Production of Monoclonal Antibody for Okadaic Acid and Its Utilization

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Production of Monoclonal Antibody for Okadaic Acid and Its Utilization in Ultrasensitive Enzyme-Linked Immunosorbent Assay and One-Step Immunochromatographic Strip Biing-Hui Liu, Chun-Tse Hung, Chuan-Chen Lu, Hong-Nong Chou, and Feng-Yih Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf404827s • Publication Date (Web): 21 Jan 2014 Downloaded from http://pubs.acs.org on January 27, 2014

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Journal of Agricultural and Food Chemistry

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Production of Monoclonal Antibody for Okadaic Acid and Its Utilization in Ultrasensitive

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Enzyme-Linked Immunosorbent Assay and One-Step Immunochromatographic Strip

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BIING-HUI LIU1, CHUN-TSE HUNG2,# , CHUAN-CHEN LU2, HONG-NON CHOU3,

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FENG-YIH YU,2,4,*

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Taiwan.

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Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei,

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School of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan

Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan.

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Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan

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Running title: Immunoassay and immunochromatographic strip of okadaic acid *Corresponding author: No.110, Sec.1, Chien Kuo N. Road, Taichung, Taiwan

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Tel : 886-4-24730022 ext 11816

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Fax: 886-4-23758184.

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E-mail:[email protected]

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＃

equal contribution to first author

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ABSTRACT

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Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes

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diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to

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OA from a hybridoma cell line, 6B1A3, which was obtained by fusion of myeloma cells

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(P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA--globulin.

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The 6B1A3 mAb is belong to the immunoglobulin G1 ( chain) isotype. Both competitive direct and

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indirect enzyme-linked immunosorbent assays (cdELISA) were established for characterization of

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the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to

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the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive

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mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed

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strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20

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seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean

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concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the

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immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based

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ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity

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and accuracy for rapid screening of OA in shellfish samples.

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Key works: Enzyme-linked immunosorbent assay; gold nanoparticle immunochromatographic strip; monoclonal antibody; okadaic acid;

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Introduction Toxic algae blooms occur regularly in many regions of the world. Okadaic acid (OA), a

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common marine biotoxin produced by Dinophysis and Prorocentrum dinoflagellates1,2, readily

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contaminates various species of bivalves, and consumption of contaminated shellfish by human

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causes gastroabdominal disturbances and diarrhetic shellfish poisoning (DSP) in the first few hours.

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However, OA has no harmful effect on bivalves after ingestion2-5. The DSP toxins, including okadaic

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acid, dinophysistoxin-1 (DTX-1), and DTX-2, are the most prevalent toxin groups in shellfish

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worldwide2-5. Okadaic acid have also induced carcinogenic, mutagenic and immunotoxic effects in

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animal studies6. The contaminated bivalves is an economic impact on the global seafood industry.

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Therefore, the European Union has limited permissible OA levels to 160 ng/g in mussels (EC No.

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2074/2005 17)7. Mouse bioassay used to be the standard reference method for OA determination [8].

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This method was been prohibited from January, 2011 due to its weak selectivity, its poor accuracy,

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and ethical concerns. Therefore, the European Union now uses alternative techniques, such as liquid

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chromatography coupled to fluorescence (LC–FLD), liquid chromatography combined with mass

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spectrometry (LC–MS)9-12. However, LC-FLD and LC-MS methods need highly qualified personnel,

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enormous sample cleanup and costly equipments.

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Many sensitive immunochemical methods have been developed for rapidly monitoring and

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quantifying OA in contaminated shellfish. In addition to proposed immunochemical techniques for

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detecting OA, an alternative enzymatic biosensor method of inhibiting protein phosphatase 2A

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(PP2A) by OA has also been established 13-25. However, most of the LC-FLD, LC-MS, enzymatic

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biosensor and even immunochemical measures are impractical for on-site detection due to their long

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incubation times, complex washing procedures, and complicated instrumentation. Therefore, rapid,

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reliable and affordable on-site detection methods are needed.

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Currently, gold nanoparticles have been applied in various biosensors and immunochromatographic strips to detect small molecular toxins15, 25. One emerging immunoassay for 3 ACS Paragon Plus Environment


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mycotoxins and phycotoxins is a immunochromatographic strip that uses conjugate antibody-colored

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gold nanoparticle as a signal reagent17, 26-27. The principle of immunochromatographic strip depends

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on the migration of test samples and gold nanoparticle-antibody conjugates along membrane strips on

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which the binding interactions occur. Because the results of the strip can be inspected visually, the

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strip provides simple and fast on-site detection in less than 10 min without proficient personnel or

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other equipments 17, 26-27. Since various levels of OA contamination have been reported in shellfish

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samples collected from different regions28-30, an effective method for on-site detecting OA in

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shellfishes is required. Therefore, this study produced a mAb against OA and successfully applied it

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to ultrasensitive ELISA and gold nanoparticle immunochromatographic strip for detecting OA in

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shellfish samples.

