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Development of a new monoclonal antibody based direct competitive enzyme-linked immunosorbent assay for detection of brevetoxins in food samples
Food Chemistry 118 (2010) 467–471
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Development of a new monoclonal antibody based direct competitive enzyme-linked immunosorbent assay for detection of brevetoxins in 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 Zhang Key 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 2008 Received in revised form 13 April 2009 Accepted 2 May 2009
Keywords: Hybridoma cell line Monoclonal antibody Enzyme-linked immunosorbent assay Brevetoxin Cross-reactivity Recovery
a b s t r a c t Brevetoxin B (PbTx-2) was covalently linked to carrier protein bovine serum albumin and human gamma globulin. 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 monoclonal antibody were studied. An direct competitive enzyme-linked immunosorbent assay (ELISA) for detection of PbTxs was developed, which showed an IC50 value of 5.3 ng mL1 with a detection limit of 0.6 ng well1. The recoveries of PbTxs from cockle (88.4%–102.3%) and oyster (89.4%–104.3%) demonstrated 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 monitoring of PbTxs in mollusk. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Brevetoxins (PbTxs) are potent marine neurotoxins produced by the planktonic red tide dinoflagellate Karenia brevis and are accumulated in filter-feeding molluscan shellfish by dietary transfer. They are lipid-soluble polycyclic polyether compounds and are the only molecules known to activate voltage sensitive sodium channels in mammals through a specific interaction with site 5 of the alpha subunit of the sodium channel (Dechraoui, Naar, Pauillac, & Legrand, 1999). Human ingestion of toxic shellfish causes neurotoxic shellfish poisoning (NSP). Although PbTxs can be used as powerful tools in neuroscience research (Naar, Branaaa, Bottein-Dechraoui, Chinain, & Pauillac, 2001), their detrimental effects on human health and negative impact on seafood industries have mainly raised global awareness. Therefore it is very important to develop a precise, sensitive, reproducible and specific detection method as an alternative to the conventional mouse intraperitoneal (i.p.) bioassay (Lewis, 1995), solid-phase extraction (SPE) assay, receptor binding assay (RBA), radioimmuno-assay (RIA), and liquid chromatography–mass spectrometry (LC–MS) (Twiner et al., 2007), competitive electro-chemiluminescence-based immunoassay (Poli et al., 2007), cell based assay (Fairey, Bottein * Corresponding author. Tel.: +86 0431 87836718. E-mail address: [email protected] (Y. Zhou). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.05.015
Dechraoui, Sheets, & Ramsdell, 2001) and in vitro assays (Plakas et al., 2008). Immunological methods based on antibodies are simple, sensitive, economical and high throughput procedure for quantifying biologically relevant compounds, but require a continuous supply of well-defined specific antibodies. To date, Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins (Naar et al., 2001) and polyclonal antisera to PbTxs have been raised in animals such as 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 high affinity, but usually available in only limited supply and require batch standardisation and continual supply of antigen for booster injections. On the other hand, the antigens (PbTxs) are very expensive, so it is restricted to detect this kind of compounds by polyclonal 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 are small molecules, haptens must be synthesised and coupled to carrier proteins to induce antibody production. In this study, we aimed at obtaining mAbs against PbTxs and developing mAb-based enzyme-linked immunosorbent assay (ELISA) with better performance using a larger number of haptens as competitors.
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(250 mL 95% ethanol + 80 mL acetic acid, distilled water to 1000 mL) with several changes until be satisfied. The pictures of the gels were taken by ultraviolet irradiation (UVI) gel auto imaging system.
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 (the purity P 95% by HPLC) were obtained from Sigma Chemicals Co. (St. Louis, MO, USA). Agarose, complete and incomplete Freund/s adjuvant, polythylene glycol-4000 (PEG), RPMI 1640, foetal bovine serum (FBS), horseradish peroxidase-conjugated goat anti-mice IgG (HRP–IgG), bovine serum albumin (BSA), human gamma globulin (HGG), HT and HAT were purchased from Express China. All other reagents were of analytical grade. The ELISA was carried out in 96-well polystyrene microtiter plates (Stripwell plate 2592, Costar, Changchun, China). Well absorbencies were read with 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, were obtained from Changchun Institute of Biological Products, Jilin province China.
