Food Chemistry 118 (2010) 467–471

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Analytical Methods

Development of a new monoclonal antibody based direct competitiveenzyme-linked immunosorbent assay for detection of brevetoxinsin food samples

Yu Zhou *, Yan-Song Li, Feng-Guang Pan, Yuan-Yuan Zhang, Shi-Ying Lu, Hong-Lin Ren, Zhao-Hui Li,Zeng-Shan Liu, Jun-Hui ZhangKey Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130062, PR China

a r t i c l e i n f o

Article history:Received 18 November 2008Received in revised form 13 April 2009Accepted 2 May 2009

Keywords:Hybridoma cell lineMonoclonal antibodyEnzyme-linked immunosorbent assayBrevetoxinCross-reactivityRecovery

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.05.015

* Corresponding author. Tel.: +86 0431 87836718.E-mail address: zhouyu69@sina.com (Y. Zhou).

a b s t r a c t

Brevetoxin B (PbTx-2) was covalently linked to carrier protein bovine serum albumin and human gammaglobulin. A monoclonal antibody against PbTx-2, which showed high cross-reactivity values with PbTx-1,PbTx-3 and PbTx-9 (more than 89%) was obtained from ascites and some characteristics of monoclonalantibody were studied. An direct competitive enzyme-linked immunosorbent assay (ELISA) for detectionof PbTxs was developed, which showed an IC50 value of 5.3 ng mL�1 with a detection limit of0.6 ng well�1. The recoveries of PbTxs from cockle (88.4%–102.3%) and oyster (89.4%–104.3%) demon-strated that the matrices of cockle and oyster where PbTxs are found do not interfere with the assay.The newly developed competitive ELISA appears to be a reliable and useful method for mass monitoringof PbTxs in mollusk.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Brevetoxins (PbTxs) are potent marine neurotoxins produced bythe planktonic red tide dinoflagellate Karenia brevis and are accu-mulated in filter-feeding molluscan shellfish by dietary transfer.They are lipid-soluble polycyclic polyether compounds and arethe only molecules known to activate voltage sensitive sodiumchannels in mammals through a specific interaction with site 5of the alpha subunit of the sodium channel (Dechraoui, Naar,Pauillac, & Legrand, 1999). Human ingestion of toxic shellfishcauses neurotoxic shellfish poisoning (NSP). Although PbTxs canbe used as powerful tools in neuroscience research (Naar, Branaaa,Bottein-Dechraoui, Chinain, & Pauillac, 2001), their detrimentaleffects on human health and negative impact on seafood industrieshave mainly raised global awareness. Therefore it is very importantto develop a precise, sensitive, reproducible and specific detectionmethod as an alternative to the conventional mouse intraperito-neal (i.p.) bioassay (Lewis, 1995), solid-phase extraction (SPE)assay, receptor binding assay (RBA), radioimmuno-assay (RIA),and liquid chromatography–mass spectrometry (LC–MS) (Twineret al., 2007), competitive electro-chemiluminescence-basedimmunoassay (Poli et al., 2007), cell based assay (Fairey, Bottein

ll rights reserved.

Dechraoui, Sheets, & Ramsdell, 2001) and in vitro assays (Plakaset al., 2008).

Immunological methods based on antibodies are simple, sensi-tive, economical and high throughput procedure for quantifyingbiologically relevant compounds, but require a continuous supplyof well-defined specific antibodies. To date, Polyclonal and mono-clonal antibodies to PbTx-2-type brevetoxins (Naar et al., 2001)and polyclonal antisera to PbTxs have been raised in animals suchas mice (Trainer & Baden, 1990), goats (Melinek, Rein, Schultz, &Baden, 1994; Poli, Rein, & Baden, 1995; Trainer & Baden, 1991),and rabbits (Levine & Shimizu, 1992). Although they exhibit highaffinity, but usually available in only limited supply and requirebatch standardisation and continual supply of antigen for boosterinjections. On the other hand, the antigens (PbTxs) are very expen-sive, so it is restricted to detect this kind of compounds by poly-clonal antisera.

