Determination of Microcystins in Fish Tissue by ELISA and MALDI-TOF MS Using a Highly Specific Single Domain Antibody

Natalia Badagian; Macarena Pírez Schirmer; Andrés Pérez Parada; Gualberto Gonzalez-Sapienza; Beatriz M Brena

doi:10.3390/toxins15020084

Determination of Microcystins in Fish Tissue by ELISA and MALDI-TOF MS Using a Highly Specific Single Domain Antibody

Toxins (Basel). 2023 Jan 17;15(2):84. doi: 10.3390/toxins15020084.

Authors

Natalia Badagian¹, Macarena Pírez Schirmer², Andrés Pérez Parada³, Gualberto Gonzalez-Sapienza², Beatriz M Brena¹

Affiliations

¹ Biochemistry Area, Department of Biosciences, Faculty of Chemistry, Universidad de la República, Av. Gral. Flores 2124, Montevideo 11800, Uruguay.
² Immunology Area, Department of Biosciences, Faculty of Chemistry, Universidad de la República, Av A. Navarro 3051, Montevideo 11600, Uruguay.
³ Technological Development Department, Centro Universitario Regional del Este, Universidad de la República, Ruta 9, Rocha 27000, Uruguay.

Abstract

The development of simple, reliable, and cost-effective methods is critically important to study the spatial and temporal variation of microcystins (MCs) in the food chain. Nanobodies (Nbs), antigen binding fragments from camelid antibodies, present valuable features for analytical applications. Their small antigen binding site offers a focused recognition of small analytes, reducing spurious cross-reactivity and matrix effects. A high affinity and broad cross-reactivity anti-MCs-Nb, from a llama antibody library, was validated in enzyme linked immunosorbent assay (ELISA), and bound to magnetic particles with an internal standard for pre-concentration in quantitative-matrix-assisted laser desorption ionization-time of flight mass spectrometry (Nb-QMALDI MS). Both methods are easy and fast; ELISA provides a global result, while Nb-QMALDI MS allows for the quantification of individual congeners and showed excellent performance in the fish muscle extracts. The ELISA assay range was 1.8-29 ng/g and for Nb-QMALDI, it was 0.29-29 ng/g fish ww. Fifty-five fish from a MC-containing dam were analyzed by both methods. The correlation ELISA/sum of the MC congeners by Nb-QMALDI-MS was very high (r Spearman = 0.9645, p < 0.0001). Using ROC curves, ELISA cut-off limits were defined to accurately predict the sum of MCs by Nb-QMALDI-MS (100% sensitivity; ≥89% specificity). Both methods were shown to be simple and efficient for screening MCs in fish muscle to prioritize samples for confirmatory methods.

Keywords: ELISA; MALDI-TOF; fish; microcystins; nanobody.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Enzyme-Linked Immunosorbent Assay
Microcystins* / analysis
Single-Domain Antibodies*
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods

Substances

Microcystins
Single-Domain Antibodies

Grants and funding

This research was funded by the following organisms: National Agency for Research and Innovation, Uruguay (ANII FMV 104085); Sectorial Commission of Scientific Research (CSIC Grupos Udelar 150725), Uruguay; Basic Sciences Development Program, PEDECIBA Química (Ministry of Education and Culture-Udelar, Uruguay). The APC was funded by Fundaquim MCs-Funds (B.M.B.; G.G.-S.). Natalia Badagian was supported by fellowships from ANII and Academic Commission of Posgraduates, UdelaR.