Highly Sensitive and Practical Fluorescent Sandwich ELISA for

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Highly Sensitive and Practical Fluorescent Sandwich ELISA for Ciguatoxins Takeshi Tsumuraya, Takeshi Sato, Masahiro Hirama, and Ikuo Fujii Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00519 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Analytical Chemistry

Highly Sensitive and Practical Fluorescent Sandwich ELISA for Ciguatoxins

Takeshi Tsumuraya,*,1 Takeshi Sato,2 Masahiro Hirama,1 and Ikuo Fujii1 1

Department of Biological Science, Graduate School of Science,

Osaka Prefecture University, Osaka 599-8570, Japan 2

Cell Science Inc., Aoba-ku, Sendai, Miyagi 989-3212, Japan

Corresponding Author *E-mail: [email protected]

Phone: +81-72-254-9835

Abstract Ciguatera fish poisoning (CFP) caused by the consumption of fish that have accumulated ciguatoxins (CTXs) affects more than 50,000 people annually. The spread of CFP causes enormous damage to public health, fishery resources, and the economies of tropical and subtropical endemic regions. The difficulty in avoiding CFP arises from the lack of sensitive and reliable analytical methods for the detection and quantification of CTXs in contaminated fish, along with the normal appearance, smell, and taste of fish contaminated with the causative toxins. Thus, an accurate, sensitive, routine, and

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portable detection method for CTXs is urgently required. We have successfully developed a highly sensitive fluorescent sandwich ELISA, which can detect, differentiate,

and

quantify

four

major

CTX

congeners

(CTX1B,

CTX3C,

51-hydroxyCTX3C, and 54-deoxyCTX1B) with a detection limit of less than 1 pg/mL. The ELISA protocol, using one microtiter plate coated with two mAbs (10C9 and 3G8), and ALP-linked 8H4, can detect any of the four CTX congeners in a single operation. CTX1B spiked into fish at the FDA guidance level of 0.01ppb CTX1B equivalent toxicity in fish from Pacific regions was also proven to be reliably detected by this ELISA. Furthermore, the efficiency of extraction/purification procedures and the matrix effect of contaminants in fish were evaluated in detail, since pretreatment and matrix effects are critical for ELISA analysis.


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Introduction Ciguatera fish poisoning (CFP) is one of the most common food-borne illnesses caused by the consumption of a variety of fish that have accumulated ciguatoxins (CTXs) through the food chain. CTXs originate in dinoflagellates of the genus Gambierdiscus.1,2 Human symptoms of CFP include severe gastrointestinal, cardiovascular, and neurological disorders, which may last for months or even years. More than 50,000 people worldwide are estimated to suffer annually from CFP, making it one of the most common types of nonbacterial food poisoning.3-5 The spread of CFP causes increasingly serious damage to public health, fishery resources, and the economies of tropical and subtropical endemic regions.6 Globalization of trade, in addition to climate change, might contribute to the spread of CFP even in temperate regions. The difficulty in avoiding CFP arises from the lack of a sensitive and reliable analytical method for detecting causative CTXs, together with the normal appearance, smell, and taste of fish contaminated with CTXs. The presence of CTXs can only be determined by analytical methods. CTXs are structurally classified as 3 nm long ladder-like polycyclic ethers.1,2,7 CTX1B and its congeners (Figure 1) are highly toxic to



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mammals, and their lethal doses by intraperitoneal injection into mice [medium lethal dose (LD50) 0.15–4 µg/kg] are much lower than those of brevetoxins, which are structurally related red-tide toxins (LD50 > 100 µg/kg), and tetrodotoxin (LD50 ~10 µg/kg), the puffer fish toxin.8-13 In addition to the traditional mouse bioassay using fish extracts, several other methods have been developed recently to detect CTXs in contaminated fish, including a neuroblastoma cell-based assay,14 radio and fluorescent receptor binding assays,12,15,16 and HPLC,17 MS18-20 and LC/MS/MS21-28 assays. However, no rapid and reliable methods were available for detecting CTXs at fisheries and even at inspection sectors for seafood. We believed that antibody-based immunoassays would likely provide the best method for accurate, sensitive, routine, and portable detection. We therefore recently prepared four specific monoclonal antibodies (mAbs) (10C9, 3D11, 8H4, and 3G8; Figure S1, Supporting Information) against either wing of several CTXs by immunizing mice with keyhole limpet hemocyanin (KLH) conjugates of rationally designed synthetic haptens in place of highly toxic natural CTXs.29-36 Our results showed that the haptenic groups require a surface area larger than 400 Å2 to induce mAbs specific for CTXs. In addition, we also established a sandwich


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ELISA utilizing two different monoclonal antibodies that bind specifically to either of the two wings of CTXs: a monoclonal antibody for the left wing of CTXs was adsorbed on a plate as a capture antibody and a horseradish peroxidase (HRP)-conjugated monoclonal antibody for the right wing was used as a detection antibody. This analytical method allowed specific detection of the four principal congeners of CTXs (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX1B), which were originally isolated in the Pacific region, characterized by Yasumoto et al.,8, 9, 11, 37, 38 and then detected also in the Atlantic,24,25 without cross-reactivity with other related marine toxins. For example, CTX1B was detected specifically at less than 0.28 ng/mL using this methodology.35 Whereas regulatory limits for CTXs in fish have not yet been issued by official organizations such as the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) of the United Nations, a guidance level of 0.01 ppb CTX1B equivalent toxicity in fish from Pacific regions was issued by the United States Food and Drug Administration (FDA).5,39 Consequently, the sensitivity of the ELISA is to be warranted to detect CTX1B at 0.01 ppb in contaminated fish. This toxicity equivalent in CTX1B is the composite toxicity of the contaminated fish in which