It is one of the key steps to generate a hybridoma cell line,which permanent secreting high affinity monoclonal antibody(mAb) against PbTxs, for the establishment of a precise, sensitive,and specific immunoenzymatic assay for PbTxs. Since PbTxs aresmall molecules, haptens must be synthesised and coupled to car-rier proteins to induce antibody production. In this study, weaimed at obtaining mAbs against PbTxs and developing mAb-basedenzyme-linked immunosorbent assay (ELISA) with better perfor-mance using a larger number of haptens as competitors.

468 Y. Zhou et al. / Food Chemistry 118 (2010) 467–471

2. Materials and methods

2.1. Materials

PbTx-1, PbTx-2, PbTx-3, PbTx-9, ciguatoxin-1 (the purity P 98%by HPLC), okadaic acid, microcystin LR and dinophysistoxin (thepurity P 95% by HPLC) were obtained from Sigma Chemicals Co.(St. Louis, MO, USA). Agarose, complete and incomplete Freund/sadjuvant, polythylene glycol-4000 (PEG), RPMI 1640, foetal bovineserum (FBS), horseradish peroxidase-conjugated goat anti-miceIgG (HRP–IgG), bovine serum albumin (BSA), human gamma glob-ulin (HGG), HT and HAT were purchased from Express China. Allother reagents were of analytical grade. The ELISA was carriedout in 96-well polystyrene microtiter plates (Stripwell plate2592, Costar, Changchun, China). Well absorbencies were readwith a MK3 microplate reader (Thermo, Shanghai, China).

2.2. Animals

Female Balb/C mice, 6- to 7-week old and 9- to 10-week old,being immunized and using to produce ascites, respectively, wereobtained from Changchun Institute of Biological Products, Jilinprovince China.

2.3. Buffers and solutions

The buffers used regularly were coating buffer, 50 mmol L�1

carbonate buffer (pH 9.5); phosphate buffered saline (PBS),10 mmol L�1 sodium phosphate buffer (pH 7.4) containing140 mmol L�1 NaCl; dilution buffer, PBS containing 0.1% (w/v) gel-atin; washing buffer (PBST), 10 mmol L�1 sodium phosphate buffer(pH 7.4) containing 140 mmol L�1 NaCl and 0.05% (v/v) Tween 20;and TMB solution, 70 lL of 0.65% H2O2, 250 lL of 10 mg mL�1 3,3/5,5/-tetramethylbenzidine (TMB) in dimethylsulfoxide (DMSO) per25 mL of phosphate citrate buffer, pH 5.4.

2.4. Preparation of PbTx–protein conjugates

Conjugates were prepared according to a modified method ofNaar et al. (2001). Briefly, A 10-fold molar excess of succinic anhy-dride solubilised in 10 mL of anhydrous pyridine was added to8 mg of crystalline PbTx-2. After incubation (6 h at 65 �C) the sol-vent was evaporated under a stream of nitrogen and the residuewas reacted with a 10-fold molar excess of tributylamine and iso-butyl chlorocarbonate as 1/10th dilutions in dry peroxide-free di-oxan for 30 min at room temperature. Then the carrier proteinHGG and BSA (the same hapten/carrier molar ratio) were addedat room temperature and incubated for more than 30 min, thatwere used for immunisation (PbTx–HGG) and antibody screening(PbTx–BSA) respectively. Conjugates were recovered by acetoneprecipitation, resuspended in 1 mL distilled water, filter sterilised(0.22 lm), dispensed into sterile tubes then freeze-dried overnightand stored at �20 �C until use.