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Materials and Methods

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Materials Okadaic acid (OA) and dinophysistoxin-1 (Fig. 1) were obtained from Taiwan Algal

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Science Co. (Taiwan). An analytical standard solution of OA at 20 g/mL was a product from

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Calbiochem (San Diego, CA). Bovine serum albumin (BSA), -globulin, gelatin, ovalbumin (OVA),

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ammonium biocarbonate, Tween 20, dimethyl sulfoxide (DMSO), 1,1-carbonyldiimidazole (CDI),

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1-ethyl-3- [3-dimethylaminopropyl]- carbodimide (EDC), and N-hydroxysuccinimide (NHS) were

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purchased from Sigma (MO, USA). Keyhole limpet hemocyanin (KLH) and anti-mouse-peroxidase

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conjugate from goat were purchased from Pierce (IL, USA). Horseradish peroxidase (HRP), BM

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Condimed H1 Hybridoma Cloning Supplement (BMH1) and the mouse mAb isotyping kit were from

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Roche (Mannheim, Germany). TMB (3, 3', 5, 5'-tetramethylbenzidine ) is a substrate solution of

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HRP from Neogen Corp ( KY, USA). Acetonitrile (HPLC grade), methanol (HPLC grade),

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ammonium sulfate and absolute ethanol were obtained from Merck (Darmstadt, Germany).

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Polyethylene glycol (PEG 1500), hypoxanthine (H), aminopterin (A), and thymidine (T) were

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purchased from Boehringer Mannheim Biochemicals (IN, USA). Microtiter plates and strips with 4 ACS Paragon Plus Environment

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various protein binding abilities were obtained from Nunc (Roskilde, Demark). The fetal calf serum

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and penicillin–streptomycin were obtained from GIBCO Laboratories (Grand Island, NY). Gold

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nanoparticles with diameters of 20 nm and 40 nm were obtained from BB International (Cardiff,

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United Kingdom). The murine myeloma cell line P3/NS–1/1–AG4–1 (NS–1) was obtained from

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Bioresources Collection and Research Center in Taiwan and virus-free BALB/c mice ( 9 to

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10-week-old, female) were ordered from National Animal Research Center (Taipei, Taiwan).

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Preparation of Different OA Conjugates.

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Conjugation of OA to -globulin. OA was conjugated to -globulin in the presence of EDC31. The

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EDC and NHS solutions (1.0 mg EDC and 1.0 mg NHS in 0.04 mL DMSO) were freshly prepared

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and then mixed with an OA solution containing 1.0 mg OA in 0.2 mL DMSO. The mixture (0.24 mL)

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was added slowly into 0.4 mL of -globulin solution (2.0 mg in 0.1 M carbonate buffer, pH 9.6), and

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then the reaction was conducted at room temperature for 2 h. Continuously, the mixture was dialyzed

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against 0.01 M phosphate buffer containing 0.15M NaCl (PBS, pH 7.5) for 3 constitutive days with

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two exchanges of PBS. Final products were lyophilized for storage at –20 oC.

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OA-KLH/OVA conjugation.

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conjugated to KLH or OVA by EDC and NHS method31. Generally, 0.5 mg of OA in 0.1 mL

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DMSO was added with 0.5 mL mg of KLH or OVA (2.5 mg in 0.1 M carbonate buffer, pH 9.6) first,

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and then EDC and NHS solutions (1.0 mg EDC and 0.75 mg NHS in 0.04 mL DMSO) were added to

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the mixed solution at room temperature with a constant stirring for 2 hours. The final product was

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dialyzed against PBS for three days and then lyophilized for storage.

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Preparation of OA-Peroxidase.

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method 26, 31. Briefly, 0.2 mg of OA in 0.1 mL of DMSO was mixed with 0.6 mg of EDC and 0.4

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mg of NHS, and then a HRP solution (0.8 mg of HRP in 0.3 mL of 0.1 M carbonate buffer, pH 9.6)

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was added. After being stirred at room temperature for 2 h, the mixture was dialyzed against PBS for

As a solid-phase antigen for indirect competitive ELISA, OA was

Conjugation of OA to HRP was achieved by the EDC and NHS



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72 h and then lyophilized.

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Monoclonal Antibody (mAb) Production.

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Immunization.

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Female BALB/c mice (9–10 weeks of age) were intraperitoneally (ip) injected with 40 g of

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OA--globulin in 0.1 mL of PBS solution, which had been emulsified with 0.1 mL of Freund’s

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complete adjuvant. Four weeks after the first injection, sequential boosters were conducted weekly

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with 40 g of immunogen in 0.1 mL of PBS without adjuvant. After each booster, blood samples

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were collected from the tail vein of each mouse and serum was isolated by centrifugation. The

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antibody specificity was determined by a competitive indirect ELISA (ciELISA) as described below

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Fusion and cloning.