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several congeners could be present. In this paper, we describe an accurate and highly sensitive sandwich ELISA protocol with a detection limit of less than 1 pg/mL using alkaline phosphatase (ALP) and a fluorescent substrate system. This method can detect any congener of the four CTXs shown in Figure 6 in a single assay using a plate onto which a mixture of the mAbs 10C9 and 3G8 is absorbed, and ALP-linked 8H4 used for fluorescent detection. CTX1B spiked into the fish Variola louti at the FDA guidance level was confirmed to be reliably detected. Furthermore, since pretreatment such as extraction/purification procedures is critical for ELISA analysis, the efficiency of these procedures and the matrix effect of contaminants were evaluated in detail.

Experimental Section General Methods.

All organic solvents used in this study were analytical grade or

higher (Nacalai Tesque, Inc., Kyoto, Japan). Water used in the experiments was Milli-Q Ultra-pure grade with 18.2 MΩ resistivity. The CTX standards (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX3C) were synthesized by Hirama and co-workers


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at Tohoku University.40-43 The buffer solutions used in sandwich ELISA were prepared as follows. Blocking buffer was prepared by dissolving 0.63 g of potassium phosphate monobasic, 5.53 g of sodium phosphate dibasic dodecahydrate, 8.77 g of sodium chloride, 100 g of sucrose, 200 mL of Blocking Reagent-N101 (NOF Corporation, Tokyo, Japan), 0.5 mL of Tween 20 (Nacalai Tesque, Inc.), and 1 mL of Proclin 300 (Sigma-Aldrich, Co., St. Louis, Missouri) in 600 mL of water and the volume was adjusted to 1 L by the addition of water. ELISA plate washing buffer concentrate (x20) was prepared by dissolving 48.5 g of tris(hydroxymethyl)aminomethane in 800 mL of water and adjusting the pH to 7.5 by the addition of hydrochloric acid. Then, to this solution, 175.3 g of sodium chloride, 11.05 g of Tween 20, and 0.5 mL of Proclin 300 were added and the volume was adjusted to 1 L by the addition of water. The ELISA plate washing buffer concentrate (x20) was diluted 20 times by adding water before use. Sample dilution buffer (D1) was prepared by the addition of 200 mL of Blocking Reagent-N102 (NOF Corporation) and 100 mL of dimethyl sulfoxide (Nacalai Tesque, Inc.) to 700 mL of Tris-buffered saline (TBS). Enzyme-conjugated antibody dilution buffer (D2) was prepared by the addition of 200 mL of Blocking Reagent-N102 and 95



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mg of magnesium chloride to 800 mL of TBS.

Detection of CTXs by Fluorescent Sandwich ELISA.

To detect CTX1B, 100 µg of

mAb 8H4 was conjugated with ALP using an AP-Antibody All-in-One Conjugation Kit (Solulink, Inc., San Diego, California), following the manufacturer’s instructions. Each well of a 96-well ELISA plate (F96 MAXISORP NUNC-IMMUNO. PLATE, Thermo Fisher Scientific, Waltham, Massachusetts) was coated with 100 µL of mAb 3G8 (10 µg/mL) in PBS overnight at 4 °C. After the mAb solution was decanted, the blocking buffer (400 mL/well) was added to each well, incubated for 1hr at room temperature, removed, and the plate was dried by centrifuge at 1500 rpm for 20 sec.

This

mAb-coated plate can be used without deterioration in the activity at least for 6 months, when the plate was kept in an aluminum pouch without air at 4 °C. After 100 µL solutions of CTX1B serially diluted with D1 buffer were added to the plate and incubated for 1 h at room temperature, the supernatant was removed from each well and the plate was washed three times with the washing buffer (400 µL/well). Each well was incubated for 1 h with a solution (100 µL) of ALP-linked 8H4 (2 µg/mL) dissolved in


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D2 buffer. After washing three times with the washing buffer (400 µL/well), each well was treated with 100 µL of a solution of AttoPhos® AP Fluorescent Substrate System (Promega, Madison, Wisconsin). The plates were left undisturbed for 15–30 min and then fluorescence intensities (excitation, λ = 435 nm; emission, λ = 555 nm) were measured using a microtiter plate reader (Varioskan Flash, Thermo Fischer Scientific, Waltham, Massachusetts).

To detect 54-deoxyCTX1B, mAb 3G8 was coated on the ELISA plate and ALP-conjugated 3D11 was used for detection in place of 8H4. To detect CTX3C, mAb 10C9 was coated on the ELISA plate in place of 3G8, and ALP-conjugated 3D11 was used for detection. To detect 51-hydroxyCTX3C, mAb 10C9 was coated on the ELISA plate, and ALP-conjugated 8H4 was used for detection. For the detection of CTX3C and 54-deoxyCTX1B, a solution (Solution 2) of Can Get Signal® Immunoreaction Enhancer Solution (Toyobo Life Science, Osaka, Japan) was used in place of D1 buffer.