2.5. Conjugate analysis

The conjugates were analysed using a modification of the meth-ods of Kamps, Carlin, and Shefield (1993). Briefly, TAE was em-ployed for electrophoresis buffer and the mixture solution of0.04% bromophenoland blue and 6.67% sucrose was used as load-ing buffer. Each sample (5 lg lL�1) 1 lL mixed with an equal vol-ume of loading buffer was applied to the gel and samples wereseparated at 230 V for 30–40 min. The gel was fixed with 20% tri-chloroacetic acid for 30 min, stained with Coomassie blue R-250for more than 2 h and destained with ethanol–acetic acid

(250 mL 95% ethanol + 80 mL acetic acid, distilled water to1000 mL) with several changes until be satisfied. The pictures ofthe gels were taken by ultraviolet irradiation (UVI) gel auto imag-ing system.

2.6. Immunization

Immunogen (PbTx–HGG 100 lg, in 0.1 mL PBS, pH 7.4) and fre-unds complete adjuvant (0.1 mL) was injected (female Balb/C mice,6- to 7-week old) at multiple subcutaneously (s.c.) sites as the firstimmunization. The subsequent injections were intraperitoneally(i.p.) with 50 lg of immunogen (0.1 mL) and the same volume Fre-unds incomplete adjuvant at the 3rd, 5th, 7th, and 9th week,respectively. After the third immunization, at the 10th day of eachinjection, the animals were bled by tail–tip cut method, and theserum was tested for its ability to bind to PbTx–BSA or the carrierprotein HGG and BSA. The animals, whose serum titres (serumantibody titres) bind to PbTx–BSA were 4 � 103 or higher were se-lected to be spleen donors for hybridoma production, and receivedintravenous injection (i.v.) boosts of 20 lg immunogen conjugatein PBS (pH 7.0). Four days after the final boost, spleens were re-moved from immunized mice.

2.7. Hybridoma screening and generation

The immunized mice spleen cells were mixed with myelomacells (SP2/0) at a 5–10:1 (spleen: myeloma) ratio in the presenceof polyethylene glycol (PEG), mol. wt 4000, and plated into 96-welltissue culture plates filled with RPMI + 20% FBS/HAT medium at37 �C in an atmosphere of 5% CO2. Specific antibody-secretinghybridoma were screened by ELISA employing PbTx–BSA as targetantigen, BSA and HGG as non-relevant control antigens. Hybrid-oma showing significant PbTx-specific inhibition were clonedthree times by limiting dilution. One clone was chosen for furtherstudy.

2.8. Ascites produce and mAb affinity determination

Hybridoma (2 � 106 cells for each mice) were injected intoabdomens of the 9- to 10-week old Balb/C mice for seven days afterliquid olefin was injected. The ascites were obtained through theneedle of a 20 mL injector about seven days later. The mAb waspurified from ascites using the method of Zhou et al. (2006).MAb subtyping was performed by an ELISA commercial kit ‘‘mousemonoclonal antibody isotyping reagents” (Sigma), and the opera-tional procedure based on its manufacturer’s instructions. Theaffinity of mAb for PbTxs was measured by a competitive enzymeimmunoassay according to the method of Dong and Wang (2002).

2.9. Protocol of direct competitive ELISA

A direct competition ELISA format was utilised to measurePbTx-2 binding and cross-reactivity (CR) to related compoundsPbTx-1, PbTx-3, PbTx-9, ciguatoxin-1, okadaic acid, microcystinLR and dinophysistoxin. Indirect ELISA was carried out as follows:(1) 200 lL of coating antigen PbTx–BSA diluted with the coatingbuffer at 0.5 lg mL�1 was added into a microtiter plate and incu-bated overnight at 4 �C. (2) Plates were washed three times using300 lL well�1 of PBST (10 mM PBS containing 0.05% Tween 20,pH 7.4) and then followed by incubation with 50 lL well�1 of stan-dard analyte (for inhibition assay) in 0.01 M PBS at different dilu-tions or sample solution or blank together with the mAb (1:8000with the dilution buffer solution, 50 lL well�1) for 30 min at37 �C. (3) After washing three times, 100 lL of HRP–IgG (workingconcentration recommend 1:4000) was then added to each welland incubated for 30 min at 37 �C. (4) The plates were washed