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The mouse showing the highest antibody specificity, 9 weeks after the initial immunization, was

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chosen for further fusion reaction. The selected mouse was primed with a total of 50 g OA--

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globulin four days before fusion, and then the mouse was euthanized on the fusion day. The entire

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spleen was removed aseptically, mashed with a glass pestle, and then passed through a sieve-tissue

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grinder kit equipped with mesh 80 (CD-1, Sigma). Spleen cell suspension at a single-cell stage was

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mixed with 1 x 107 of myeloma cells. The cells were centrifuged, suspended in 0.2 mL of HT

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medium, and then 1 mL of PEG 1500 was dropped into the cell pellet within one minute. To dilute

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the PEG1500, 15 mL of HT medium was added gradually within 10 min. After centrifugation at 300

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g for 5 min, the pelleted cells were resuspended in the HAT medium containing hypoxanthine,

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aminopterin, thymidine plus BMH1 (final concentration 10%) and plated into 96-well tissue culture

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plates. The plates were fed with freshly prepared HAT medium every five days. When the cell

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colonies reached at least half-confluence in the well, ciELISA was applied to screen hybridomas

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secreting specific antibodies against OA. Finally, hybridoma cells from three wells were found to be

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positive and were further cloned by the limiting dilution method32. 6 ACS Paragon Plus Environment

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Ascites fluid generation Female BALB/c mice, 10 weeks old, were injected with pristine (0.5 mL, ip); seven days later, 2

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x 106 hybridoma cells suspended in DMEM were injected (ip). Three weeks after the injection,

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ascites were collected every other day and centrifuged at room temperature for 5 min. IgG antibodies

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in the supernatant fluid were precipitated with 50% of ammonium sulfate solution twice. Purified

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antibodies were stored at –70°C for future usage.

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Characterization of Monoclonal Antibodies

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Determination of mAb isotype. Identification of specific immunoglobulin was carried out by a mouse mAb isotyping kit (Roche,

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Germany) according to the manufacturer’s protocol.

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Competitive Indirect ELISA (ciELISA)

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The process of ciELISA was conducted primarily according to Yu et al.31 with a slight

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modification. OA-OVA (0.1 mL) was coated on each well of a microtiter plate which then was

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kept at 4 oC overnight. After the plate had been washed four times with Tween-PBS, 0.17 mL of

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gelatin-PBS (0.17 mL per well; 0.1% gelatin in PBS) was poured into the wells and incubated at 37

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o

C for 30 min. The plate was washed again and 0.05 mL of OA standard with concentrations from

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0.01 to 100 ng/mL in PBS or extracted sample solutions were added to each well, and then the

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anti-OA mAb (25 ng/mL in PBS, 0.05 mL per well) was added to all wells and incubated at 37 °C

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for 50 min. The plate was washed again and 0.1 mL of goat anti-mouse IgG-HRP conjugate (1:20000

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dilution) was added and incubated at 37°C for 45 min. The plate was washed and 0.1 mL of TMB

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substrate solution

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hydrochloric acid (0.1 mL, 1 N) was added to stop the reaction. Vmax ELISA reader (Molecular

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Devices Co. ; Menlo Park, CA) was used to determine the absorbance value at 450 nm

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cdELISA.

(Neogen Corp) was added and waited the color development for 10 min, then



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The process of cdELISA was conducted primarily according to Liu et al. 26 with a slight

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modification. The anti-OA mAb from ascites in PBS (1:10000 dilution; 1 g/mL) was used to coat

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the plate (0.1 mL/well). After a series of washing and blocking steps, OA standards (concentrations

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from 0.01 to 100 ng/mL) or samples together with the OA-HRP conjugate (10 ng/mL in PBSl) were

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added and incubated at 37oC for 50 min. The plate was washed with PBS-Tween and incubated with

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TMB solution (Neogen Corp) at room temperature in the dark for 10 min. The absorbance at 450 nm

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was determined in the Vmax automatic ELISA reader

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cdELISA of Shellfish Samples Contaminated with OA. Twenty shellfish samples, including clam, scallop, mussel and oyster, were obtained from local

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stores for OA level determination. Briefly, five gram of each sample (whole body) was homogenized

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with 20 mL of extraction solvent (methanol/water, 80/20, v/v) for 5 min24,33 and then centrifugaed at

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14000 g for 10 min. One mL of supernatant fluid was mixed with 1 mL of hexane by vortex for 1

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min, and then 3 mL of chloroform was added. After centrifugation for 5 min, the lower chloroform

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layer was aspirated and dried under rotary vacuum evaporator. The dried sample was reconstituted

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with 1 mL of PBS buffer for cdELISA analysis or for the immunostrip assay.

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Preparation of Antibody-Gold Nanoparticle Probe For conjugation, OA mAbs were dialyzed in boric acid-borax buffer (pH 8) for 24 h at 4 oC and

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the gold nanoparticle solution was adjusted to pH 9.0 with 0.1 M K2CO3 (pH 11.5)27, 28. 5 L of

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anti-OA mAb ( 1 g/L) was added dropwise to 2 mL of gold nanoparticle solution with gentle

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stirring. After 1 h reaction at room temperature, 10% (w/v) filtered BSA was added to block the

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reaction for 30 min and then centrifuged at 19000 g for 30 min at 4oC. The pellets containing OA

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mAb-gold nanoparticle conjugate were resuspended in 450 L of 20 mM Tris-buffered saline (pH

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8.0) containing 1% BSA and 0.1% sodium azide and stored at 4 oC for the further usage. 8 ACS Paragon Plus Environment

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Preparation of Immunochromatographic Strip An Easypack Developer's Kit is composed of three pads (sample, conjugate release and

