Evaluation of different competitive immunoassays for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry

Abstract

Haptens (i.e. biomolecules which molecular weight is lower than 10 kDa) determination by inductively coupled plasma mass spectrometry (ICP-MS) is usually performed by means of competitive immunoassays. In these immunoassays, analyte quantification is indirectly carried out using two different tracer species (i.e. antibodies or antigen-protein conjugates). However, the benefits (and drawbacks) derived from using a given tracer species have not been systematically investigated so far. The goal of this work is to evaluate the influence of the tracer species employed in competitive immunoassays on the analytical figures of merit for aflatoxin M₁ (AFM₁) determination in milk samples. To this end, three different strategies have been developed and evaluated, namely: (i) antibody binding inhibition assay (ABIA); (ii) capture inhibition assay (CIA); and (iii) capture bridge inhibition assay (CBIA). Experimental results show that the use of the antibody as tracer species (as in the ABIA approach) affords better analytical figures of merit for AFM₁ determination than using the antigen-protein conjugate as the tracer one (as in the CIA and CBIA strategies). The limit of detection afforded by ABIA strategy (i.e. 0.1 ng kg^-1) for AFM₁ determination was 1000-fold and 50-fold lower regarding the CIA and CBIA strategies, respectively. In the case of the ABIA approach, the characteristics of the metal nanoparticle label employed to detect the tracer species is critical on the analytical figures of merit. However, when the hapten-protein conjugates are used as tracer species, immunocomplex formation is severely hampered by steric effects caused by the protein moiety and, consequently, the characteristics of the metal nanoparticle label is not critical in the immunoassay performance. The different immunoassay strategies were successfully validated for AFM₁ determination in milk samples using a certified reference material of whole milk powder (ERM-BD283) according to European Conformity guidelines for analytical methods of food contaminants and mycotoxins. Compared to ELISA, the immunoassay developed for AFM₁ determination in milk samples improve limits of detection up to 10-fold.

Keywords: Aflatoxin M(1); Competitive immunoassay; Hapten; Inductively coupled plasma mass spectrometry; Nanoparticle.