Cross-reactivity of the Fluorescent Sandwich ELISA.

Other marine toxins, such



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as brevetoxin A, brevetoxin B, maitotoxin and okadaic acid, in addition to the four CTX congeners, were also analyzed by the present fluorescent sandwich ELISA following the procedures described above.

Detection of Four CTX Congeners by a Single ELISA Protocol.

Each well of a

96-well ELISA plate (NUNC MAXISORP) was coated with 100 µL of a mixture of mAbs 3G8 (10 µg/mL) and 10C9 (10 µg/mL) in PBS overnight at 4 °C. After the mAb solution wad decanted, the blocking buffer (400 mL/well) was added to each well and incubated for 1 hr at room temperature.

The blocking buffer was removed and the

plate was dried by centrifuge at 1500 rpm for 20 sec, and serially diluted solutions (100 µL) of CTX1B, CTX3C, 51-hydroxyCTX3C, or 54-deoxyCTX3C in D1 buffer were added to the ELISA plate and incubated for 1 h at room temperature. Then, the supernatant was removed from each well and the plate was washed three times with washing buffer (400 µL/well). Each well was incubated for 1 h with a solution (100 µL) of ALP-conjugated 8H4 (2 µg/mL) dissolved in D2 buffer. After washing three times with washing buffer (400 µL/well), each well was treated with 100 µL of a solution of


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AttoPhos® AP Fluorescent Substrate System (Promega). The plate was left undisturbed for 15–30 min, then fluorescence intensity (excitation, λ = 435 nm; emission, λ = 555 nm) was measured using a microtiter plate reader (Varioskan Flash, Thermo Fischer Scientific).

Detection of CTX1B Spiked into Fish Flesh.

Flesh of fish (Variola louti), which

was caught in Okinawa and claimed to be free of CTXs using LC-MS/MS analysis,22 was spiked with CTX1B (0.01 ppb) to confirm that the ELISA analysis is applicable for the detection of CTX1B at the FDA guidance level. The flesh (5 g) was spiked with a solution (0.5 mL) of 50 pg of pure CTX1B in PBS/dimethyl sulfoxide (9/1). The flesh sample was then homogenized in 15 mL of acetone and centrifuged at 3000 rpm for 10 min. The acetone solution was decanted. The flesh residue was extracted with acetone as above one more time, then the combined acetone solution was concentrated to provide an aqueous residue, which was extracted twice with 5 mL of diethyl ether. The combined ether solution was evaporated and the residue was dissolved in 1.5 mL of methanol/water (9/1), then the aqueous methanol solution was defatted twice with 3 mL



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of hexane and evaporated under reduced pressure. The residue was dissolved in 7 mL of ethyl acetate/methanol (9/1) and passed through a Florisil cartridge (500 mg/6 mL, Solid Phase Extraction Cartridges InterSep® FL-PR, GL Sciences Inc., Tokyo, Japan). The cartridge was eluted with 7 mL of ethyl acetate/methanol (9/1) and the combined eluate was evaporated under reduced pressure. The residue was further dissolved in 3 mL of acetonitrile and applied to a PSA cartridge (200 mg/3 mL, Solid Phase Extraction Cartridges InterSep® PSA, GL Sciences Inc.). The cartridge was eluted with 3 mL of methanol and the eluate was concentrated under reduced pressure. The residue was dissolved in 1 mL of D1 buffer and 100 µL of the solution was diluted with 900 µL of D1 buffer. Both 100 µL of the original and the diluted solutions were analyzed as described above on a 3G8-coated ELISA plate.

Evaluation of the Matrix Effect at Each Step of the Extraction/Purification Procedure.

To evaluate the matrix effect at each step of extraction/purification, we

spiked pure CTX1B into fish (Variola louti) flesh at the FDA guidance level, and each extract was analyzed by the new ELISA protocol. The fish flesh (10 g) was spiked with


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a solution (1 mL) of 100 pg of pure CTX1B in PBS/dimethyl sulfoxide (9/1). The flesh sample was extracted twice by homogenizing in 30 mL of acetone and centrifuging at 3000 rpm for 10 min. One fourth of the combined acetone solution was evaporated, and the residue was dissolved in 1 mL of D1 buffer (sample A). The rest of the acetone solution was concentrated to provide an aqueous residue, which was extracted twice with 7.5 mL of diethyl ether. One third of the combined ether solution was evaporated, and the residue was dissolved in 1 mL of D1 buffer (sample B). The rest of the ether solution was evaporated, and the residue was dissolved in 1.5 mL of methanol/water (9/1) and then defatted twice with 3 mL of hexane. Half of the aqueous methanol solution was evaporated under reduced pressure, and the residue was dissolved in 1 mL of D1 buffer (sample C). The other half of the aqueous methanol solution was concentrated, and the residue was dissolved in 7 mL of ethyl acetate/methanol (9/1). This latter solution was passed through a Florisil cartridge (500 mg/6 mg, Solid Phase Extraction Cartridges InterSep® FL-PR, GL Sciences Inc.). The cartridge was eluted with 7 mL of ethylacetate/methanol (9/1) and the combined eluate was evaporated under reduced pressure. The residue was then dissolved in 3 mL of acetonitrile and applied to



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a PSA cartridge (200 mg/3 mg, Solid Phase Extraction Cartridges InterSep® PSA, GL Sciences Inc.). The cartridge was eluted with 3 mL of methanol and the eluate was concentrated under reduced pressure. The residue was dissolved in 1 mL of D1 buffer (sample D). All sample solutions (samples A–D: 100 µL) were analyzed by the ELISA as described above.

