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Sensitivity improvement of an immuno-detection method for azaspiracids based on the use of microspheres coupled to a flow-fluorimetry system

María Fraga Corral1*, Natalia Vilariño1*, M. Carmen Louzao1* and Luis M. Botana1*
  • 1 University of Santiago de Compostela, Department of Pharmacology, Spain

1. INTRODUCTION Azaspiracids (AZAs) are lypophilic marine toxins known to produce AZA poisoning (AZP). AZP is the most recent human syndrome related to the ingestion of toxin-contaminated shellfish meat which symptoms are similar to those produced by diarrheic shellfish poisoning (DSP) (Furey et al., 2010). Since this first human intoxication by AZAs reported in 1995 (McManhon, 1996), their presence has been widely described along European coasts and punctually in Chile, Morocco and Japan (Ueoka et al., 2009;Furey et al., 2010). To protect human health, a maximum content of 160 µg AZA equivalents/kg in shellfish meat has been regulated in many countries (EC, 2004). The implementation of these legal regulations demands the development and validation of AZA detection methods. Bioassays have not been fully validated, imply some technical disadvantages and raise ethical issues related to the use of laboratory animals (Holland, 2008). The commission decision EC 15/2011 established the progressive substitution of bioassays by liquid chromatography-mass spectrometry (LC-MS) (EC, 2011). However LC-MS methods need to be validated, involve expensive instruments, highly trained personnel and specific standards. Currently, the development of screening methods offers a fast, simple alternative to reduce the number of samples to be analyzed by expensive and ethically questionable methodologies. Actually, multiplexed screening methods, in which several groups of toxins are simultaneously detected in one sample, represent an efficient option to save time and reagents. Flow-fluorimetry technology has been widely employed in clinical and research fields for the development of multiplexed assays in which different analytes present in the same sample are simultaneously detected (Kellar and Iannone, 2002). Luminex technology uses microsphere classes with different spectral properties and surface carboxyl groups for covalent ligand attachment. Each microsphere class becomes specific for an analyte through the immobilization of a specific ligand. A fluidics system separates individual microspheres where respective red and green lasers distinguish microsphere class and quantify the attached compound. Multiplexing is provided by the incubation of a sample with multiple classes of analyte-specific microspheres simultaneously. The aim of this work is to optimize a single, semi-quantitative microsphere-based immuno-detection method for AZAs using the Luminex technology. This work, based on a previously published assay for AZA (Rodriguez et al., 2014), contributes two main improvements, an important increase of assay sensitivity, and a reduction of protocol duration preserving sensitivity. These results will be relevant to future development of a multiplexed marine toxin detection method. 2. MATERIAL AND METHODS 2.1. Materials Standard material of azaspiracid-1 (AZA-1) was obtained from CIFGA (Lugo, Spain). N-hydroxysuccinimide (NHS), ethylenediamine and ethanolamine were from Sigma-Aldrich (Madrid, Spain). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was from Pierce (Rockford, IL). The anti-azaspiracid monoclonal antibody (mAb 8F4) was obtained as previously described (Frederick et al., 2009). Phycoerythrin Goat Anti-Mouse Ig (PE-Ab) was from Invitrogen (Eugene, OR). Carboxylated microspheres LC10027-01 were from Luminex Corporation (Austin, TX). Luminex disposable material was from Millipore (Billerica, MA). Reagent grade solvents and buffer constituents were used. Phosphate-buffered saline solution (PBS) was 130 mM NaCl, 10 mM NaPO4, pH 7.4. 2.2. Toxin or binding protein immobilization on the microsphere surface AZA-1 was covalently attached to the carboxylated surface of LC10027-01 microsphere class as described in Rodríguez et al. (Rodriguez et al., 2014). A slightly modified protocol was followed. Briefly, 2x106 microspheres were activated using a EDC/NHS solution. Later ethylenediamine in borate buffer was added for 1 h. The free NHS-ester groups were inactivated by ethanolamine-HCl for 20 min. Pre-activation of toxin involved 50 µg of free-AZA-1 in DMSO diluted with 40 µL of EDC/NHS in sodium acetate buffer. Ethanolamine was removed and pre-activated toxin was added to microspheres and allowed to react for 4 h. At the end of the immobilization, microspheres were washed with PBS and stored in PBS with 0.01 % sodium azide at 4 ˚C in the dark. 2.3. Microsphere-based inhibition immunoassay for AZAs The detection method was based on a competition immunoassay performed in three steps. The procedure started with a incubation of 60 µL of AZA-1 calibration solution (ranging from 1 pM to 30 nM) with 60 µL of 14 pg/mL mAb 8F4. After 1 h, competition step began with the transfer of 100 µL of this mixture to a microtiter filter plate containing previously washed 2x103 AZA-1-coated microspheres for 1h. Finally, after a washing step, mAb 8F4 bound to microspheres was quantified with 100 µL of 0.5 µg/mL PE-Ab during 1h. 2.4. Quantification of binding signals PE-fluorescence attached to the surface of the toxin-microspheres was quantified with a Luminex 200™ analyzer (LuminexCorp, Austin, TX). Microspheres were classified with a 635 nm laser and PE-fluorescence was quantified with a 532 nm laser. The acquisition volume was 75 µl and the minimum number for bead count was 100. 