2.6. Immunization Immunogen (PbTx–HGG 100 lg, in 0.1 mL PBS, pH 7.4) and freunds complete adjuvant (0.1 mL) was injected (female Balb/C mice, 6- to 7-week old) at multiple subcutaneously (s.c.) sites as the first immunization. The subsequent injections were intraperitoneally (i.p.) with 50 lg of immunogen (0.1 mL) and the same volume Freunds incomplete adjuvant at the 3rd, 5th, 7th, and 9th week, respectively. After the third immunization, at the 10th day of each injection, the animals were bled by tail–tip cut method, and the serum was tested for its ability to bind to PbTx–BSA or the carrier protein HGG and BSA. The animals, whose serum titres (serum antibody titres) bind to PbTx–BSA were 4 103 or higher were selected to be spleen donors for hybridoma production, and received intravenous injection (i.v.) boosts of 20 lg immunogen conjugate in PBS (pH 7.0). Four days after the final boost, spleens were removed from immunized mice. 2.7. Hybridoma screening and generation
2.3. Buffers and solutions 1
The buffers used regularly were coating buffer, 50 mmol L carbonate buffer (pH 9.5); phosphate buffered saline (PBS), 10 mmol L1 sodium phosphate buffer (pH 7.4) containing 140 mmol L1 NaCl; dilution buffer, PBS containing 0.1% (w/v) gelatin; washing buffer (PBST), 10 mmol L1 sodium phosphate buffer (pH 7.4) containing 140 mmol L1 NaCl and 0.05% (v/v) Tween 20; and TMB solution, 70 lL of 0.65% H2O2, 250 lL of 10 mg mL1 3,3/ 5,5/-tetramethylbenzidine (TMB) in dimethylsulfoxide (DMSO) per 25 mL of phosphate citrate buffer, pH 5.4. 2.4. Preparation of PbTx–protein conjugates Conjugates were prepared according to a modified method of Naar et al. (2001). Briefly, A 10-fold molar excess of succinic anhydride solubilised in 10 mL of anhydrous pyridine was added to 8 mg of crystalline PbTx-2. After incubation (6 h at 65 °C) the solvent was evaporated under a stream of nitrogen and the residue was reacted with a 10-fold molar excess of tributylamine and isobutyl chlorocarbonate as 1/10th dilutions in dry peroxide-free dioxan for 30 min at room temperature. Then the carrier protein HGG and BSA (the same hapten/carrier molar ratio) were added at room temperature and incubated for more than 30 min, that were used for immunisation (PbTx–HGG) and antibody screening (PbTx–BSA) respectively. Conjugates were recovered by acetone precipitation, resuspended in 1 mL distilled water, filter sterilised (0.22 lm), dispensed into sterile tubes then freeze-dried overnight and stored at 20 °C until use. 2.5. Conjugate analysis The conjugates were analysed using a modification of the methods of Kamps, Carlin, and Shefield (1993). Briefly, TAE was employed for electrophoresis buffer and the mixture solution of 0.04% bromophenoland blue and 6.67% sucrose was used as loading buffer. Each sample (5 lg lL1) 1 lL mixed with an equal volume of loading buffer was applied to the gel and samples were separated at 230 V for 30–40 min. The gel was fixed with 20% trichloroacetic acid for 30 min, stained with Coomassie blue R-250 for more than 2 h and destained with ethanol–acetic acid
The immunized mice spleen cells were mixed with myeloma cells (SP2/0) at a 5–10:1 (spleen: myeloma) ratio in the presence of polyethylene glycol (PEG), mol. wt 4000, and plated into 96-well tissue culture plates filled with RPMI + 20% FBS/HAT medium at 37 °C in an atmosphere of 5% CO2. Specific antibody-secreting hybridoma were screened by ELISA employing PbTx–BSA as target antigen, BSA and HGG as non-relevant control antigens. Hybridoma showing significant PbTx-specific inhibition were cloned three times by limiting dilution. One clone was chosen for further study. 2.8. Ascites produce and mAb affinity determination Hybridoma (2 106 cells for each mice) were injected into abdomens of the 9- to 10-week old Balb/C mice for seven days after liquid olefin was injected. The ascites were obtained through the needle of a 20 mL injector about seven days later. The mAb was purified from ascites using the method of Zhou et al. (2006). MAb subtyping was performed by an ELISA commercial kit ‘‘mouse monoclonal antibody isotyping reagents” (Sigma), and the operational procedure based on its manufacturer’s instructions. The affinity of mAb for PbTxs was measured by a competitive enzyme immunoassay according to the method of Dong and Wang (2002). 