Development of sandwich ELISA for testing bovine β-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes

He, Shengfa; Li, Xin; Gao, Jinyan; Tong, Ping

doi:10.1016/j.foodchem.2017.01.060

Cited by 51 publications

(23 citation statements)

References 28 publications

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“…Therefore, 120 min was selected for the capture antibody to antigen interaction. A similar situation obtained in the development of ELISA for detecting CD8α and bovine β‐lactoglobulin (He et al, 2017; Zhang et al, 2016). Furthermore, the incubation time of bound rMan i1 to the detection antibody was optimized.…”

Section: Resultssupporting

confidence: 62%

“…The two layers of antibodies can be either polyclonal antibody or monoclonal antibody, and they play an important role in the sensitivity and specificity of sELISA. For instance, sELISA based on two different polyclonal antibodies from rabbit for detecting β‐lactoglobulin, a major allergen in cows' milk, has a detection limit of 1.96 ng/ml and good reliability in the analysis of food samples, such as milk, whey, or yoghurt (He, Li, Gao, Tong, & Chen, 2017). Moreover, IgY polyclonal antibody is also attracting attention because a large amount of IgY antibodies can be harvested from eggs without sacrificing animals (Chu, Lin, Chen, Chen, & Wen, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Development of monoclonal antibody‐based sandwich ELISA for detecting major mango allergen Man i1 in processed foods

Tsai

Yin

Chen

et al. 2021

Journal of Food Safety

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Mango (Mangifera indica L.) is a popular tropical fruit but also an allergenic source. In Taiwan, the packaging of any food product that contains mango must display an allergen warning. Therefore, establishing a reliable method for detecting mango residues in food products is important. In this study, a sandwich enzyme‐linked immunosorbent assay (sELISA) was developed to detect the major mango allergen Man i1. First, five batches of polyclonal and monoclonal antibodies against Man i1 were generated and used to identify the optimal antibody pair for sELISA. Using 2G5 mAb as the capture antibody and anti‐rMan i1 pAb as the detection antibody yielded the strongest assay signal. The limit of detection (LOD) and limit of quantification (LOQ) of the developed ELISA are estimated to be 3.9 and 21.2 ng/ml rMan i1, respectively, after optimization. The assay exhibits good specificity to mango and offers good precision and reproducibility since the calculated coefficient of variation (CV) of the intra‐ and inter‐assays were 5.5–11.7% and 7.4–12.4%, respectively. Finally, 23 commercial food products were evaluated using the developed sELISA and the results of this assay closely agreed (95.7%) with those of the Western blot. Thus, the developed sELISA provides a potential method for detecting mango allergens in foods for protecting people with mango allergy from the accidental ingestion of mango.

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Section: Resultssupporting

confidence: 62%

Section: Introductionmentioning

confidence: 99%

Development of monoclonal antibody‐based sandwich ELISA for detecting major mango allergen Man i1 in processed foods

Tsai

Yin

Chen

et al. 2021

Journal of Food Safety

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“…Upon arrival, the primary weight of each rabbit was recorded and primed in the Animal House, Faculty of Veterinary Medicine, Universiti Putra Malaysia for seven days. Rabbit immunization was carried out according to antibody production guideline from Canadian Council on Animal Care and He et al ( 2017) with some modifications [29,30]. For primary immunization (injection 1), 200 µg of peptide-KLH was dissolved in 1 mL of saline and mixed with Titermax Gold Adjuvant at 1:1 (v:v) ratio.…”

Section: Immunization Of Animalsmentioning

confidence: 99%

Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein

et al. 2020

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The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10−3 μM compared to 9.046 × 10−3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.

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“…In addition, the lot-to-lot variances could lead to the inaccurate recovery of target analytes [39]. It should be noted that, similar to Tween-20, studies have reported the advantages of gelatin for providing the best positive to negative ratio [40] and improving the ELISA sensitivity significantly [41]. Also, Rajasekariah et al [42] showed that the blocking efficiency from gelatin increased as a function of its concentration.…”

Section: Effect Of Protein and Non-protein Blockersmentioning

confidence: 99%

“…This is because these non-protein blockers are polymers that are known for their ability to coat hydrophobic surfaces [36,55]. The usage of PEG improved both positive and negative signals, which led to a decreased assay sensitivity [40]. Their blocking deficiency was also reported by Huber et al [52], who found that PVP and PEG could not replace protein blockers in the ELISA development of food allergen detection.…”

Section: Effect Of Protein and Non-protein Blockersmentioning

confidence: 99%

Non-Specific Binding and Cross-Reaction of ELISA: A Case Study of Porcine Hemoglobin Detection

Jiang

Albo

et al. 2021

Foods

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Different types of enzyme-linked immunosorbent assays (ELISA) have been widely used to control food safety and quality. To develop an accurate and reproducible ELISA, false immunodetection results caused by non-specific binding (NSB) and cross-reaction must be prevented. During the case study of sandwich ELISA development for the detection of porcine hemoglobin (PHb), several critical factors leading to NSB and cross-reaction were found. First, to reduce the NSB of the target analyte, the selection of microplate and blocker was discussed. Second, cross-reactions between enzyme-labeled secondary antibodies and sample proteins were demonstrated. In addition, the function of (3-aminopropyl)triethoxysilane (APTES) was evaluated. Overall, this study highlights the essence of both antibody and assay validation to minimize any false-positive/negative immunodetection results.

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Development of sandwich ELISA for testing bovine β-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes

Cited by 51 publications

References 28 publications

Development of monoclonal antibody‐based sandwich ELISA for detecting major mango allergen Man i1 in processed foods

Development of monoclonal antibody‐based sandwich ELISA for detecting major mango allergen Man i1 in processed foods

Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein

Non-Specific Binding and Cross-Reaction of ELISA: A Case Study of Porcine Hemoglobin Detection

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