toxins Article
A Novel Sensitive Cell-Based Immunoenzymatic Assay for Palytoxin Quantitation in Mussels Marco Pelin 1 ID , Silvio Sosa 1 , Valentina Brovedani 1 , Laura Fusco 1 Aurelia Tubaro 1, * 1 2
*
ID
, Mark Poli 2 and
Department of Life Sciences, University of Trieste, 34127 Trieste, Italy;
[email protected] (M.P.);
[email protected] (S.S.);
[email protected] (V.B.);
[email protected] (L.F.) U.S. Army Medical Research Institute of Infectious Diseases, Ft. Detrick, MD 21701-5011, USA;
[email protected] Correspondence:
[email protected]; Tel.: +39-040-558-8835; Fax: +39-040-557-3215
Received: 26 July 2018; Accepted: 10 August 2018; Published: 14 August 2018
Abstract: The marine algal toxin palytoxin (PLTX) and its analogues are some of the most toxic marine compounds. Their accumulation in edible marine organisms and entrance into the food chain represent their main concerns for human health. Indeed, several fatal human poisonings attributed to these compounds have been recorded in tropical and subtropical areas. Due to the increasing occurrence of PLTX in temperate areas such as the Mediterranean Sea, the European Food Safety Authority (EFSA) has suggested a maximum limit of 30 µg PLTX/kg in shellfish meat, and has recommended the development of rapid, specific, and sensitive methods for detection and quantitation of PLTX in seafood. Thus, a novel, sensitive cell-based ELISA was developed and characterized for PLTX quantitation in mussels. The estimated limits of detection (LOD) and quantitation (LOQ) were 1.2 × 10−11 M (32.2 pg/mL) and 2.8 × 10−11 M (75.0 pg/mL), respectively, with good accuracy (bias = 2.5%) and repeatability (15% and 9% interday and intraday relative standard deviation of repeatability (RSDr), respectively). Minimal interference of 80% aqueous methanol extract allows PLTX quantitation in mussels at concentrations lower than the maximum limit suggested by EFSA, with an LOQ of 9.1 µg PLTX equivalent/kg mussel meat. Given its high sensitivity and specificity, the cell-based ELISA should be considered a suitable method for PLTX quantitation. Keywords: Palytoxin; mussels; cell-based ELISA Key Contribution: A novel cell-based immunoenzymatic assay (cell-based ELISA) for palytoxin (PLTX) quantitation was set up and characterized for its sensitivity, accuracy, reproducibility, and specificity to quantify PLTX in mussels.
1. Introduction Palytoxin (PLTX), a complex marine poly-ol toxin, is one of the most toxic natural compounds. The discovery of PLTX dates back to the 1960s, when, in a tide pool of Hana Bay (Maui Island, Hawaii), Prof. Paul Helfrich collected samples of a toxic soft coral, subsequently identified as Palythoa toxica. Ten years later, the chemical structure of PLTX isolated from this coral was reported [1]. Later, PLTX and a series of its analogues were also identified in other Zoantharia belonging to the genera Palythoa [2–7] and Zoanthus [8], in benthic dinoflagellates of the genus Ostreopsis [9–15], and in cyanobacteria of the genus Trichodesmium [16]. Only a few of these analogues have been studied from a biological and chemical point of view, including (i) 42-hydroxy-PLTX (42S-OH-50S-PLTX), isolated from P. toxica [2,17], and its stereoisomer (42S-OH-50R-PLTX), isolated from P. tuberculosa [3]; (ii) ostreocin-D (OST-D) and Toxins 2018, 10, 329; doi:10.3390/toxins10080329
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its analogues, produced by Ostreopsis siamensis [11,18–20]; and (iii) ovatoxin-a (OVTX-a), the most abundant PLTX analogue, produced by Ostreopsis cf. ovata in the Mediterranean Sea [21–23]. The main public health concern associated with these toxins is their presence in marine organisms and potential entrance into the human food chain. Indeed, PLTXs have been detected in porifera and polychaete worms as well as in other edible species, including crustaceans, mollusks (gastropods, bivalves, and cephalopods), and echinoderms (sea urchins, starfishes) [8,24,25]. Moreover, consumption of PLTX-contaminated fish or crabs has been associated with cases of fatal human poisoning in tropical and subtropical areas [26–29]. On the other hand, adverse effects in humans attributed to PLTX along the Mediterranean and Atlantic coasts of Portugal have been associated with inhalation and/or cutaneous exposure to marine aerosol and/or direct exposure to seawater during Ostreopsis blooms [30,31]. In particular, signs and symptoms in the respiratory tract, including dyspnea associated with fever >38 ◦ C, as well as conjunctivitis and dermatitis have been reported [13,14,24,27,30,32,33]. In these areas, Ostreopsis has been recorded since the early 1970s [34], and in the last decade PLTXs have been detected both in microalgae and in edible marine organisms, but no foodborne poisonings attributed to these toxins have yet been documented. Despite their high toxicity, PLTXs are not regulated as seafood or environmental contaminants. However, the European Food Safety Authority (EFSA) has