Y. Zhou et al. / Food Chemistry 118 (2010) 467–471 469

again, and 100 lL well�1 of TMB solution was added. (5) After incu-bation for 15 min at 37 �C, the reaction was stopped by adding50 lL of 2 M H2SO4 well�1. (6) The absorbance was measured at450 nm and recorded. Standard curves were obtained by plottingabsorbance against the logarithm of analyte concentration.

2.10. Spiking of mollusk extracts

Non-toxic cockle Austrovenus stuchibuli and oyster Crassostreavirginica were purchased from a local supermarket. The tissuewas thoroughly disrupted in dimethyl sulfoxide (50% w/v) andthen centrifuged at 1500 g for 15 min. The supernatant was passedsequentially through a 100 mm nylon mesh, Whatman No. 1 filterand then finally through a Whatman GF/B filter yielding a slightlyopalescent yellowcolored extract. Standard analyte of PbTx-2,PbTx-1, PbTx-3 and PbTx-9 was incorporated respectively at therequired concentrations using vortexing. Recoveries were deter-mined at concentrations of 4, 40 and 400 ng/g of whole shellfishmeat (n = 5) by the use of non-toxic cockle or oyster homogenatesspiked with standard analytes.

In order to see if the under- or overestimation in recoveries canbe rationalised by the matrix effect of the extract of the mollusks,unspiked cockles or oysters were extracted with 10 mL of dimethylsulfoxide as described above and standard analyte of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 was spiked respectively into the extract toobtain standard curves. The standard curve in the buffer was alsoobtained.

3. Results and discussion

3.1. Analysis of the conjugates

Conjugates were prepared according to the procedure describedin Section 2. In nondenaturing agarose gel electrophoresis, themore negative charged protein migrates further in the gel towardsthe anode (+) than the more positive charged protein. The couplingof protein with protein modifier and haptens will induce thechanges of the protein charge, therefore the migration of carrierprotein, treated protein and hapten-carrier protein will be differ-ent. Fig. 1 illustrates the results of nondenaturing agarose gel elec-trophoresis for conjugates. The net charge of PbTx–HGG (lane 3)becomes more negative than that of treated HGG (lane 2) anduntreated HGG (line 1), so it migrates to a greater extent towardsthe anode (+), and the migration of PbTx–HGG and treated HGG

Fig. 1. Analysis of PbTx–HGG and PbTx–BSA conjugations by non-denaturingagarose gel electrophoresis. Lane 1. HGG, lane 2: treated HGG (the procedure wasperformed according to Section 2, only PbTxs was not added), lane 3: PbTx–HGGconjugates, lane 4: BSA, lane 5: treated BSA (the procedure was performedaccording to Section 2, only PbTxs was not added), lane 6. PbTx–BSA conjugates.

became decentralized. The conjugate band migrations were differ-ent from those of treated proteins and carrier proteins alone. Thenet charge of PbTx–BSA (lane 6) becomes more negative, soPbTx–BSA migrates to a greater extent towards the anode (+) thanthat of treated BSA and untreated BSA, and the migration of PbTx–BSA and treated BSA became decentralized. The results indicatedthat PbTx was coupled with BSA and HGG successfully.

3.2. Antisera titres of immunized mice and screening of hybridoma

Like most other marine toxins, PbTx, as hapten, has no immuno-genicity and must be conjugated to a protein carrier to add immu-nogenicity. A previous study indicated that the titres of producedantibody for the hapten changed according to the carrier proteinused in the conjugation and different mice (Kentaro, Yonekazu, &Tamao, 1999). In the present study, six mice were immunized byPbTx–HGG. Table 1 shows the results of serum titres of immunizedmice. There are four mice whose serum titres were 4 � 103 higher.No. 6 mouse was selected to be spleen donors for hybridoma pro-duction. Hybridoma supernatants were screened by ELISA for bind-ing to target and control antigens in parallel, then positive cloneswere checked for PbTx-2 reactivity by competitive inhibition ELI-SA. A screened hybridoma cell line designated 2C4, was establishedafter being subcloned for 3 cycles by ‘‘limiting dilution”.