Direct Evaluation of the Matrix Effect of Each Extract.

To evaluate the matrix

effect more directly, pure CTX1B was added at the FDA guidance level to each residue obtained by the above extraction/purification procedures and analyzed by the fluorescent sandwich ELISA. Variola louti flesh (10 g) was extracted twice by homogenizing in 30 mL of acetone. The mixture was centrifuged at 3000 rpm for 10 min and the acetone solution was separated from the flesh residue. One fourth of the acetone solution was concentrated (sample E). The remainder of the acetone solution was concentrated and the residue was extracted twice with 7.5 mL of diethyl ether. One third of the ether solution was concentrated (sample F) and the rest of the ether solution was evaporated. The residue was dissolved in 1.5 mL of methanol/water (9/1) and


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defatted twice with 3 mL of hexane. Half of the aqueous methanol solution was evaporated under reduced pressure (sample G), the rest of the aqueous methanol solution

was

concentrated

and

the

residue

was

dissolved

in

7

mL of

ethylacetate/methanol (9/1) and passed through a Florisil cartridge (500 mg/6 mL, Solid Phase Extraction Cartridges InterSep® FL-PR, GL Sciences Inc.). The cartridge was eluted with 7 mL of ethyl acetate/methanol (9/1) and the combined eluate was evaporated under reduced pressure. The residue was dissolved in 3 mL of acetonitrile and applied to a PSA cartridge (200 mg/3 mL, Solid Phase Extraction Cartridges InterSep® PSA, GL Sciences Inc.). The cartridge was eluted with 3 mL of methanol and the eluate was concentrated under reduced pressure (sample H). To each sample solution (samples E–H) was added a 1 mL solution of 25 pg of pure CTX1B in D1 buffer and then 100 µL of each solution was analyzed by the ELISA.

Results and Discussion Highly Sensitive Fluorescent Sandwich ELISA of CTXs. We used four available mAbs that bind to CTXs with high affinity (Figure S1,



Supporting

Information):

10C9

binds

to

the

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left

wing

of

CTX3C

and

51-hydroxyCTX3C,32 3G8 binds to the left wing of CTX1B and 54-deoxyCTX1B,35 3D11 binds to the right wing of CTX3C and 54-deoxyCTX1B,32 and 8H4 binds to the right wing of CTX1B and 51-hydroxyCTX3C.33 Using these mAbs, we previously developed a direct sandwich ELISA for specific detection of the four major congeners of CTXs (Figure 2(A)). The wells of a microtiter plate were directly coated with mAb (10C9 or 3G8) for the left wing and mAb (3D11 or 8H4) for the right wing was conjugated with horseradish peroxidase (HRP). This conventional sandwich ELISA protocol using o-phenylenediamine (OPD) as a colorimetric substrate detected CTXs in a dose-dependent manner. For example, CTX1B was detected at less than 0.28 ng/mL using mAb 3G8 and HRP-conjugated 8H4.35 This sensitivity is insufficient for detecting CTX1B at the FDA guidance level in fish (0.01 ppb of CTX1B equivalent).5,39 We therefore embarked on improving the sensitivity of our sandwich ELISA protocol. We first attempted a luminescent sandwich ELISA system using ALP as an enzyme and disodium

3-(4-methoxyspiro{1,2-dioxetane-3,2’-(5’-chloro)tricyclo[3,3,1,13,7]decan}-

4-yl)phenyl phosphate (CSPD®) as a luminescent substrate. However, the background


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was high and the luminescent signal was unstable, giving unsatisfactory results. After considerable experimentation, we found that a combination of ALP as an enzyme and 2’-(2-benzothiazoyl)-6’-hyrodxybenzothiazole phosphate (BBTP) as a fluorescent substrate is optimal (Figure 2(B)): the BBT anion, which is the product of the reaction of BBTP with ALP, has an unusually large Stokes’ shift leading to lower levels of background fluorescence and thus higher detection sensitivity. The present new ELISA protocol can detect and quantify CTXs with exceptionally high sensitivity, as described below. The wells of a microtiter plate were directly coated with mAb 3G8 and mAb 8H4 was conjugated with ALP using catalytic HydraLink® chemistry.44 Following the sandwich ELISA protocol with BBTP as a fluorescent substrate, CTX1B was detected in a dose-dependent manner (Figure 3). The detection sensitivity of the sandwich ELISA was significantly improved using ALP and BBTP and the detection limit was demonstrated to be 0.16 pg/mL (quantification limit: 0.49 pg/mL). Similarly, the fluorescent sandwich ELISA protocol using a combination of 10C9 coated on a microtiter plate, together with ALP-linked 8H4 and BBTP, allowed the detection of