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absorbent pads), glass fiber, cover tape and nitrocellulose (NC) membrane plates (pore sizes of 5 and

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15 M) are purchased from Advanced Microdevices P. Ltd. (Ambala Cantt, India). With the aid of

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Double Axes Programmable Control Printer (model P-602 from Troy Technology, Taiwan), the test

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and control zones of NC membrane were drawn with 0.5 L of OA-KLH (0.8 mg/mL) conjugate and

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0.5 L of rabbit-anti-mouse IgG antibody (1 mg /mL), respectively, and then dried for 10 min at

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room temperature. Subsequently, the OA mAb-gold nanoparticle conjugate (5 L/strip) was added to

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the conjugate release pad and dried at 37 oC for 5-10 min. The release pad was first pasted on the

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plate by over-crossing 4 mm with the NC membrane. Then, the sample pad was further pasted on the

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plate by over-crossing 6 mm with the release pad. Finally, the top of the membrane sheet was pasted

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on absorbent pad. The whole assembled sheet was cut lengthways with an automatic cutter and

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divided into strips (5 mm × 75 mm)27, 28.

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Assay of OA in Shellfish Samples with Immunochromatographic Strip Three hundred microliters of sample extract solutions as mentioned above or various

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concentrations of OA analytical standard (0-50 ng/mL) were poured into the wells of microtiter plate.

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Subsequently, one immunochromatographic strip was dipped into per well vertically. The extracted

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samples or OA standard solution migrated upward the membrane by capillary action. Ten minutes

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later after color development, test results on the strip were determined visually.

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Results

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Monoclonal Antibody Production

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Mice were injected with OA--globulin and boosted for 4 times with the same antigen, the 9 ACS Paragon Plus Environment


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mouse with serum that had the highest affinity for OA was sacrificed for a hybridoma screening.

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Following the NS-1/spleen cell fusion and growing, the ciELISA with OA-OVA as a coated reagent

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was used for screening the hybridoma cells, which secreted mAbs specific to OA. In the about 500

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examined wells, three clones displayed strong positive color results in the ciELISA; of these, the

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clone 6B1 had the highest affinity for OA. Therefore, the clone 6B1 culture was aspirated from the

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fusion well and subjected to limiting dilution for a single hybridoma selection. After limiting dilution

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and ciELISA screening, clone 6B1A3, which showed the highest affinity for OA, was used to

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generate culture supernatant and ascites fluid.

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Antibody Characterization.

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Isotype Determination. The isotype of the mAb, secreted from hybridoma cell line 6B1A3, was

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identified as immunoglobulin G1, -light chain.

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Competitive direct and indirect ELISA. The specificity of 6B1A3 mAb was measured by both

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cdELISA and ciELISA. As shown in Fig. 2A, the concentrations causing 50% inhibition (IC50) of

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binding of OA-HRP to the mAb by OA and DTX-1, were calculated to be 0.077 and 1.870 ng/mL,

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respectively in cdELISA. The ciELISA revealed similar results for OA-OVA coated onto the

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microplate wells to serve as solid-phase antigen. The IC50 values for mAb binding to OA-OVA by

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free OA and DTX-1 were 0.58, and 2.0 ng/mL, respectively (Fig. 2B). However, other phycotoxins

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such as domoic acid and saxitoxin, even at concentrations as high as 10 g/mL, did not inhibit the

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mAb binding to the OA-enzyme/protein conjugates (OA-HRP or OA-OVA) in either ELISA system,

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Assay of OA in Shellfishes with mAb-based cdELISA. Twenty shellfish samples were purchased from local supermarket and subjected to cdELISA to measure OA contamination; Table 1 presents the measurement results. The proposed detection 10 ACS Paragon Plus Environment

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system revealed that ten of the twenty examined samples were OA-positive. Sample 7 showed the

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highest OA levels (0.517 ng/mL; 2.068 ng/g). The OA levels of sample 9 and 13 are also higher than

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1.0 ng/g. The OA levels in the other seven positive samples were lower than 1.0 ng/g. However, all

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the oyster samples are free of OA contamination.

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Assmbly of Immunochromatographic Strip The mAb-gold nanoparticle conjugates were used to fabricate an immunochromatographic strip,

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in which OA in the sample solution competes with the OA-KLH conjugate on the membrane for the

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antibody-gold nanoparticle label. Figure 3 is a schematic description of the immunochromatographic

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strip test format. A sufficient OA levels in the sample solution occupied all the antibody binding sites

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of the antibody-gold nanoparticle conjugates, which prevented the limited antibody-gold nanoparticle

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conjugates from binding with the OA-KLH conjugate in the test zone. The absence of the red line in

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the test zone indicated a positive result (Fig. 3). In contrast, When OA is absent in sample solution,

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the antibody-gold nanoparticle conjugate trapped and bound by the OA-KLH conjugate to form a red

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line in the test zone indicated a negative result (Fig. 3). The control zone was designed with

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rabbit-anti-mouse secondary antibody to confirm that the assay had been performed properly;

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regardless of the presence of OA, the control zone on the membrane will show one red line (Fig. 3).

