Multimeric recombinant antibody (scFv) for ELISA detection of allergenic walnut. An alternative to animal antibodies

Madrid, Raquel; Cruz, Silvia de la; García-García, Aina; Alcocer, Marcos; González, Isabel; Garcı́a, Teresa; Martı́n, Rosario

doi:10.1016/j.jfca.2018.01.017

Cited by 11 publications

(6 citation statements)

References 34 publications

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“…The capability to identify and detect biological molecules such as disease biomarkers with high sensitivity and specificity is of great importance to the pathology, diagnosis, and treatment of diseases. As a conventional and mature detection technology with the advantages of low cost, easy operation, and specific recognition, the ELISA provides an accurate, rapid, and efficient method to quantitatively measure target analytes in biological samples and has been widely used in clinical diagnosis and laboratories. − Based on the antigen–antibody recognition and enzymatic reaction, conventional methods for quantitative analysis in ELISA catalyze the decomposition of a substrate such as 3,3′,5,5′-tetramethylbenzidine (TMB) into colored products by the enzyme-linked antibodies such as horseradish peroxidase (HRP) for visual quantification or spectrophotometric analysis, which can only detect targets at picogram or higher levels. − This limited sensitivity hinders the widespread application of ELISA , in the detection of those disease-related biomarkers at lower concentrations. Currently, a number of diagnostic technologies such as lateral flow assays (LFAs), − gold immunoassays (CGAs), − electrochemistry, − surface-enhanced Raman scattering, − and so on have been intensively explored to provide complementary approaches to conventional ELISA.…”

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confidence: 99%

Optical Microfiber Reader for Enzyme-Linked Immunosorbent Assay

Sun

Huang

et al. 2019

Anal. Chem.

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In clinical diagnosis, accurate and reliable measurement technologies for the detection of disease biomarkers at ultralow concentrations can provide guidance for the initiation of treatment and potentially improve survival for patients. Here, we demonstrate an optical microfiber reader for enhanced analytical sensitivity in enzyme-linked immunosorbent assays (ELISA) that enables the detection of tiny changes of the refractive index (RI) induced by the catalyzed oxidation of substrate, owing to the strong interaction between the evanescent field and surrounding medium. By employing the microfiber reader for the C-reaction protein (CRP) and interleukin-6 (IL-6) assays after the enzymatic signal amplification in ELISA, we experimentally investigate the biosensing capacity of the device. As a result, log–linear relations of CRP and IL-6 detection in PBS and human serum between the concentration and spectral response were obtained at both nanogram and picogram levels, respectively, and anti-CRP/HRP detection as low as 9.75 pg/mL was achieved, which was undetectable by the conventional spectrophotometry. With a stable, accurate, and color-free detection capacity, this optical microfiber reader has a promising prospect in early disease diagnosis and clinical treatment.

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confidence: 99%

Optical Microfiber Reader for Enzyme-Linked Immunosorbent Assay

Sun

Huang

et al. 2019

Anal. Chem.

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“…The specificity and sensitivity of the multimeric scFv ELISA for the detection of walnut were previously assessed [ 15 ]. The LOD was 1616 mg kg −1 , and some cross-reactivity was found with pecan (2.25%) and almond (0.35%), but not with the remaining tree nuts analyzed.…”

Section: Resultsmentioning

confidence: 99%

“…The JrBSF scFv was isolated from a phagemid synthetic library of human scFv fragments (Tomlinson I, Source Bioscience, Nottingham, UK) by phage display against a walnut protein extract [ 15 ]. However, the walnut protein fraction recognized by the phage-antibody was not identified.…”

Section: Resultsmentioning

confidence: 99%

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Survey of Commercial Food Products for Detection of Walnut (Juglans regia) by Two ELISA Methods and Real Time PCR

Madrid

García-García

Cabrera

et al. 2021

Foods

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Labeling of food allergens in accordance with legal regulations is important to protect the health of allergic consumers. The requirements for detecting allergens in foods involve adequate specificity and sensitivity to identify very small amounts of the target allergens in complex food matrices and processed foods. In this work, one hundred commercial samples were analyzed for walnut detection using three different methods: a sandwich enzyme-linked immunosorbent assay (ELISA) kit based on polyclonal antibodies, a direct ELISA using a recombinant multimeric scFv, and a real time PCR. The most sensitive method was real time PCR followed by sandwich ELISA kit and multimeric scFv ELISA. There was agreement between the three methods for walnut detection in commercial products, except for some heat-treated samples or those that contained pecan. The walnut ELISA kit was less affected by sample processing than was the multimeric scFv ELISA, but there was cross-reactivity with pecan, producing some false positives that must be confirmed by real time PCR. According to the results obtained, 7.0 to 12.6% of samples (depending on the analytical method) contained walnut but did not declare it, confirming there is a risk for allergic consumers. Moreover, there was one sample (3.7%) labelled as containing walnut but that tested negative for this tree nut. Genetic and immunoenzymatic techniques offer complementary approaches to develop a reliable verification for walnut allergen labeling.

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“…The biopanning processes were performed as described in Madrid et al [ 19 ], but using roasted pistachio, cashew, and peanut whole or defatted extracts, as shown in Table 2 .…”

Section: Methodsmentioning

confidence: 99%

Phage Displayed Domain Antibodies (dAb) for Detection of Allergenic Pistachio Proteins in Foods

Madrid

García-García

González

et al. 2020

Foods

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Pistachio nuts (Pistacia vera) have been consumed by past and present-day civilizations because of their organoleptic characteristics and potential health benefits. However, they can also produce moderate to severe IgE-mediated reactions in allergic individuals. In this work, we report the isolation of the first recombinant antibodies against pistachio nut, produced without animal immunization, to be used in immunoassays for detection of allergenic pistachio in food products. Several phage display biopanning strategies were evaluated to screen the human-based domain antibody library (dAb) in search for pistachio-specific probes. The clone producing the PVF4 phage-dAb was finally selected, and it does not cross-react with cashew despite the phylogenetic proximity with pistachio. Western blot and matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-TOF/TOF) analysis demonstrated that this clone recognised a unique band of ∼22 kDa related to the basic subunit of pistachio 11S globulin (allergen Pis v 2). The PVF4 phage-dAb allowed detection of pistachio in a food matrix with a limit of detection (LOD) of 3983 mg kg-1 in an indirect phage-enzyme-linked immunosorbent assay (ELISA). The ELISA method developed was used to assess applicability of the PVF4 phage-dAb for analysis of 77 commercial food products.

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Multimeric recombinant antibody (scFv) for ELISA detection of allergenic walnut. An alternative to animal antibodies

Abstract: Walnuts are classified as an important allergenic ingredient that can cause severe reactions in sensitized individuals. To prevent unintended exposure to products containing walnut, food manufacturers have the responsibility to declare its presence in packaged foods.

Cited by 11 publications

References 34 publications

Optical Microfiber Reader for Enzyme-Linked Immunosorbent Assay

Optical Microfiber Reader for Enzyme-Linked Immunosorbent Assay

Survey of Commercial Food Products for Detection of Walnut (Juglans regia) by Two ELISA Methods and Real Time PCR

Phage Displayed Domain Antibodies (dAb) for Detection of Allergenic Pistachio Proteins in Foods

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