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51-hydroxyCTX3C with a detection limit of 0.1 pg/mL (quantification limit: 0.3 pg/mL, Figure 4(A)). However, fluorescent sandwich ELISA using mAb 3D11 for the right wing led to unsatisfactory results: ELISA with a combination of 10C9 for the left wing and ALP-linked 3D11 gave only a poor detection limit (ca. 2 pg/mL) of CTX3C, and the background was high, possibly due to the relatively low affinity (KD = 122 nM) of 3D11 for CTX3C.32 Thus for improving detection, we added Can Get Signal® Immunoreaction Enhancer Solution to the fluorescent sandwich ELISA reaction system. This additive was found to significantly improve the detection limit (0.090 pg/mL, quantification limit: 0.27 pg/mL, Figure 4(B)) as well as the background. Similarly, the addition of Can Get Signal® Immunoreaction Enhancer Solution to the ELISA using a combination of 3G8 coated on a microtiter plate and ALP-linked 3D11 significantly improved the detection of 54-deoxyCTX1B, with a detection limit of 0.11 pg/mL (quantification limit: 0.32 pg/mL, Figure 4(C)). In contrast, this additive did not improve the detection limits in fluorescent ELISAs using the combination of 3G8 and ALP-linked 8H4 for CTX1B and the combination of 10C9 and ALP-linked 8H4 for 51-hydroxyCTX3C.


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Cross-reactivity and Specificity of the Fluorescent Sandwich ELISA. The cross-reactivities of the newly developed fluorescent sandwich ELISA protocols were investigated against other structurally related marine toxins such as brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin, as shown in Figure S2, Supporting Information). There was no cross-reactivity observed with these marine toxins. Next, the specificities of the ELISA protocols against the other CTX congeners were examined (Figure 5). Apparently, each ELISA protocol strictly discriminated between CTXs with and without the A-ring dihydroxybutenyl substituent, namely, between the CTX1B series and the CTX3C series. Thus, the capture antibody 3G8 specifically recognized the presence of the A ring dihydroxybutenyl substituent (CTX1B and 54-deoxyCTX1B in Figure 5(A) and 5(D)) and mAb 10C9 strictly recognized the A ring without the dihydroxybutenyl substituent (CTX3C and 51-hydroxyCTX3C in Figure 5(B) and 5(C)). More interestingly, ALP-linked 8H4 appears to be tolerant to the absence of the



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M-ring hydroxy group (CTX1B vs. 54-deoxyCTX1B in Figure 5(A); 51-hydroxyCTX3C vs. CTX3C in Figure 5(C)), whereas free 8H4 in a homogeneous aqueous solution showed more strict discrimination between 51-hydroxyCTX3C (Kd = 13.8 nM) and CTX3C (Kd = 3.2 µM).32-36 On the other hand, ALP-linked 3D11 still showed some discrimination, as shown in Figures 5(B) and 5(D). These results suggested that ALP-linked 8H4, rather than ALP-linked 3D11, should be useful for a sandwich ELISA designed to recognize the right half of CTXs irrespective of the presence of the M-ring hydroxy group (vide infra).

Detection of Four CTX Congeners by a Single Sandwich ELISA Protocol. For practical purposes, detection of CTX irrespective of congener is the most important. We therefore developed a more convenient ELISA protocol to detect any of the four major CTX congeners (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX3C) by a single operation using one microtiter plate. The wells of the plate were coated with a mixture of 10C9 and 3G8, and ALP-linked 8H4 was used as the detection antibody. Following the fluorescent sandwich ELISA protocol using BBTP


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as a fluorescent substrate, CTX1B was detected effectively in a dose-dependent manner (Figure 6). The other three CTX congeners were also detected similarly, and it is noteworthy that these congeners provided very similar fluorescence intensity curves as the CTX concentration changed (Figure 6). These results indicated that this ELISA protocol can detect any of the four CTXs in a single operation.

Detection of CTX1B Spiked into Fish Flesh. Fish flesh (Variola louti) spiked with 0.01 ppb CTX1B was extracted and analyzed following the new ELISA protocol. The concentrations of CTX1B in the final extract and in the 10-fold dilution of the final extract were 17 pg/mL and 20 pg/mL, respectively, after correction by the dilution factor. These results indicated that 34% and 40% of the originally spiked CTX1B were recovered. Thus, CTX1B spiked in fish flesh at the FDA guidance level was reliably detected by the present sandwich ELISA after extraction using a conventional procedure. To simplify the extraction procedure, we next clarified the recovery of CTX1B, step by step at each extraction/purification step, since contaminants such as lipids,