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Characterizaion of Immunochromatographic Strip for OA. Different concentrations of OA analytical standard solution (0-50 ng/mL) were subjected to

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immunochromatographic strip test. This assay required no more than 10 min, and the detection limit

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of strip test for OA approached 5 ng/mL (Fig. 4). At least 5 measurements were examined by the

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strip detection system to define the cutoff level for each selected concentration. When an OA levels

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are higher than 5 ng/mL, all antibody-gold nanoparticle conjugates were bound by free OA, which

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restricted the antibody-gold nanoparticle conjugates to bind with the OA-KLH in the test zone of the 11 ACS Paragon Plus Environment


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membrane. Finally, rabbit anti-mouse secondary antibody captured antibody-gold nanoparticle

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conjugates in the control zone which resulted in only one red line on the membrane.

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Analysis of OA in Shellfish Samples by Immunochromatographic Strip Assay The strip was further applied to examine OA contamination shellfishes. Table 1 shows the

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analytical results. Ten contaminated shellfish samples with OA levels ranging from 0.051 ng/mL to

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0.517 ng/mL, according to cdELISA results, (Table 1) had two red lines on the immunostrip

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membranes, which indicated a negative outcome (Fig 5). However, in one spiked sample (S)

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containing 5.0 ng/mL of OA in shellfish extract, one red line disappeared in the test zone, which

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indicated that the sample was OA-positive (Fig 5).

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Discussion Since OA is a nonimmunogenic phycotoxin with low molecular weight, conjugating OA to a

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protein carrier is needed to contribute its immunogenicity. Our laboratory has been successfully

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produced a polyclonal antibody for OA using OA--globulin conjugate as an antigen, and established

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a sensitive ELISA for detecting OA. Since this study obtained a hybridoma clone secreting mAb

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specific to OA, the supply of mAb for OA is theoretically unlimited. Both mAbs produced from

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culture supernatant and mouse ascites could be adopted in the cdELISA. However, rabbit-anti-mouse

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Fc secondary antibody must be coated onto the microplate before adding the supernatant. In contrast,

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cdELISA can be run by directly coating the microplate with mAb purified from ascites.

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The mAb obtained from 6B1A3 showed a better affinity (IC50 value) for OA compared to

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DTX-1 in both cd- and ciELISA systems. The IC50 value of the mAb-based cdELISA for OA was

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0.077 ng /mL, and the detection limit of OA in PBS solution in the cdELISA approximated 0.004

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ng/mL based on 90% confidence interval at 10% of inhibition of binding of OA-HRP conjugate.

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Therefore, the proposed ELISA method is about 2 orders of magnitude higher than that of the

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mAb-based ELISA reported by Lu et al.16 (IC50 and detection limits of 4.4 and 0.45 ng /mL, 12 ACS Paragon Plus Environment

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respectively) and that of Kreuzer et al.14 with an IC50 of 100 ng/mL.

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After using a combination of water/methanol (20:80, v/v) to extract OA from shellfishes, 1 mL

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of clear supernatant solution was diluted with 9 mL of before cdELISA analysis. The analytical

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results showed that all examined samples were slightly OA positive because the matrix components

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caused interference in the cdELISA. Therefore, a clean-up procedure was incorporated into the

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sample preparation procedure. Washing the methanolic sample extracts with hexane effectively

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eliminated the lipid in the extract solution to avoid the matrix interference24. The cdELISA analysis

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of shellfishes demonstrated that ten of the twenty shellfish samples had a low-level contamination

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with OA (Table 1). In all samples, OA was lower than 2.068 ng/g, which was approximately 80-fold

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below the regulatory limit of EU which is 160 ng/g of OA in shellfish samples.7 This concentration is

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consistent with a previous report by Lee et al.29 that gastropod samples collected in Korea had OA

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concentrations up to 21.5 ng/g but is lower that reported by Ciminiello, et al.28 with OA

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concentrations of 100-200 ng/g.

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To establish an assay that could be broadly used for field detection of OA contaminated

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shellfish sample, this study further developed a mAb-based rapid immunochromatographic strip for

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faster and simpler screening compared to cdELISA. The mAb-based immunochromatographic strip

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established in this study for on-site OA determination had a limit of detection about 5.0 ng/mL. In Lu

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et al.17, a lateral flow assay developed for OA analysis had a detection limit of 30~50 ng/mL, which

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may not be sensitive enough for practical sample test. Several factors also correlated with the

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effectiveness of an immunochromatographic strip include the size of the gold nanoparticles, the

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membrane pore size, and the test line of OA-protein conjugates. Therefore, both 20 nm and 40 nm

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gold particles were adopted to prepare of mAb-gold particle conjugates. On the nitrocellulose

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membrane, the 40 nm particles provided better visibility compared to the 20 nm gold particles,

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probably due to their optimal size and their lower steric hindrance between conjugation of 40 nm

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gold particles and antibody. To test the effectiveness of 5 and 15 m membrane pore size, in the 13 ACS Paragon Plus Environment


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membrane pore size of 15 m, the red gold nanoparticles provided a more obvious visual effect and

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also a faster migration during the process. Finally, 15 m membrane pore size and 40 nm gold

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particles were selected for all immunochromatographic strips. Either OA-KLH or OA-OVA is drawn

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onto the membrane test zone to compare their effectiveness in binding OA in the sample with the

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antibody-gold nanoparticle conjugate. Compared to OA-OVA, the OA-KLH proved to be a suitable

332

protein conjugate because coating OA-KLH onto the test zone of a strip obtained a stronger red color

333

line in the strips and also had a lower detection limit of 5 ng/mL compared with 20 ng /mL in which

334

coated with OA-OVA onto the test line. (data not shown).