suggested a maximum limit of 30 µg PLTX/kg of shellfish meat [35]. Moreover, given the significant concerns for public health due to the expanding distribution of PLTXs, EFSA has recommended the development of suitable methods to detect these toxins in seafood. In addition to liquid chromatography–mass spectrometry (LC-MS) based chemical methods [36,37], both structural and functional assays are currently available for PLTX. Among these are the hemolytic assay [38–42], the lactate dehydrogenase-based hemolytic biosensor [43], and methods based on PLTX binding to Na+ /K+ ATPase [44,45]. However, these methods suffer from insufficient sensitivity, significant matrix effects, low toxin recovery, and/or other limitations for routine use. Among the structural assays, sensitive, inexpensive, and easy-to-use immunoassays have been set up [46–49]. Recently, antibody-based biosensors have also been developed as innovative and highly sensitive analytical methods to detect and quantify PLTXs. In particular, a surface plasmon resonance (SPR) biosensor using a murine monoclonal anti-PLTX antibody was set up by Yakes et al. [50], while Zamolo et al. developed a sensitive electrochemiluminescence-based sensor combining the specificity provided by anti-PLTX antibodies and the electric conductivity of carbon nanotubes [51]. Recently, Fraga et al. set up a cytometry immunoassay based on the competitive binding of a monoclonal anti-PLTX antibody between PLTX immobilized on microspheres and PLTX in solution [52]. Another recently developed biosensor for PLTX detection is an immunoenzymatic assay based on biolayer interferometry coupled with a competitive binding assay through an enzyme-linked aptamer [53]. Recently, we demonstrated the ability of specific anti-PLTX antibodies to measure and characterize the binding of PLTX to cultured cells [54]. Using ouabain as a well-known antagonist of PLTX effects in vitro, this binding seems to occur on Na+ /K+ ATPase expressed on the cell surface. Given the high-affinity binding of PLTX to cells and the ability of a monoclonal anti-PLTX antibody to efficiently and simply quantify bound PLTX, a novel cell-based immunoenzymatic assay (cell-based ELISA) for PLTX quantitation was set up and characterized for its sensitivity, accuracy, reproducibility, and specificity. This novel method was further characterized for its suitability to quantify the toxin in mussels. 2. Results 2.1. Development and Optimization of the Cell-Based ELISA The cell-based ELISA was developed starting from the protocol for the characterization of PLTX binding to cultured cells reported by Pelin et al. [54]. The assay was then optimized through the following steps: (i) choosing the most sensitive cell line for PLTX binding, (ii) choosing the fixative
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solutions and temperature of cell incubation with the antibodies, (iii) choosing the sequence of fixation andand exposure to PLTX, (iv)(iv) choosing the cell fixation exposure to PLTX, choosing theblocking blockingagent, agent,and and(v) (v) choosing choosing the the primary antibody dilution. 2.1.1. PLTX PLTX Binding 2.1.1. Binding on on Different Different Cultured Cultured Cells Cells PLTX binding on cultured cells was evaluated using a panel of of different different cell cell lines. lines. Cells were ◦ C and the toxin binding was subsequently evaluated as described exposed to PLTX for 10 min at 37 °C in the Materials and Methods section. The obtained results for each cell model were normalized on curves of PLTX binding for each the protein content of each sample. Figure 1 shows the saturation curves cell model (panel A). From these curves, Kd values and maximal binding were calculated, and their −–10 10 M distribution was ofof 8.18.1 ××1010 was analyzed analyzed in inthe thebox boxplot plotof ofFigure Figure11(panels (panelsBBand andC). C).AAmedian medianKd Kd −10 9 M) and a median maximal binding of 0.015 (interquartile –9−M) (interquartile 2.4××1010 (interquartilerange range==2.2 2.2×× 10 10–10 toto2.4 and a median maximal binding of 0.015 (interquartile 0.0095 to to 0.02738) 0.02738) were were calculated. calculated. Binding Binding parameters parameters varied varied between between the the different different cell cell lines, lines, range == 0.0095 and HaCaT cells were the most sensitive cell line, as confirmed by the Kd values and maximal maximal binding −10 M and 0.043, respectively). On the contrary, the less sensitive cell models for PLTX (1.4 × 10–10 × 10 M and 0.043, respectively). On the contrary, the less sensitive cell models for PLTX binding −9 M; maximal binding = 0.009) and MCF-7 cells (Kd = not detectable; were HepG2 were HepG2 cells cells(Kd (Kd== 6.5 6.5× × 10–9 M; maximal binding = 0.009) and MCF-7 cells (Kd = not detectable; 0.003). For For these these reasons, reasons, the the HaCaT HaCaT cell cell line line was chosen as the most sensitive maximal binding == 0.003). model to set up the cell-based ELISA.