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proteins, and/or blood in fish flesh may affect the present fluorescent sandwich ELISA through their matrix effects. Variola louti flesh (10 g) was spiked with 100 pg of CTX1B, which corresponds to the FDA guidance level of 0.01 ppb (= 10 ng/kg). Extracts at different purification step was collected for analysis by ELISA. First, one fourth of the acetone solution (corresponding to 2.5 g of the flesh) was removed, evaporated, and dissolved in 1 mL of the buffer (sample A). Second, one third of the ether solution was treated as above (sample B), and third, half of the aqueous methanol solution was removed, evaporated, defatted with hexane, and dissolved in 1 mL of the buffer (sample C). Finally, the residue eluted by passing MeOH through the PSA cartridge was dissolved in 1 mL of the buffer (sample D). These samples were analyzed by the sandwich ELISA. The concentrations were 2.3 pg/mL (9% recovery of CTX1B spiked) for sample A, 2.5 pg/mL (10%) for sample B, 2.7 pg/mL (11%) for sample C, and 8.0 pg/mL (32%) for sample D after correction for the dilution factor. In the earlier stages of the extraction procedure, the detected concentrations of CTX1B were significantly reduced to about 10% of the originally spiked concentration, possibly due to the matrix effects of contaminants.


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Furthermore, to confirm the matrix effect of the contaminants on the ELISA more directly, we prepared extracts of Variola louti flesh (10 g) following the above procedure without adding CTX1B, and then a solution of 25 pg of pure CTX1B in D1 buffer was added afterward to each extract, before conducting the ELISA. The acetone extract dissolved in 1 mL of D1 buffer (sample E), the ether extract dissolved in 1 mL of D1 buffer (sample F), the aqueous methanol extract (after washing with hexane) dissolved in 1 mL of D1 buffer (sample G), and the residue of PSA-eluate (MeOH) dissolved in 1 mL of D1 buffer (sample H) were analyzed by the fluorescent sandwich ELISA. The concentrations of CTX1B were 3.2 pg/mL (sample E), 5.3 pg/mL (sample F), 7.7 pg/mL (sample G), and 25 pg/mL (sample H). Thus, 13%, 21%, 31%, and 100% of the added CTX1B was detected for samples E, F, G, and H, respectively. These results clearly demonstrated significant matrix effects of contaminants in fish flesh in samples E, F, and G (in decreasing order). However, since the present fluorescent sandwich ELISA is highly sensitive, simple extraction by homogenizing in acetone, followed by centrifugation, is useful as a pretreatment procedure for the detection of fish contaminated with CTX1B at the FDA guidance level.



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We should remark that the present method is superior in terms of LOD, sensitivity, specificity, and sample-to-answer time, compared with other established methods (See Table S1, Supporting Information).

Conclusions We have successfully developed an accurate and highly sensitive fluorescent sandwich ELISA for CTX congeners which shows no cross-reactivity with related marine toxins. This ELISA can detect and differentiate four major CTX congeners (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX1B) with a detection limit (LOD) of less than 1 pg/mL. The sensitivity of this ELISA is sufficiently high to detect fish contaminated with CTX1B at the FDA guidance level (0.01 ppb) following a conventional extraction procedure. Furthermore, the proposed single ELISA protocol, using one microtiter plate coated with a mixture of 10C9 and 3G8, and ALP-linked 8H4, can detect any of the four CTXs. We demonstrated that simple extraction in acetone by homogenization, followed by centrifugation without any conventional, tedious, successive extraction/purification procedure, is satisfactory for detecting CTX1B at the


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FDA guidance level in fish using this ELISA. It should be noted that this method will be useful not only for the prevention of CFP, but also for epidemiological and physiological studies. We are currently developing the present ELISA protocol into portable friendly ELISA kits.

Associated Content Supporting Information. The supporting Information is available free of charge on the ACS Publication website. Synthetic haptens conjugated with keyhole limpet hemocyanin (KLH) and anti-CTX mAbs produced by immunizing mice with the hapten-KLH conjugates (synthetic antigens) and combinations of mAbs used for the fluorescent sandwich ELISA to detect the four major CTX congeners. Cross-reactivity of fluorescent sandwich ELISA with other marine toxins. LOD, Sample-to-answer time, sample preparation, and specificity found in sandwich ELISA, LC-MS/MS, receptor binding assay, and cell-based assay.



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Author Information Corresponding Author *E-mail: [email protected] ORCID Takeshi Tsumuraya: 0000-0001-8979-2160 Notes The authors declare no competing financial interest.

Acknowledgment. We are very grateful to Dr. Naomasa Oshiro for his valuable discussion and his generous gift of Variola louti flesh.


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Figure Captions Figure

1.

Structures

of

the

major

CTX

congeners:

CTX1B,

CTX3C,

51-hydroxyCTX3C, and 54-deoxyCTX1B, where n shows the number of methylenes (CH2) in the E-ring.

Figure 2. Schematic diagram of the sandwich ELISA of CTXs. Antibody (blue) specifically bound against the left wing of CTX (red) is immobilized on the plate and antibody (orange) against the right wing is conjugated with horseradish peroxidase (HRP, red) (A) or alkaline phosphatase (ALP, blue) (B).

Figure 3. Fluorescent sandwich ELISA of CTX1B using 3G8 coated on a microtiter plate and ALP-linked 8H4. The results are expressed as mean fluorescence intensity (excitation, λ = 435 nm; emission, λ = 555 nm) ± SD of three experiments.