335

Results of sample analysis acquired by the immunochromatographic strip were in good

336

agreement with those acquired by ELISA (Table 1). The test strip can be directly immersed into 300

337

L of sample extraction solution and the test can be performed in 10 min. Therefore, the strip test is

338

effective for on-site OA detection not only in terms of accuracy and sensitivity, but also in terms of

339

labor and speed. This study obtained a novel hybridoma cell line for secreting mAb against OA and

340

successfully applied it to an ultrasensitive ELISA and gold nanoparticle immunostrip for detecting

341

OA in shellfish. The IC50 value of ELISA was 0.077 ng/mL, and the gold nanoparticle immunostrip

342

had a visual detection limit of 5.0 ng/mL. The OA immunostrip is a semiquantitative, rapid and

343

visual assay that uses gold nanoparticles as a color tracer to provide visual indication to examine OA

344

in shellfish samples within 10 min. This strip will be conceivably applicable for on-site detection of

345

OA in seafood industry.

346 347

Abbreviation used

348

A, aminopterin; BMH1, BM Condimed H1 Hybridoma Cloning Supplement (BMH1); BSA, bovine

349

serum albumin; CDI, 1,1-carbonyldiimidazole; Dulbeco Modified Eagle’s Medium, DMEM; DMSO,

350

dimethyl sulfoxide; EDC, 1-ethyl-3 [3-dimethylaminopropyl] carbodimide; ELISA, enzyme-linked

351

immunosorbent assay; cdELISA, competitive direct ELISA; ciELISA, competitive indirect ELISA; 14 ACS Paragon Plus Environment

Page 15 of 27


352

H, hypoxanthine; iELISA, indirect ELISA; HRP, horseradish peroxidase; KLH, keyhole limpet

353

hemocyanin; mAb, momoclonal antibody; MES, 2-N-morpholinoethane-sulfonic acid; NHS,

354

N-hydroxysuccinimide; NS-1, P3/NS–1/1–AG4–1; PBS, Phosphate buffer containing 0.15M NaCl;

355

PEG 1500, Polyethylene glycol; OVA, ovalbumin; T, thymidine; TMB, 3, 3', 5,

356

5'-tetramethylbenzidine.

357 358 359

Acknowledgments This work was supported by grants NSC 100-2923-B-040-001-MY2 and 101-2313-B-040-005

360

from the National Science Council of the Taiwan. Ted Knoy is appreciated for his editorial

361

assistance.

362 363 364 365 366 367 368 369 370 371 372 373 374 375 376



Page 16 of 27

377

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(2) Scoging, A.; Bahl, M. Diarrhetic shellfish poisoning in the UK. Lancet 1998, 352, 117.

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shellfish poisoning (DSP) toxins in clams (Ruditapes decussatus) from Tunis north lagoon.

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outbreaks linked to mussels contaminated with okadaic acid and ester dinophysistoxin-3 in

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France, June 2009. Euro. Surveill. 2011, 16, Article 3

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(5) Li, A.; Ma, J.; Cao, J.; McCarron, P. Toxins in mussels (Mytilus galloprovincialis) associated with diarrhetic shellfish poisoning episodes in China. Toxicon. 2012, 60, 420-425.

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(6) Carvalho, P. S.; Catian, R.; Moukha, S.; Matias, W. G.; Creppy, E. E. Comparative study of

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(9) Louppis, A. P.; Badeka, A. V.; Katikou, P.; Paleologos, E. K.; Kontominas, M.G. Determination

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of okadaic acid, dinophysistoxin-1 and related esters in Greek mussels using HPLC with

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fluorometric detection, LC-MS/MS and mouse bioassay. Toxicon. 2010, 55, 724-733.

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(10) Suzuki, T.; Quilliam, M. A. LC-MS/MS analysis of diarrhetic shellfish poisoning (DSP) toxins, 16 ACS Paragon Plus Environment

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okadaic acid and dinophysistoxin analogues, and other lipophilic toxins. Anal. Sci. 2011, 27,

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571-584.

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(11) Stobo, L. A.; Lacaze, J. P.; Scott, A. C.; Gallacher, S.; Smith, E. A.; Quilliam, M. A. Liquid

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chromatography with mass spectrometry--detection of lipophilic shellfish toxins. J. AOAC Int.

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2005, 88, 1371-82.

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(12) Draisci, R.; Croci, L.; Giannetti, L.; Cozzi, L.; Lucentini, L.; De Medici, D.; Stacchini, A.

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Comparison of mouse bioassay, HPLC and enzyme immunoassay methods for determining

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diarrhetic shellfish poisoning toxins in mussels. Toxicon. 1994, 32, 1379-1384.