2.9. Protocol of direct competitive ELISA A direct competition ELISA format was utilised to measure PbTx-2 binding and cross-reactivity (CR) to related compounds PbTx-1, PbTx-3, PbTx-9, ciguatoxin-1, okadaic acid, microcystin LR and dinophysistoxin. Indirect ELISA was carried out as follows: (1) 200 lL of coating antigen PbTx–BSA diluted with the coating buffer at 0.5 lg mL1 was added into a microtiter plate and incubated overnight at 4 °C. (2) Plates were washed three times using 300 lL well1 of PBST (10 mM PBS containing 0.05% Tween 20, pH 7.4) and then followed by incubation with 50 lL well1 of standard analyte (for inhibition assay) in 0.01 M PBS at different dilutions or sample solution or blank together with the mAb (1:8000 with the dilution buffer solution, 50 lL well1) for 30 min at 37 °C. (3) After washing three times, 100 lL of HRP–IgG (working concentration recommend 1:4000) was then added to each well and incubated for 30 min at 37 °C. (4) The plates were washed
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again, and 100 lL well1 of TMB solution was added. (5) After incubation for 15 min at 37 °C, the reaction was stopped by adding 50 lL of 2 M H2SO4 well1. (6) The absorbance was measured at 450 nm and recorded. Standard curves were obtained by plotting absorbance against the logarithm of analyte concentration. 2.10. Spiking of mollusk extracts Non-toxic cockle Austrovenus stuchibuli and oyster Crassostrea virginica were purchased from a local supermarket. The tissue was thoroughly disrupted in dimethyl sulfoxide (50% w/v) and then centrifuged at 1500 g for 15 min. The supernatant was passed sequentially through a 100 mm nylon mesh, Whatman No. 1 filter and then finally through a Whatman GF/B filter yielding a slightly opalescent yellowcolored extract. Standard analyte of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 was incorporated respectively at the required concentrations using vortexing. Recoveries were determined at concentrations of 4, 40 and 400 ng/g of whole shellfish meat (n = 5) by the use of non-toxic cockle or oyster homogenates spiked with standard analytes. In order to see if the under- or overestimation in recoveries can be rationalised by the matrix effect of the extract of the mollusks, unspiked cockles or oysters were extracted with 10 mL of dimethyl sulfoxide as described above and standard analyte of PbTx-2, PbTx1, PbTx-3 and PbTx-9 was spiked respectively into the extract to obtain standard curves. The standard curve in the buffer was also obtained. 3. Results and discussion 3.1. Analysis of the conjugates Conjugates were prepared according to the procedure described in Section 2. In nondenaturing agarose gel electrophoresis, the more negative charged protein migrates further in the gel towards the anode (+) than the more positive charged protein. The coupling of protein with protein modifier and haptens will induce the changes of the protein charge, therefore the migration of carrier protein, treated protein and hapten-carrier protein will be different. Fig. 1 illustrates the results of nondenaturing agarose gel electrophoresis for conjugates. The net charge of PbTx–HGG (lane 3) becomes more negative than that of treated HGG (lane 2) and untreated HGG (line 1), so it migrates to a greater extent towards the anode (+), and the migration of PbTx–HGG and treated HGG