Figure 4. Fluorescent sandwich ELISA of 51-hydroxyCTX3C (A) using 10C9 coated on a microtiter plate and ALP-linked 8H4, CTX3C (B) using 10C9 coated on a



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microtiter plate and ALP-linked 3D11, and 54-deoxyCTX1B (C) using 3G8 coated on a microtiter plate and ALP-linked 3D11. Can Get Signal® Immunoreaction Enhancer Solution was used for the detection of CTX3C and 54-deoxyCTX1B. The results are expressed as mean fluorescence intensity (excitation, λ = 435 nm; emission, λ = 555 nm) ± SD of three experiments.

Figure 5. Specificity of fluorescent sandwich ELISA systems for the four CTX congeners (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX1B). The results are expressed as mean fluorescence intensity (excitation, λ = 435 nm; emission, λ = 555 nm) ± SD of three experiments. (A) Specificity of fluorescent sandwich ELISA designed to detect CTX1B using 3G8 coated on a microtiter plate and ALP-linked 8H4. (B) Specificity of fluorescent sandwich ELISA designed to detect CTX3C using 10C9 coated on a microtiter plate and ALP-linked 3D11. (C) Specificity of fluorescent sandwich ELISA designed to detect 51-hydroxyCTX3C using 10C9 coated on a microtiter plate and ALP-linked 8H4. (D) Specificity of fluorescent sandwich ELISA designed to detect 54-deoxyCTX1B using 3G8 coated on a microtiter plate and


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ALP-linked 3D11.

Figure 6. Detection of all of the four CTXs congeners (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX1B) by the single fluorescent sandwich ELISA protocol using one microtiter plate coated with a mixture of 10C9 and 3G8, and ALP-linked 8H4. The results are expressed as mean fluorescence (excitation, λ = 435 nm; emission, λ = 555 nm) ± SD of three experiments.



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References

(1) Yasumoto, T.; Murata, M. Chem. Rev. 1993, 93, 1897-1909. (2) Yasumoto, T. Chem. Rec. 2001, 1, 228-242. (3) Scheuer, P. J. Tetrahedron 1994, 50, 3-18. (4) Lewis, R. J. Toxicon 2001, 39, 97-106. (5) Dickey, R. W.; Plakas, S. M. Toxicon 2010, 56, 123-136. (6) Bagnis, R. Bull. Soc. Pathol. Exot. 1992, 85, 412-414. (7) Michio M.; Yasumoto, T. Nat. Prod. Rep., 2000, 17, 293-314. (8) Murata, M.; Legrand, A. M.; Ishibashi, Y.; Yasumoto, T. J. Am. Chem. Soc. 1989, 111, 8929-8931. (9) Murata, M.; Legrand, A.-M.; Ishibashi, Y.; Fukui, M.; & Yasumoto, T. J. Am. Chem. Soc. 1990, 112, 4380-4386. (10) Lewis, R. J.; Sellin, M.; Poli, M. A.; Norton, R. S.; MacLeod, J. K.; & Sheil, M. M. Toxicon 1991, 29, 1115-1127. (11) Satake, M.; Murata, M.; Yasumoto, T. Tetrahedron Lett. 1993, 34, 1975-1978.


Page 31 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(12) Dechraoui, M.-Y.; Naar, J.; Paullac, S.; Legrand, A.-M. Toxicon 1999, 37, 125-143. (13) Lewis, R. J.; Vernoux, J.-P.; Brereton, I. M.; J. Am. Chem. Soc. 1998, 120, 5914-5920. (14) Manger, R. L.; Leja, L. S.; Lee, S. Y.; Hungerford, J. M.; Hokama, Y.; Dickey, R. W.; Granade, H. R.; Lewis, R.; Yasumoto, T.; Wekell, M. M. J. AOAC Int. 1995, 78, 521-527. (15) Poli, M. A.; Lewis, R. J.; Dickey, R. W.; Musser, S. M.; Buckner, C. A.; Carpenter, L. G. Toxicon 1997, 35, 733-741. (16) Hardison, D. R.; Holland, W. C.; McCall, J. R.; Bourdelais, A. J., Baden, D. G.; Darius, H. T.; Chinain, M.; Tester, P. A;; Shea, D.; Quintana, H. A. F; Morris, J. A., Jr.; Litaker, R. W. PLOS ONE 2016, DOI:10.1371/journal.pone.0153348. (17) Yasumoto, T.; Fukui, M.; Sasaki, K.; Sugiyama, K. J. AOAC Int. 1995, 78, 574-582. (18) Lewis, R. J.; Jones, A. Toxicon 1997, 35, 159-168. (19) Lewis, R. J.; Jones, A.; Vermoux, J.-P. Anal. Chem. 1999, 72, 247-250.