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(13) Hokama, Y. Recent methods for detection of seafood toxins: recent immunological methods for ciguatoxin and related polyethers. Food Addit. Contam. 1993, 10, 71-82.

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(14) Kreuzer, M. P.; O’Sullivan, C. K.; Guilbault, G. G. Development of an ultrasensitive

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immunoassay for rapid measurement of okadaic acid and its isomers. Anal. Chem. 1999, 71,

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(15) Lamas, N. M.; Stewart, L.; Fodey, T.; Higgins, H. C.; Velasco, M. L.; Botana, L. M.; Elliott, C.

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T. Development of a novel immunobiosensor method for the rapid detection of okadaic acid

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contamination in shellfish extracts. Anal. Bioanal. Chem. 2007, 389, 581-587.

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(16) Lu, S. Y.; Zhou, Y.; Li, Y. S.; Lin, C.; Meng, X. M.; Yan, D. M.; Li, Z. H.; Yu, S. Y.; Liu, Z. S.;

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Ren, H. L., Production of monoclonal antibody and application in indirect competitive ELISA for

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detecting okadaic acid and dinophytoxin-1 in seafood. Environ. Sci. Pollut. Res. Int. 2011, 19,

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2619-2626.

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(17) Lu, S.Y.; Lin, C.; Li, Y. S.; Zhou, Y.; Meng, X. M.; Yu, S. Y.; Li, Z. H.; Li, L., Ren, H. L.; Liu,

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Z.S. A screening lateral flow immunochromatographic assay for on-site detection of okadaic acid

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in shellfish products. Anal. Biochem. 2012, 422, 59-65.

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(18) Matsuura, S.; Hamano, Y.; Kita, H.; Takagaki, Y. Preparation of mouse monoclonal antibodies to okadaic acid and their binding activity in organic solvents. J. Biochem. 1993, 114, 273-278. 17 ACS Paragon Plus Environment


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okadaic acid in mussels. Toxicon. 1999, 37, 909-922.

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(20) Stewart, L. D.; Hess, P.; Connolly, L.; Elliott, C. T. Development and single-laboratory

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validation of a pseudofunctional biosensor immunoassay for the detection of the okadaic acid

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group of toxins. Anal. Chem. 2009, 81, 10208-10214.

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monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their

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toxicity equivalence factors. Toxicon. 2009, 54, 491-498.

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detection of okadaic acid and esters in Portuguese bivalves. Toxicon. 1999, 37, 1565-1577.

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investigation of analytical conditions and sample matrix on assay performance. J. AOAC Int.

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(25) Paek, S. H.; Lee, S. H.; Cho, J. H.; Kim, Y. S. Development of rapid one-step immunochromatographic assay. Methods 2000, 22, 53-60. (26) Liu, B. H., Tsao, Z. J., Wang, J. J., Yu, F. Y. Development of a monoclonal antibody against

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ochratoxin A and its application in enzyme-linked immunosorbent assay and gold nanoparticle

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immunchromatographic strip. Anal. Chem. 2008, 80, 7029-7035.

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(27) Tsao, Z. J.; Liao Y. C.; Liu, B. H.; Su, C. C.; Yu, F. Y. Development of a monoclonal antibody

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against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal

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gold immunostrip. J. Agric. Food Chem. 2007, 55, 4921-4927.

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Investigation of the toxin profile of Greek mussels Mytilus galloprovincialis by liquid

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chromatography-mass spectrometry. Toxicon. 2006, 47, 174-181.

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(29) Lee, K. J.; Mok, J. S.; Song, K. C.; Yu, H.; Lee, D. S.; Jung, J. H.; Kim, J. H. First detection and

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seasonal variation of lipophilic toxins okadaic acid, dinophysistoxin-1, and yessotoxin in Korean

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gastropods. J. Food Prot. 2012, 75, 2000-2006.

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(30) Vale, P.; Veloso, V.; Amorim, A. Toxin composition of a Prorocentrum lima strain isolated from the Portuguese coast. Toxicon. 2009, 54, 145–152. (31) Yu, F. Y.; Liu, B. H.; Chou, H. R.; Chu, F. S. Development of a sensitive ELISA for the determination of microcystins in algae. J. Agri. Food Chem. 2002, 50, 4176- 4182. (32) Oi, V. T.; Herzenberg, L. A. In Selected Methods in Cellular Immunlogy: Mishell, B. B.; Shiigi, S. M. Eds.; Freeman: San Francisco, CA, 1980; pp 351-371. (33) Kelly, S. S.; Bishop, A, G.; Carmody, E. P.; James, K. J. Isolation of dinophysistoxin-2 and the

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high-performance liquid chromatographic analysis of diarrhetic shellfish toxins using

465

derivatisation with 1-bromoacetylpyrene. J. Chromatoatog. A. 1996, 749, 33-40.

466 467 468 469 470 471 472 473 474



475 476

Page 20 of 27

FIGURE CAPTIONS Figure 1. Structures of OA and DTX-1.

477 478

Figure 2. A. Cross-reactivity of anti-OA antibodies with OA () and DTX-1 () in a cdELISA.