2.5. Data analysis Calibration curves for the microsphere-based method were fitted using GraphPad Prism 5.0 by a four-parameter logistic equation obtained with a nonlinear regression fitting procedure: Y=Rhi+(Rlo-Rhi)/(1+10^((LogIC50-X)*HillSlope)), where Rhi is the response at infinite concentration, Rlo is the response at 0 concentration, IC50 is the half maximal inhibitory concentration and X is the logarithm of concentration. 3. RESULTS The inhibition assay consisted of the competition of AZA-1 attached to the microspheres with free AZA-1 in calibration solution for binding to mAb 8F4. This specific antibody bound to the microspheres was quantified using a PE-Ab. Optimization of the method was based on immobilization of AZA-1 instead of AZA-2, and adjustment of mAb 8F4 dilutions and incubation times. 3.1. Optimization of AZA-1 immobilization Immobilization of AZA-1 on the microsphere surface was the first optimization step. A higher number of microspheres (2x106) was used to perform the immobilization of a different toxin, AZA-1, in spite of the synthetic AZA-2 used in Rodríguez et al.(Rodriguez et al., 2014). 3.2. Optimization of the immuno-detection method for AZA-1 Optimization of the inhibition assay was done following previously published protocols for other marine toxins (Fraga et al., 2013). With the future aim of multiplexing, some experimental conditions were maintained constant: 1 h incubation times and 0.5 µg/mL PE-Ab concentration. In these conditions several mAb 8F4 concentrations were tested (from 1.4 ng/mL to 140 pg/mL). The final mAb 8F4 concentration (140 pg/mL) was selected considering the Max/non-specific binding signal (Min) ratio (60), IC50 of 1.1 nM and dynamic range (IC20-IC80) of 0.2 - 2.9 nM (Figure 1A). To achieve the maximum sensitivity of the assay a longer protocol was fully optimized. An overnight incubation (ON, 16 h) was used during the competitive step combined with higher antibody dilutions. Different mAb 8F4 concentrations, from 280 to 2.8 pg/mL were tested. The highest sensitivity was provided by a 1h-incubation of free AZA-1 in the calibration solution (1 pM-50 nM) with 14 pg/mL mAb 8F4, followed by an ON incubation with 2x103 AZA-coated microspheres. In this conditions a Max/Min ratio of 200, IC50 of 0.3 nM and dynamic range of 0.07-0.98 nM were obtained (Figure 1B). 4. DISCUSSION These results demonstrate the high capability in terms of sensitivity of the microsphere-based immuno-detection assay for AZAs. The immobilization of AZA-1 instead of the synthetic AZA-2 used in Rodríguez et al (Rodriguez et al., 2014), combined with a lower mAb 8F4 concentration provided a remarkable improvement of sensitivity. The ON protocol used in Rodríguez et al. (Rodriguez et al., 2014) displayed a similar IC50 than the new short assay (around 1 nM) while the new ON protocol provided an IC50 5-fold more sensitive (0.3 nM). Therefore, the new short assay allows a reduction of the experimental time. Additionally, the increase of sensitivity could help to avoid shellfish matrix interferences. Previously published works using immunoassays for the detection of phycotoxins present in shellfish avoided matrix interference by further extract dilution in combination with an increase of assay sensitivity (Fraga et al., 2012;Fraga et al., 2013). The extraction protocol described by Rodríguez et al. (Rodriguez et al., 2014) will probably be suitable for this newly optimized AZA-detection method since many reagents are the same and the higher sensitivity will allow higher extract dilution. Considering the extraction protocol recovery, sensitivity of the current assay and the regulated limit, shellfish extracts could be diluted up to 1:30 or 1:150 (v/v) for detection with the short or long protocols, respectively. Additionally, mAb 8F4 was demonstrated to recognize AZA-2 and AZA-3 with cross-reactivities of 42 and 138 %, respectively. Presumably, this optimized assay will detect these analogs with similar cross-reactivity. The sensitivity of the microsphere-based assay for AZAs is enough to detect these compounds at the regulated levels in shellfish. This microsphere-based multi-detection method provides an easy-to-perform, highly sensitive and rapid method for the detection of AZAs. It could be included in a multi-detection method, which would allow time and sample volume saving due to the simultaneous detection of different toxin classes. The assay could be used in a single or multiplexed format as a semi-quantitative screening tool useful to reduce the number of analysis to be performed by confirmatory, quantitative methods.