3.3. The characteristics of mAb

The hybridoma cells were expanded and injected into Balb/Cmouse abdomen. The mAb was obtained from ascites and purifiedby the caprylic/ammonium sulphate precipitation (CA–AS) meth-od. The protein concentration of ascites was 24.5 mg mL�1, calcu-lated as following equation (Liu, Cai, Wang, & Li, 1999):Concentration (mg mL�1) = 1.45 � OD280–0.74 � OD260. HereOD280 is the absorption value of ascites at 280 nm, OD260 is theabsorption value of ascites at 260 nm. The ELISA titres of ascitesand purified mAb were 3.2 � 105 and 6.4 � 105, respectively. ThemAb secreted by 2C4 belongs to the IgG1 class.

The average affinity of mAb was 0.82 � 109 M�1 calculated asfollowing equation (Dong & Wang, 2002):

Ka ¼ n� 12ðn½Ab0� � ½Ab�Þ

ð1Þ

Here n is the concentration time of two different concentrationplate coating antigens in one group, [Ab0] and [Ab] are the mAbconcentrations (ng L�1) correspond to 50% of maximum absorptionvalues of two different concentration plate coating antigens.

The CR was calculated according to the following equation:

CR% ¼ standardIC50

cross-reactantIC50ð2Þ

Table 1Titres of antisera from the immunized mice by indirect ELISAa.

Mouse no. Titers of antiserum

PbTx–HGGb PbTx–BSAc BSAc HGGc

1 1:8000 Negative 1:160002 1:400 1:200 1:80003 1:16000 Negative 1:640004 1:8000 Negative 1:320005 1:4000 1:400 1:16006 1:32000 Negative 1:64000

a The titrations by indirect ELISA were performed for the serum of mice after thefourth booster.

b As immunogen.c As plate-coating antigen.

470 Y. Zhou et al. / Food Chemistry 118 (2010) 467–471

IC50 is PbTx concentration reducing the ELISA maximum re-sponse to 50%. CR (%) of PbTx-2 was defined as 100%. The CR ofthe mAb with some other marine toxins is shown in Table 2. ThemAb showed high CR values with PbTx-1, PbTx-3 and PbTx-9,low CR values with ciguatoxin-1 and okadaic acid, and no signifi-cant CR values with microcystin LR and dinophysistoxin in the di-rect competitive ELISA. Therefore the mAb secreted by 2C4 is class-selective and has potential to be used for broad spectrum assay ofPbTx-1, PbTx-2, PbTx-3 and PbTx-9. It is expensive and time con-suming to produce the brevetoxin antiserum, which depends on

Table 2Specificity of the mAb to different marine toxins in the competitive ELISAa usingPbTx–BSA as coating antigen.

Compound IC50 (ng mL�1)b CR (%)

PbTx-2 6.4 100.00PbTx-1 6.57 97.45PbTx-3 5.31 120.63PbTx-9 7.15 89.56Ciguatoxin-1 175.82 3.64Okadaic acid 216.22 2.96Microcystin LR #c <0.1Dinophysistoxin #c <0.1

a Antigen coating solution was made in PBS at 500 ng mL�1 and final ascitesdilution was 1:64,000 in PBS-T.

b Data represent the means of five experiments.c The IC50 cannot be evaluated.

Fig. 2. Direct competition ELISA curves for PbTxs in matrix extract of cockle and oyster. Stby direct competition ELISA, (c) PbTx-3 in extract by direct competition ELISA, (d) PbTx-9in text.

the continual supply of animals and brevetoxins, and it is difficultto get the uniform antiserum due to the individual difference ofanimals. The uniform mAb can be readily obtained which dosenot need brevetoxins.