became decentralized. The conjugate band migrations were different from those of treated proteins and carrier proteins alone. The net charge of PbTx–BSA (lane 6) becomes more negative, so PbTx–BSA migrates to a greater extent towards the anode (+) than that of treated BSA and untreated BSA, and the migration of PbTx– BSA and treated BSA became decentralized. The results indicated that 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 immunogenicity and must be conjugated to a protein carrier to add immunogenicity. A previous study indicated that the titres of produced antibody for the hapten changed according to the carrier protein used in the conjugation and different mice (Kentaro, Yonekazu, & Tamao, 1999). In the present study, six mice were immunized by PbTx–HGG. Table 1 shows the results of serum titres of immunized mice. There are four mice whose serum titres were 4 103 higher. No. 6 mouse was selected to be spleen donors for hybridoma production. Hybridoma supernatants were screened by ELISA for binding to target and control antigens in parallel, then positive clones were checked for PbTx-2 reactivity by competitive inhibition ELISA. A screened hybridoma cell line designated 2C4, was established after being subcloned for 3 cycles by ‘‘limiting dilution”. 3.3. The characteristics of mAb The hybridoma cells were expanded and injected into Balb/C mouse abdomen. The mAb was obtained from ascites and purified by the caprylic/ammonium sulphate precipitation (CA–AS) method. The protein concentration of ascites was 24.5 mg mL1, calculated as following equation (Liu, Cai, Wang, & Li, 1999): Here Concentration (mg mL1) = 1.45 OD280–0.74 OD260. OD280 is the absorption value of ascites at 280 nm, OD260 is the absorption value of ascites at 260 nm. The ELISA titres of ascites and purified mAb were 3.2 105 and 6.4 105, respectively. The mAb secreted by 2C4 belongs to the IgG1 class. The average affinity of mAb was 0.82 109 M1 calculated as following equation (Dong & Wang, 2002):
Ka ¼
n1 0 2ðn½Ab ½AbÞ
ð1Þ
Here n is the concentration time of two different concentration plate coating antigens in one group, [Ab0 ] and [Ab] are the mAb concentrations (ng L1) correspond to 50% of maximum absorption values of two different concentration plate coating antigens. The CR was calculated according to the following equation:
CR% ¼
standardIC50 cross-reactantIC50
ð2Þ
Table 1 Titres of antisera from the immunized mice by indirect ELISAa.
Fig. 1. Analysis of PbTx–HGG and PbTx–BSA conjugations by non-denaturing agarose gel electrophoresis. Lane 1. HGG, lane 2: treated HGG (the procedure was performed according to Section 2, only PbTxs was not added), lane 3: PbTx–HGG conjugates, lane 4: BSA, lane 5: treated BSA (the procedure was performed according to Section 2, only PbTxs was not added), lane 6. PbTx–BSA conjugates.
Mouse no.
Titers of antiserum
PbTx–HGGb
PbTx–BSAc
BSAc
HGGc
1 2 3 4 5 6
1:8000 1:400 1:16000 1:8000 1:4000 1:32000
Negative 1:200 Negative Negative 1:400 Negative
1:16000 1:8000 1:64000 1:32000 1:1600 1:64000
a The titrations by indirect ELISA were performed for the serum of mice after the fourth booster. b As immunogen. c As plate-coating antigen.
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IC50 is PbTx concentration reducing the ELISA maximum response to 50%. CR (%) of PbTx-2 was defined as 100%. The CR of the mAb with some other marine toxins is shown in Table 2. The mAb showed high CR values with PbTx-1, PbTx-3 and PbTx-9, low CR values with ciguatoxin-1 and okadaic acid, and no significant CR values with microcystin LR and dinophysistoxin in the direct competitive ELISA. Therefore the mAb secreted by 2C4 is classselective and has potential to be used for broad spectrum assay of PbTx-1, PbTx-2, PbTx-3 and PbTx-9. It is expensive and time consuming to produce the brevetoxin antiserum, which depends on
Table 2 Specificity of the mAb to different marine toxins in the competitive ELISAa using PbTx–BSA as coating antigen. Compound
IC50 (ng mL1)b
CR (%)
PbTx-2 PbTx-1 PbTx-3 PbTx-9 Ciguatoxin-1 Okadaic acid Microcystin LR Dinophysistoxin
6.4 6.57 5.31 7.15 175.82 216.22 #c #c
100.00 97.45 120.63 89.56 3.64 2.96 <0.1 <0.1
a Antigen coating solution was made in PBS at 500 ng mL1 and final ascites dilution was 1:64,000 in PBS-T. b Data represent the means of five experiments. c The IC50 cannot be evaluated.