479

B. Cross-reactivity of anti-OTA antibodies with OA () and DTX-1 () as determined by a

480

ciELISA. All data were obtained based on the average of three sets of experiments. The absorbance

481

of the control, A0, with no toxin present, was 2.0.

482 483

Figure 3. The schematic illustration of immunochromatographic strip. C, control zone (rabbit

484

anti-mouse IgG); T, test zone (OA-KLH).

485 486

Figure 4. Detection limit of OA with immunochromatographic strip. Different concentrationof OA

487

analytical standard (0-50 ng /mL) was dissolved in PBS. A concentration higher than 5 ng/mL of

488

OA caused a disappearance of a red line at the test zone.

489 490

Figure 5. Detection of OA with immunochromatographic strip in control (C), spiked (S) and 10

491

shellfish samples. A spiked strip containing 5 ng/mL of OA in shellfish extract indicates that a red

492

line disappeared in the test zone, verifying that it is OA positive. All samples containing OA lower

493

than 5 ng/mL showed two red lines on the membrane indicating that they are OA negative.

494 495


Page 21 of 27


Table 1. ELISA and Immunochroatographic Strip Analysis of OA in Shellfish Samples

496

ELISA a

497

(ng/mL  SD)

Immunochromatographic stripa

(ng/g SD) b

498 499

No.

500

1.

clam

0.051 0.008

0.204 0.032

―

501

2.

clam

0.143 0.005

0.5720.020

―

502

3.

mussel

0.106  0.004

0.424  0.016

―

503

4

clam

ND

ND

504

5

oyster

ND

ND

505

6

scallop

0.196  0.018

0.784  0.072

―

506

7.

clam

0.517  0.004

2.068  0.016

―

507

8.

clam

0.195  0.012

0.780  0.048

―

508

9.

clam

0.307  0.012

1.228 0.048

―

509

10. oyster

ND

ND

510

11 scallop

ND

ND

511

12 scallop

ND

ND

512

13. clam

513

14 . mussel

ND

ND

514

15. clam

ND

ND

515

16. scallop

516

17. crab

517

18.

518

19. oyster

ND

ND

20. oyster

ND

ND

519 520 521 522

clam

0.388  0.005

0.216  0.026 ND

1.552  0.020

0.864  0.104

―

―

ND

0.112  0.023

0.448 0.092

―

a

Each sample was extracted twice and each extract was analyzed in triplicate.

b

One mL extract solution contains 0.25 g of shellfish samples.

c

ND, not detectable

523 524 525 526



527

R1

R2

R3

R4

OA

CH3

H

H

H

DTX-1

CH3 CH3

H

H

528 529 530

Figure 1. Structures of Okadaic acid (OA) and dinophysistoxin-1(DTX-1)

531 532 533 534 535 536 537 538 539 540 541 542 543


Page 22 of 27

Page 23 of 27

544


(A)

545

% of Binding

100

OA DTX-1

80

546

60

547

40

548

20 549

0 550

0.001

0.01

0.1

1

10

100

Toxin concentration, ng/mL 551

(B) 100

% of Binding

552

557

OA DTX-1

80

553

60

554

40

555

20

556

0

0.001

0.01

0.1

1

10

100

558

Toxin concentration, ng/mL 559 560 561

Figure 2. A. Cross-reactivity of anti-OA antibodies with OA () and DTX-1 () in a cdELISA. B.

562

Cross-reactivity of anti-OTA antibodies with OA () and DTX-1 () as determined by a ciELISA.

563

All data were obtained based on the average of three sets of experiments. The absorbance of the

564

control, A0, with no toxin present, was 2.0.

565 566 567 568 569 23 ACS Paragon Plus Environment


Page 24 of 27

570 571 572 573 574 575 576 577 578 579 580 581 582

Figure 3. Schematic illustration of immunochromatographic strip for OA. C, control zone (Rabbit

583

anti-mouse IgG); T, test zone (OA-KLH).

584 585 586 587 588 589 590 591 592 593 594 24 ACS Paragon Plus Environment

Page 25 of 27


0 0.1

595

1

5 10 20

50 ng/mL

596 597 598

C

599

T

600 601

－

－

－

＋

＋

＋

＋

602 603 604 605

Figure 4. Detection limit of OA with immunochromatographic strip. A series of dilution (0-50 ng

606

/mL) of certificated standard OA was dissolved in PBS. A concentration higher than 5 ng/mL of

607

OA caused a disappearance of a red line at the test zone.

608 609 610 611 612 613 614 615 616 617 618 619 620 621 25 ACS Paragon Plus Environment


Page 26 of 27

622 623 624 625 626 627

C T

628 629 630 631 632 633 634

Figure 5. Detection of OA with immunochromatographic strip in control (C), spiked (S) and 10

635

shellfish samples. A spiked strip containing 5 ng/mL of OA in shellfish extract indicates that a red

636

line disappeared in the test zone, verifying that it is OA positive. All samples containing OA lower

637

than 5 ng/mL showed two red lines on the membrane indicating that they are OA negative.

638 639 640 641 642 643 644 645 646


Page 27 of 27


647 648

Graphic for manuscript 649


Production of Monoclonal Antibody for Okadaic Acid and Its Utilization

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