Immunorecognition magnetic supports for the development of an electrochemical immunoassay for azaspiracid detection in mussels

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

“…Differences between the two approaches were not significant (t=0.292, P=0.774). In comparison with the competitive colorimetric immunoassay previously reported by our group [25], where magnetic beads were used as antibody immobilisation supports, it was possible to use lower antiserum and tracer concentrations, which could explain the lower LODs achieved in these approaches.…”

“…Prior to the analysis of mussel samples, AZA-1 calibration curves using a blank certified reference mussel tissue matrix (CRM-Zero-Mus) were performed to evaluate matrix effects on the immunoassays. A matrix concentration of 20 mg/mL mussel matrix was chosen according to the protocol for lipophilic toxins extraction in shellfish (100 mg of matrix in 1 mL of MeOH) and its subsequent dilution to 20% v/v MeOH, a percentage that had been previously demonstrated not to interfere with the assay [25]. No significant differences were observed between the calibration curves performed in buffer and in 20 mg/mL mussel matrix with 20% v/v MeOH, neither in the protein G-based immunoassay (t=0.24, P=0.81) nor in the immunoassay based on the avidin-biotin interaction (t=0.32, P=0.77) (Fig.…”

“…These percentages higher than 100% are not surprising taking into account the different recognition principles between analytical methods. While NRC values are obtained by physicochemical approaches, the immunoapproaches provide responses based on the structural recognition of the toxins by the antibody, which can differ between analogues and the assay configuration [25]. Consequently, the application of cross-reactivity factors (CRFs) to each individual AZA analogue concentration can contribute to better understand the correlation between the different analytical methods.…”

“…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].…”

“…Bioaffinity immobilisation, mainly based on the avidin-biotin interaction and the affinity of protein A/G for immunoglobulins (IgGs), provides an attractive method for the controlled and stable surface-tethering of antibodies. In our previous work [25], the anti-AZA PAb was immobilised on protein G-coated magnetic beads and a competitive step using peroxidase-labelled AZA (AZA-HRP) was performed. The use of magnetic beads facilitated performance of the assay in suspension, thus allowing rapid assay kinetics, but mass transfer limitations were observed when the immunocomplexes were subsequently anchored on the electrode surface to perform electrochemical detection.…”