophage ◽

Mimic Peptide

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.

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Graphene Oxide and Fluorescent Aptamer Based Novel Biosensor for Detection of 25-hydroxyvitamin D3

10.21203/rs.3.rs-334003/v1 ◽

2021 ◽

Author(s):

Ritika Gupta ◽

Sunaina Kaul ◽

Vishal Singh ◽

Sandeep Kumar ◽

Nitin Kumar Singhal

Keyword(s):

Vitamin D ◽

Graphene Oxide ◽

Limit Of Detection ◽

Low Cost ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Detection Methods ◽

Hydroxyvitamin D ◽

25 Hydroxyvitamin D ◽

Sensitive Sensor

Abstract For maintaining the healthy metabolic status, vitamin D is a beneficial metabolite stored majorly in its pre-activated form, 25-hydroxyvitamin D3 (25(OH)D3). Due to its important role in bone strengthening, the study was planned to quantify 25(OH)D3 levels in our blood. Quantification techniques for 25(OH)D3 are costly thus requiring a need for a low cost, and sensitive detection methods. In this work, an economic, and sensitive sensor for the detection of 25(OH)D3 was developed using aptamer and graphene oxide (GO). Aptamer is an oligonucleotide, sensitive towards its target, whereas, GO with 2D nanosheets provides excellent quenching surface. Aptamer labeled with fluorescein (5’, 6-FAM) is adsorbed by π -π interaction on the GO sheets leading to quenching of the fluorescence due to Förster resonance energy transfer (FRET). However, in the presence of 25(OH)D3, a major portion of aptamer fluorescence remains unaltered, due to its association with 25(OH)D3. However, in the absence, aptamer fluorescence gets fully quenched. Fluorescence intensity quenching was monitored using fluorescence spectrophotometer and agarose gel based system. The limit of detection of 25(OH)D3 by this method was found to be 0.15 µg/mL. Therefore, this method could come up as a new sensing method in the field of vitamin D detection.

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Interpretations of Antibody Responses toSalmonella enterica Serotype Enteritidis gm Flagellin in Poultry Flocks Are Enhanced by a Kinetics-Based Enzyme-Linked Immunosorbent Assay

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.5.4.550-555.1998 ◽

1998 ◽

Vol 5 (4) ◽

pp. 550-555 ◽

Cited By ~ 8

Author(s):

Patrick L. McDonough ◽

Richard H. Jacobson ◽

John F. Timoney ◽

Ahmed Mutalib ◽

David C. Kradel ◽

...

Keyword(s):

Immunosorbent Assay ◽

High Sensitivity ◽

High Specificity ◽

Enzyme Linked Immunosorbent Assay ◽

Analytical Sensitivity ◽

Department Of Agriculture ◽

Trace Back ◽

Elisa Format ◽

Commercial Poultry ◽

Computer Controlled

ABSTRACT Many regulatory and diagnostic programs for the detection ofSalmonella enterica serotype Enteritidis infection in commercial poultry flocks have relied on rapid Pullorum agglutination tests to screen birds because of the shared antigens of S. enterica Enteritidis and S. enterica Pullorum and Gallinarum; however, the use of the enzyme-linked immunosorbent assay (ELISA) format affords better analytical sensitivity than crude agglutination tests. In this study, we adapted our earlier conventional indirect ELISA, using gm flagellin as the antigen, to a kinetics-based, computer-controlled ELISA (KELA). The KELA was used to screen for flagellin antibody from three commercial flocks: (i) a large flock involved in a U.S. Department of Agriculture trace back from a humanS. enterica Enteritidis foodborne outbreak (n = 3,209), (ii) a flock infected with the endemicS. enterica Enteritidis serotype but which also had multiple other salmonella serotypes (n = 65), and (iii) an S. enterica Pullorum-infected flock (n = 12). The first flock (S. entericaEnteritidis prevalence of 2.45% based on culture) provided a field test of the KELA and allowed the calculation of diagnostic sensitivity (D-Sn) and diagnostic specificity (D-Sp). With a cutoff of 10 (used for screening flocks [i.e., high sensitivity]), the KELA has a D-Sn of 95.2% and a D-Sp of 18.5%; with a cutoff of 140 (used in confirmatory flock testing [i.e., high specificity]), the KELA has a D-Sn of 28.0% and a D-Sp of 99.1%. We found that with a cutoff of 60 (D-Sn = 63.1%; D-Sp = 91.6%), we could eliminate reactions in the KELA caused by other non-S. enterica Enteritidis salmonellae. The KELA was also compared to two commercial rapid Pullorum tests, the Solvay (D-Sn = 94.9%; D-Sp = 55.5%) and the Vineland (D-Sn = 62.0%; D-Sp = 75.3%).

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