Figure 1
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Acknowledgements

FEDER cofunded-grants:from CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad,AGL2012-40185-CO2-01,Consellería de Cultura, Educación e Ordenación Universitaria,GRC2013-016,Axencia Galega de Innovación, Spain,ITC-20133020SINTOX,IN852A 2013/16-3MYTIGAL.From CDTI under ISIP Programme, Spain,IDI-20130304 APTAFOOD.From EU’s 7th Framework Programme managed by Research Executive Agency(FP7/2007-2013)under grant agreement Nos. 265409µAQUA, 315285CIGUATOOLS,312184PHARMASEA

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Keywords: azaspiracids, Marine Toxins, microsphere-based array, flow-fluorimetry system, screening method

Conference: IMMR | International Meeting on Marine Research 2014, Peniche, Portugal, 10 Jul - 11 Jul, 2014.

Presentation Type: Oral Presentation

Topic: SEAFOOD TECHNOLOGY

Citation: Fraga Corral M, Vilariño N, Louzao M and Botana L (2014). Sensitivity improvement of an immuno-detection method for azaspiracids based on the use of microspheres coupled to a flow-fluorimetry system. Front. Mar. Sci. Conference Abstract: IMMR | International Meeting on Marine Research 2014. doi: 10.3389/conf.fmars.2014.02.00166

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Received: 12 May 2014; Published Online: 18 Jul 2014.

* Correspondence:
Miss. María Fraga Corral, University of Santiago de Compostela, Department of Pharmacology, Lugo, Lugo, 27002, Spain, maria.fraga@usc.es
Dr. Natalia Vilariño, University of Santiago de Compostela, Department of Pharmacology, Lugo, Lugo, 27002, Spain, natalia.vilarino@usc.es
Dr. M. Carmen Louzao, University of Santiago de Compostela, Department of Pharmacology, Lugo, Lugo, 27002, Spain, mcarmen.louzao@usc.es
Prof. Luis M. Botana, University of Santiago de Compostela, Department of Pharmacology, Lugo, Lugo, 27002, Spain, luis.botana@usc.es