Figure 1. Palytoxin a Figure 1. Palytoxin(PLTX) (PLTX) binding binding evaluated evaluated on on aa panel panel of of different different cell cell lines, lines, detected detected by by a monoclonal mouse anti-PLTX antibody targeted by horseradish peroxidase (HRP)-conjugated antimonoclonal mouse anti-PLTX antibody targeted by horseradish peroxidase (HRP)-conjugated anti-mouse mouse immunoglobulin G. (A) Saturation ofbinding. PLTX binding. Boxshowing plots showing (B) distribution immunoglobulin G. (A) Saturation curves curves of PLTX Box plots (B) distribution of Kd of Kd values and (C) maximal bindings obtained by the binding assay forResults PLTX. are Results are expressed values and (C) maximal bindings obtained by the binding assay for PLTX. expressed as mean as mean ± SE experiments of three experiments performed in triplicate. ± SE of three performed in triplicate.
2.1.2. and Fixing Fixing Agents Agents 2.1.2. Incubation Incubation Temperature Temperature and To improve sensitivity, sensitivity, the the assay assay was was carried carried out out exposing exposing HaCaT HaCaT cells cells to to PLTX, PLTX, varying varying the the To improve fixing agents and incubation temperature (37–60 °C) with the primary and secondary antibodies. An ◦ fixing agents and incubation temperature (37–60 C) with the primary and secondary antibodies. increased signal (optical density, OD) waswas observed with increased incubation temperature up up to 50 An increased signal (optical density, OD) observed with increased incubation temperature to °C, which subsequently decreased at higher temperatures (Figure 2). This trend was recorded ◦ 50 C, which subsequently decreased at higher temperatures (Figure 2). This trend was recorded also also varying the thefollowing following cell fixing 4% paraformaldehyde (PFA), PFA and 1% varying cell fixing agents:agents: 4% paraformaldehyde (PFA), 4% PFA and4% 1% glutaraldehyde, glutaraldehyde, formalin neutral-buffered neutral-buffered formalin (NBF) (Figure 2).(NBF) (Figure 2). At the optimal temperature of 50 °C, the highest colorimetric reaction signal was recorded using 4% PFA as fixing agent, followed by NBF (significant differences starting from 1.1 × 10−9 M PLTX as compared to the data recorded using 4% PFA, p < 0.05) and 4% PFA + 1% glutaraldehyde (significant
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At the optimal temperature of 50 ◦ C, the highest colorimetric reaction signal was recorded using 4% PFA followed by NBF (significant differences starting from 1.1 × 10−9 M PLTX as Toxins 2018,as 10,fixing x FOR agent, PEER REVIEW 4 of 15 compared to the data recorded using 4% PFA, p < 0.05) and 4% PFA + 1% glutaraldehyde (significant 10 M PLTX as compared to the data recorded using 4% PFA, differencesstarting startingfrom from ×−1010 differences 1.21.2 × 10 M−PLTX as compared to the data recorded using 4% PFA, p < 0.01) ◦ C were chosen as the optimal fixing agent and incubation p < 0.01) (Figure 2D). Thus, 4% PFA and 50 (Figure 2D). Thus, 4% PFA and 50 °C were chosen as the optimal fixing agent and incubation temperaturewith withantibodies. antibodies. temperature
Figure of of thethe cell-based ELISA. Influence of temperature during cell incubation with Figure2.2.Optimization Optimization cell-based ELISA. Influence of temperature during cell incubation the on the signal, using (A) (A) 4% 4% paraformaldehyde (PFA), (B)(B) 4%4%PFA withantibodies the antibodies onassay the assay signal, using paraformaldehyde (PFA), PFA++1% 1% glutaraldehyde, glutaraldehyde,or or(C) (C)neutral-buffered neutral-bufferedformalin formalin(NBF) (NBF)as asfixative fixativesolutions. solutions.(D) (D)Influence Influenceof ofthe thethree three fixative with antibody antibody incubation incubationatat50 50◦°C. Eachpoint pointrepresents representsmean mean± fixativesolutions solutions on on the the assay assay signal signal with C. Each ±SE SEof ofthree threeexperiments. experiments. Statistical Statistical differences: differences: ** p < < 0.05; ** p < 0.01; *** p < 0.001 (two-way ANOVA 0.05; ** p < 0.01; *** p < 0.001 (two-way ANOVA and andBonferroni Bonferronipost posttest). test).