Page 32 of 42

(20) Yasumoto, T.; Igarashi, T.; Legrand, A.-M.; Cruchet, P.; Chinain, M.; Fujita, T.; Naokki, H. J. Am. Chem. Soc. 2000, 122, 4988-4989. (21) Lewis, R. J.; Yang, A.; Jones, A. Toxicon 2009, 54, 62-66. (22) Yogi, K.； Oshiro, N.； Inafuku, Y.； Hirama, M.； Yasumoto, T. Anal. Chem. 2011， 83, 8886-8891. (23) Yogi, K; Oshiro, N.; Matsuda, S.; Sakugawa, S.; Matsuo, T.; Yasumoto, T. Food Hygiene and Safety Science 2013, 54, 385-391. (24) Otero, P.; Pérez, S.; Alfonso, A.; Vale, C.; Rodríguez, P.; Gouveia, N. N.; Gouveia, N.; Delgado, J.; Vale, P.; Hirama, M.; Ishihara, Y.; Molgó, J.; Botana, L. M. Anal. Chem. 2010, 82, 6032-6039. (25) Silva, M; Rodriguez, I., Barreiro, A.; Kaufmann, M.; Neto, A. I.; Hassouani, M.; Sabour, B.; Alfonso, A.; Botana, L. M. Vasconcelos, V. Toxins 2015, 7, 3740-3757. (26) Hamilton, B.; Hurbungs, M.; Vernoux, J.-P.; Jones, A.; Lewis, R. J. Toxicon 2002, 40, 685-693. (27) Hamilton, B.; Hurbungs, M.; Jones, A.; Lewis, R. J. Toxicon 2002, 40, 1347-1353.


Page 33 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(28) Diogene, J; Reverte, L,; Rambla-Alegre, M.; del Rio, V.; de la Iglesia, Pl; Campas, M.; Plalcios, O.; Flores, C.; Caixach, J.; Ralijanona, C.; Razanajatovo, I.; Pirog, A.; Magalon, H.; Arnich, N.; Turquet, J.; Sci. Rep. 2017, 7, 8240, doi:10.1038/s41598-017-08682-8. (29) Oguri, H.; Tanaka, S.; Hishiyama, S.; Oishi, T.; Hirama, M.; Tsumuraya, T.; Tomioka, Y.; Mizugaki, M. Synthesis 1999, Special Issue, 1431-1436. (30) Nagumo, Y.; Oguri, H.; Shindo, Y.; Sasaki, S.; Oishi, T.; Hirama, M.; Tomioka, Y.; Mizugaki, M.; Tsumuraya, T. Bioorg. Med. Chem. Lett. 2001, 15, 2037-2040. (31) Nagumo, Y.; Oguri, H.; Tsumoto, K.; Shindo, Y.; Hirama, M.; Tsumuraya, T.; Fujii, I.; Tomioka, Y.; Mizugaki, M.; Kumagai, I. J. Immunol. Methods 2004, 289, 137-146. (32) Oguri, H.; Hirama, M.; Tsumuraya, T.; Fujii, I.; Maruyama, M.; Uehara, H.; Nagumo, Y. J. Am. Chem. Soc. 2003, 125, 7608-7612. (33) Tsumuraya, T.; Fujii, I.; Inoue, M.; Tatami, A.; Miyazaki, K.; Hirama, M. Toxicon 2006, 48, 287-294. (34) Tsumuraya, T.; Fujii, I.; Hirama, M. Toxicon 2010, 56, 797-803.



Page 34 of 42

(35) Tsumuraya, T.; Takeuchi, K.; Yamashita, S.; Fujii, I.; Hirama, M. Toxicon 2012, 60, 348-357. (36) Tsumuraya, T.; Fujii, I.; Hirama, M. J. AOAC Int. 2014, 97, 373-379. (37) Satake, M.; Fukui, M.; Legrand, A. –M.; Cruchet, P.; Yasumoto, T. Tetrahedron Lett. 1998, 39, 1197-1198. (38) Yasumoto, T.; Igarashi, T.; Legrand, A. –M.; Cruchet, P.; Cinain, M.; Fujite, T.; Naoki, H. J. Am. Chem. Soc. 2000, 122, 4988-4989. (39) U. S. Food and Drug Administration, 2011, Fish and fishery products hazards and controls guidance. 4th Ed. Diane Publishing Co., Philadelphia. (40) Hirama, M.; Oishi, T.; Uehara, H.; Inoue, M.; Maruyama, M.; Oguri, H.; Satake, M. Science 2001, 294, 1904-1907. (41) Inoue, M.; Miyazaki, K.; Uehara, H.; Maruyama, M.; Hirama, M. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 12013-12018. (42) Inoue, M.; Miyazaki, K.; Ishihara, Y.; Tatami, A.; Ohnuma, Y.; Kawada, Y.; Komano, K.; Yamashita, S.; Lee, N.; Hirama, M.; J. Am. Chem. Soc. 2006, 128, 9352.


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(43) Yamashita, S.; Ishihara Y.; Morita, H.; Uchiyama, J.; Takeuchi, K.; Inoue, M.; Hirama, M.; J. Nat. Prod. 2011, 74, 357-364. (44) Dirksen, A.; Hackeng, T. M.; Dawson, P. E.; Angew. Chem. Int. Ed. Engl. 2006, 45, 7581-7584.



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for TOC only


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Figure 1 114x78mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2 400x212mm (144 x 144 DPI)


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Figure 3 264x238mm (144 x 144 DPI)



Figure 4 415x156mm (144 x 144 DPI)


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Figure 5 306x200mm (144 x 144 DPI)



Figure 6 323x233mm (144 x 144 DPI)


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Highly Sensitive and Practical Fluorescent Sandwich ELISA for

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