Monoclonal Antibodies with Orthogonal Azaspiracid Epitopes

Frederick, Michael O.; Marin, Sandra De Lamo; Janda, Kim D.; Nicolaou, K. C.; Dickerson, Tobin J.

doi:10.1002/cbic.200900201

Cited by 15 publications

(15 citation statements)

References 70 publications

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“…2) and a lower LoD than shorter protocols (Table 1). Moreover, this inhibition immunoassay has a high sensitivity compared to the sensitivity of previously published immunoassays for AZAs or AZA fragments [28,31]. …”

Section: Resultsmentioning

confidence: 99%

“…Ethylenediamine and sodium azide were obtained from Fluka (Steinheim, Germany). Phycoerythin (PE) Goat Anti-Mouse IgG was purchased from Invitrogen (Eugene, Oregon), and the anti-azaspiracid monoclonal antibody (mAb) 8F4 was produced as previously described [28]. Sodium chloride, acetic acid, methanol, sodium acetate anhydrous and sodium phosphates were obtained from Panreac (Barcelona, Spain).…”

Section: Methodsmentioning

confidence: 99%

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Microsphere-based immunoassay for the detection of azaspiracids

Rodríguez

Vilariño

Louzao

et al. 2014

Analytical Biochemistry

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Azaspiracids (AZAs) are a group of lipophilic toxins discovered in mussels from Ireland in 1995 following a human poisoning incident. Nowadays the regulatory limit for AZAs in many countries is set at 160 Fg of azaspiracid equivalents per kg of shellfish meat. In this work a microsphere-based immunoassay has been developed for the detection of AZAs using a Luminex system. This method is based on the competition between AZA-2 immobilized onto the surface of microspheres and free AZAs for the interaction with a monoclonal anti-azaspiracid antibody (mAb 8F4). In this inhibition immunoassay the amount of mAb 8F4 bound to AZA-2-microspheres was quantified using a phycoerythrin-labeled anti-mouse antibody, and the fluorescence was measured with a Luminex analyzer. Simple acetate/methanol or methanol extractions yielded final extracts with no matrix interferences and adequate recovery rates of 86.5% and 75.8%, respectively. In summary, this work presents, a sensitive and easily performed screening method capable of detecting AZAs at concentrations below the range of the European regulatory limit using a microsphere/flow cytometry system.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Microsphere-based immunoassay for the detection of azaspiracids

Rodríguez

Vilariño

Louzao

et al. 2014

Analytical Biochemistry

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“…LC-MS techniques have been optimized for the detection of azaspiracids [48,49], although quantification and identification is possible only for those compounds that have available certified standards. Azaspiracid-specific antibodies have been produced recently, and shown to bind several azaspiracid analogs (Aza-1, 2, 3 and 6) [94,95]. Competition and sandwich ELISAs were designed with these antibodies and a synthetic fragment of the azaspiracid molecule that is conserved for many analogs.…”

Section: Azaspiracidsmentioning

confidence: 99%

Use of Biosensors as Alternatives to Current Regulatory Methods for Marine Biotoxins

Botana¹,

Vilariño²,

Alfonso³

et al. 2012

Springer Protocols Handbooks

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“…Nonetheless, the lack of commercially available anti-AZA Abs has hindered the development of immunosensors, as well as immunoassays, for the detection of AZAs. To date, only a monoclonal antibody (MAb) raised using AZA-1 [21] and a polyclonal antibody (PAb) raised using a synthetic fragment of AZA [22] have been reported. While the MAb was used in the development of a flow fluorimetry-based immunoassay [23], the anti-AZA PAb has been used in the development of an enzyme-linked immunosorbent assay (ELISA) [24] and an electrochemical immunoassay using magnetic beads as immunorecognition supports [25].…”

Section: Introductionmentioning

confidence: 99%

Detection of azaspiracids in mussels using electrochemical immunosensors for fast screening in monitoring programs

Leonardo

Kilcoyne

Samdal

et al. 2018

Sensors and Actuators B: Chemical

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Given the widespread occurrence of azaspiracids (AZAs) it is clearly necessary to advance in simple and low-cost methods for the rapid detection of these marine toxins in order to protect seafood consumers. To address this need, electrochemical immunosensors for the detection of AZAs based on a competitive direct immunoassay using peroxidase-labelled AZA as a tracer were developed. An anti-AZA polyclonal antibody was immobilised in a controlled and stable manner on protein G or avidin-coated electrodes. Experimental conditions were first optimised using colorimetric immunoassays on microtitre plates, providing intermediate products already applicable to the accurate detection of AZAs. Then, transfer of the protein G and avidin-biotin interaction-based immunoassays to 8-electrode arrays provided compact and miniaturised devices for the high-throughput detection of AZAs. The low amounts of immunoreagents required as well as the potential for reusability of the avidin-biotin interaction-based immunosensors represented significant economic savings as well as a contribution to sustainability. The electrochemical immunosensors enabled the quantification of all regulated AZAs below the regulatory limit, as well as a broad range of other toxic AZA analogues (from 63 ± 3 to 2841 ± 247 µg AZA-1 equiv./kg for the protein G-based immunosensor and from 46 ± 2 to 3079 ± 358 µg AZA-1 equiv./kg for the avidin-biotin interaction-based immunosensor). The good agreement between the results obtained by the immunosensors and LC-MS/MS in the analysis of naturally contaminated mussel samples demonstrated the easy implementation of electrochemical immunosensors for routine analysis of AZAs in food safety monitoring programs.

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Monoclonal Antibodies with Orthogonal Azaspiracid Epitopes

Cited by 15 publications

References 70 publications

Microsphere-based immunoassay for the detection of azaspiracids

Microsphere-based immunoassay for the detection of azaspiracids

Use of Biosensors as Alternatives to Current Regulatory Methods for Marine Biotoxins

Detection of azaspiracids in mussels using electrochemical immunosensors for fast screening in monitoring programs

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