2.1.3. 2.1.3.Sequence Sequenceof ofCell CellFixation Fixationand andExposure Exposureto toPLTX PLTX The ofchanging changingthethe sequence of fixation cell fixation and exposure PLTX also The possibility possibility of sequence of cell and exposure to PLTXtowas also was evaluated: evaluated: the cell-based ELISA was carried out fixing HaCaT cells with 4% PFA for 30 min before the cell-based ELISA was carried out fixing HaCaT cells with 4% PFA for 30 min before exposure to exposure PLTX orthe exposing the toxin beforeasfixation, as described in the Materials and PLTX or to exposing cells to the thecells toxintobefore fixation, described in the Materials and Methods Methods section.3A Figure shows the concentration-dependent PLTX detection recorded section. Figure shows3A the concentration-dependent curve for curve PLTX for detection recorded in the two in the two conditions; the optimal condition consists in cell exposure to PLTX before fixation, since conditions; the optimal condition consists in cell exposure to PLTX before fixation, since the inverted the inverted sequence dramatically decreased the as OD values, as expected. sequence dramatically decreased the OD values, expected. 2.1.4. 2.1.4.Blocking BlockingAgent Agent The of different differentblocking blockingagents agents assay signal also evaluated. 3B The influence influence of onon thethe assay signal was was also evaluated. FigureFigure 3B shows shows PLTX calibration curve using obtained three blocking agents. signal The highest signal using was a PLTXacalibration curve obtained threeusing blocking agents. The highest was recorded recorded using a Tris-borate buffercontaining (TBB) solution containing serum (HS) blocking a Tris-borate buffer (TBB) solution 10% horse serum 10% (HS) horse as a blocking agent.asAasignificant agent. A significant decrease in signal wasfrom recorded 1.2using × 10−10 M PLTX using a Trisdecrease in signal was recorded starting 1.2 × starting 10−10 Mfrom PLTX a Tris-buffer saline (TBS) buffer saline (TBS) solution containing 0.2% Tween 20 and 1% or 2% dried milk powder. solution containing 0.2% Tween 20 and 1% or 2% dried milk powder. 2.1.5. Primary Antibody Dilution To further increase the signal/background ratio, the influence of different dilutions of the primary antibody on the assay signal was evaluated. As shown in Figure 3C, no significant differences were observed among three primary antibody dilutions tested (1:750, 1:1500, and 1:3000).
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2.1.5. Primary Antibody Dilution To further increase the signal/background ratio, the influence of different dilutions of the primary Toxins 2018, 10, x FOR PEER REVIEW 5 of 15 antibody on the assay signal was evaluated. As shown in Figure 3C, no significant differences were observed among three primary antibody dilutions tested (1:750, 1:1500, and 1:3000). Thus, the Thus, the cell-based ELISA was subsequently carried out using the highest dilution (1:3000; 0.5 µg/mL cell-based ELISA was subsequently carried out using the highest dilution (1:3000; 0.5 µg/mL final final concentration) of the primary antibody. concentration) of the primary antibody.
Figure 3. Optimization ELISA. (A)(A) Temporal change between the the fixation phase (4% Figure Optimizationofofthe thecell-based cell-based ELISA. Temporal change between fixation phase −8 M); (B)−8influence of three blocking on PFA)PFA) and and cell treatment withwith PLTXPLTX (4.1 ×(4.1 10−11 (4% cell treatment ×–1.0 10−×1110 –1.0 × 10 M); (B) influence of threeagents blocking agents on signal; the assay (C) of influence primarydilution antibody onsignal. the assay signal. point the assay (C)signal; influence primaryofantibody ondilution the assay Each pointEach represents represents ± experiments. SE of three experiments. Statistical ** differences: 0.01;(two-way *** p < 0.001 (two-way mean ± SE mean of three Statistical differences: p < 0.01; *****pp
