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1.
Toxins (Basel) ; 15(2)2023 01 17.
Article in English | MEDLINE | ID: mdl-36828400

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.


Subject(s)
Microcystins , Single-Domain Antibodies , Animals , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Microcystins/analysis , Enzyme-Linked Immunosorbent Assay
2.
Methods Mol Biol ; 2446: 531-546, 2022.
Article in English | MEDLINE | ID: mdl-35157292

ABSTRACT

Over the last two decades, the variable domains from heavy chain-only antibodies in camelids (nanobodies) have emerged as valuable immunoreagents for analytical and diagnostic applications. One prominent use of nanobodies is for the detection of small molecules due to their ease of production, resistance to solvents used in sample extraction, facile genetic manipulation, and small size. These last two properties make it possible to produce biotinylated nanobodies in vivo, which can be loaded in an orientated manner on magnetic beads covered with avidin, creating high-density immunoadsorbenpi twbch ""ts. The method described here details the use of nanobody-based adsorbents to concentrate small molecular weight analytes for subsequent quantitative analysis by MALDI-TOF mass spectrometry. Quantitation requires the inclusion of an internal standard (IS), a compound with properties similar to those of the analyte, enabling compensation for uneven distribution during crystallization of the MALDI-TOF matrix. Since nanobody generation against small compounds requires conjugation to carrier proteins, the same conjugation chemistry can be used to synthesize the IS. By design the IS cross reacts with the capture nanobody and can be preloaded in the immunoadsorbent, facilitating quantitative detection of the target compound.


Subject(s)
Single-Domain Antibodies , Immunoglobulin Heavy Chains , Immunomagnetic Separation , Magnetic Phenomena , Single-Domain Antibodies/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
3.
Anal Chem ; 91(15): 9925-9931, 2019 08 06.
Article in English | MEDLINE | ID: mdl-31291093

ABSTRACT

Here we present a new analytical method where immunoconcentration of the analyte is coupled to quantitative matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis allowing in minutes the identification and highly sensitive quantitation of microcystins (MCs) as model targets. The key element is a site-specific in vivo biotinylated nanobody of broad cross-reactivity with microcystins. The single biotin moiety at the C-terminus and the small size of the nanobody (15 kDa) enable its oriented and tightly packed immobilization on magnetic beads, providing a highly efficient capture of the toxin. The binding capacity of the bioadsorbent is partially loaded with an easily synthesized internal standard for MS quantitation. After capture, the beads are directly dispensed on the MALDI-TOF MS target enabling the identification and sensitive quantitation of the microcystin (MC) congeners. Since salts and contaminants are removed during the concentration step, no cleanup or other sample treatments are needed. The method was validated with a large number of water and serum samples with excellent precision and recovery at quantitation limits of 0.025 µg/L of MC.


Subject(s)
Microcystins/analysis , Single-Domain Antibodies/immunology , Animals , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Biotinylation , Cattle , Humans , Immunomagnetic Separation , Kinetics , Limit of Detection , Microcystins/blood , Microcystins/immunology , Microcystins/standards , Reference Standards , Single-Domain Antibodies/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/standards
4.
Sci Total Environ ; 697: 134210, 2019 Dec 20.
Article in English | MEDLINE | ID: mdl-32380631

ABSTRACT

Freshwater harmful algal blooms produce a broad array of bioactive compounds, with variable polarity. Acute exposure to cyanotoxins can impact the liver, nervous system, gastrointestinal tract, skin, and immune function. Increasing evidence suggests chronic effects from low-level exposures of cyanotoxins and other associated bioactive metabolites of cyanobacterial origin. These sundry compounds persist in drinking and recreational waters and challenge resource managers in detection and removal. A systematic approach to assess the developmental toxicity of cyanobacterial metabolite standards was employed utilizing a robust and high throughput developmental Danio rerio embryo platform that incorporated a neurobehavioral endpoint, photomotor response. Subsequently, we applied the platform to cyanobacterial bloom surface water samples taken from temperate recreational beaches and tropical lake subsistence drinking water sources as a model approach. Dechorionated Danio rerio embryos were statically immersed beginning at four to six hours post fertilization at environmentally relevant concentrations, and then assessed at 24 h and 5 days for morbidity, morphological changes, and photomotor response. At least one assessed endpoint deviated significantly for exposed embryos for 22 out of 25 metabolites examined. Notably, the alkaloid lyngbyatoxin-a resulted in profound, dose-dependent morbidity and mortality beginning at 5 µg/L. In addition, hydrophobic components of extracts from beach monitoring resulted in potent morbidity and mortality despite only trace cyanotoxins detected. The hydrophilic extracts with several order of magnitude higher concentrations of microcystins resulted in no morbidity or mortality. Developmental photomotor response was consistently altered in environmental bloom samples, independent of the presence or concentration of toxins detected in extracts. While limited with respect to more polar compounds, this novel screening approach complements specific fingerprinting of acutely toxic metabolites with robust assessment of developmental toxicity, critical for chronic exposure scenarios.


Subject(s)
Bacterial Toxins/toxicity , Cyanobacteria , Harmful Algal Bloom , Microcystins/toxicity , Zebrafish , Animals , Bathing Beaches , Embryo, Nonmammalian/physiopathology , Lakes , Risk Assessment , Uruguay
5.
Anal Chem ; 89(12): 6800-6806, 2017 06 20.
Article in English | MEDLINE | ID: mdl-28494149

ABSTRACT

Owing to their reproducibility, stability, and cost-effective production, the recombinant variable domains of heavy-chain-only antibodies (VHHs) are becoming a salient option as immunoassay reagents. Recently, there have been several reports describing their application to the detection of small molecules (haptens). However, lacking the heavy-light chain interface of conventional antibodies, VHHs are not particularly apt to bind small analytes and failures are not uncommon. Here we describe the construction of a VHH phage display library against the cyanobacterial hepatotoxin microcystin LR and its selection using competitive panning and two novel panning strategies. The outcome of each strategy was evaluated by a large-scale screening using in vivo biotinylated nanobodies. The three methods selected for different nonoverlapping subsets of VHHs, allowing one to optimize the immunodetection of the toxin. The best results were obtained by promoting the isolation of VHHs with the slowest koff (off-rate selection). Among these, the biotinylated nanobody A2.3 performed in ELISA with excellent recovery and high sensitivity, IC50 = 0.28 µg/L, with a limit of detection that is well below the most rigorous guidelines for the toxin. While it may be case-specific, these results highlight the importance of exploring different panning strategies to optimize the selection of antihapten nanobodies.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Microcystins/analysis , Single-Domain Antibodies/immunology , Amino Acid Sequence , Animals , Antigen-Antibody Reactions , Biotinylation , Camelids, New World , Haptens/immunology , Leukocytes, Mononuclear/metabolism , Limit of Detection , Marine Toxins , Microcystins/metabolism , Peptide Library , Sequence Alignment , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism
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