3.4. Extraction recovery

In order to understand the under- and overestimation in recov-ery values, unspiked cockles or oysters were extracted with 10 mLof dimethyl sulfoxide and standard analyte of PbTx-2, PbTx-1,PbTx-3 and PbTx-9 was spiked respectively into the extract to ob-tain standard curves. The standard curves obtained in the matrixextract are presented in Fig. 2. No remarkable matrix extract inter-secting appears in standard curves of buffer, cockle and oyster ma-trix extract.

Recoveries of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 extractedfrom tissue homogenates spiked at three dose levels are shownin Table 3. Recoveries of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 were98.7%–104.3%, 92.9%–101.2%, 95.4%–103.2% and 88.4%–99.3%,respectively and the recoveries of analytes from cockle (88.4%–102.3%) and oyster (89.4%–104.3%) were very similar, which is con-sistent with the corresponding standard curves in the matrix ex-tract intersecting the standard curve in the buffer (Fig. 2). Theresults demonstrated that the matrices of cockle and oyster wherePbTxs are found do not interfere remarkably with the assay. Theoptimised ELISA detection limit was calculated by taking the meanabsorbance value of 10 blank wells plus 2 times standard devia-tions of the mean. The detection limit was found to be

andard curve (a) PbTx-2 in extract by direct competition ELISA, (b) PbTx-1 in extractin extract by direct competition ELISA. The details of assay protocol were described

Table 3Rcoveries of toxins from spiked homogenates of whole meat of shellfisha.

Toxin Concentration (ng/g) Recovery (%) of toxins from

Cockle Oyster

PbTx-2 4 102.1 ± 1.3 98.7 ± 2.440 99.7 ± 0.8 104.3 ± 0.3

400 101.3 ± 0.6 102.2 ± 2.7PbTx-1 4 98.4 ± 1.3 92.9 ± 3.2

40 101.2 ± 2.1 99.5 ± 2.2400 97.8 ± 0.7 95.4 ± 3.4

PbTx-3 4 95.4 ± 1.6 103.2 ± 3.140 99.3 ± 2.5 101.8 ± 2.3

400 102.3 ± 0.9 98.5 ± 4.1PbTx-9 4 88.4 ± 3.1 92.3 ± 3.3

40 98.6 ± 0.4 89.4 ± 2.9400 99.3 ± 1.2 96.2 ± 0.8

Results are mean ± SD (n = 5).a Assay conditions were described in the text.

Y. Zhou et al. / Food Chemistry 118 (2010) 467–471 471

0.6 ng well�1, close to the sensitivity of immunochromatographicassay (Zhou et al., 2009), higher than electrochemi-lumines-cence-based competitive displacement immunoassay (Poli et al.,2007) and lower than ELISA (Naar et al., 2001). The assay couldbe accomplished within 1.5 h, less than that of other immunoas-says (Naar et al., 2001; Twiner et al., 2007).

4. Conclusions

We produced a monoclonal antibody which shows high-affinityagainst PbTxs, and developed monoclonal antibody-based compet-itive ELISA for detection of PbTxs. The IC50 value of the optimiseddirect competitive ELISA was 5.3 ng mL�1 with a detection limit of0.6 ng well�1. The antibody showed high CR with PbTx-1, PbTx-3and PbTx-9, negligible CR with ciguatoxin-1 and okadaic acid,and no signigcant CR with microcystin LR and dinophysistoxin.Recoveries of PbTxs from spiked cockle and oyster ranged 88.4%–102.3% and 89.4%–104.3%, respectively. The newly developed com-petitive ELISA seems to be a useful method for monitoring PbTxs inmollusk.

Acknowledgement

This work was supported by the National Natural Science Foun-dation of China (No. 30771657).

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