the continual supply of animals and brevetoxins, and it is difficult to get the uniform antiserum due to the individual difference of animals. The uniform mAb can be readily obtained which dose not need brevetoxins. 3.4. Extraction recovery In order to understand the under- and overestimation in recovery values, unspiked cockles or oysters were extracted with 10 mL of dimethyl sulfoxide and standard analyte of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 was spiked respectively into the extract to obtain standard curves. The standard curves obtained in the matrix extract are presented in Fig. 2. No remarkable matrix extract intersecting appears in standard curves of buffer, cockle and oyster matrix extract. Recoveries of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 extracted from tissue homogenates spiked at three dose levels are shown in Table 3. Recoveries of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 were 98.7%–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 consistent with the corresponding standard curves in the matrix extract intersecting the standard curve in the buffer (Fig. 2). The results demonstrated that the matrices of cockle and oyster where PbTxs are found do not interfere remarkably with the assay. The optimised ELISA detection limit was calculated by taking the mean absorbance value of 10 blank wells plus 2 times standard deviations of the mean. The detection limit was found to be
Fig. 2. Direct competition ELISA curves for PbTxs in matrix extract of cockle and oyster. Standard curve (a) PbTx-2 in extract by direct competition ELISA, (b) PbTx-1 in extract by direct competition ELISA, (c) PbTx-3 in extract by direct competition ELISA, (d) PbTx-9 in extract by direct competition ELISA. The details of assay protocol were described in text.
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Table 3 Rcoveries of toxins from spiked homogenates of whole meat of shellfisha.
References
Toxin
Concentration (ng/g)
PbTx-2
4 40 400 4 40 400 4 40 400 4 40 400
Dechraoui, M. Y., Naar, J., Pauillac, S. A., & Legrand, M. (1999). Ciguatoxins and brevetoxins, neurotoxic polyether compounds active on sodium channels. Toxicon, 37, 125–143. Dong, Z. W., & Wang, Y. (2002). Antibody engineering. Beijing: Beijing Medical University Press. p. 281. Fairey, E. R., Bottein Dechraoui, M. Y., Sheets, M. F., & Ramsdell, J. S. (2001). Modification of the cell based assay for brevetoxins using human cardiac voltage dependent sodium channels expressed in HEK-293 cells. Biosensors & Bioelectronics, 16, 579–586. Kamps, C., Carlin, R. G., & Shefield, L. (1993). Analysis of hapten-carrier protein conjugates by nondenaturing gel electrophoresis. Journal of Immunological Methods, 164, 245–253. Kentaro, K., Yonekazu, H., & Tamao, N. (1999). Production and characterization of a monoclonal antibody against domoic acid and its application to enzyme immunoassay. Toxicon, 37, 1579–1589. Levine, L., & Shimizu, Y. (1992). Antibodies to brevetoxin B: Serologic differentiation of brevetoxin B and brevetoxin A. Toxicon, 30, 411–418. Lewis, R. J. (1995). Detection of ciguatoxins and related benthic dinoagellate toxins: In vivo and in vitro methods. Manual on Harmful Marine Microalgae, 923, 135–161. Liu, X. B., Cai, M. Y., Wang, X., & Li, X. R. (1999). One simple and efficient method for purification of IgG McAb from mice ascites: Caprylic acid/ammonium sulfate precipitation. Journal of Sichuan University (Medical Science Edition), 30, 455–456. Melinek, R., Rein, K. S., Schultz, D. R., & Baden, D. G. (1994). Brevetoxin PbTx-2 immunology: differential epitope recognition by antibodies from two goats. Toxicon, 32, 883–890. Naar, J., Branaaa, P., Bottein-Dechraoui, M. Y., Chinain, M., & Pauillac, S. (2001). Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins using minute amount of hapten-protein conjugates obtained in a reversed micellar medium. Toxicon, 39, 869–878. Plakas, S. M., Jester, E. L. E., El Said, K. R., Granade, H. R., Abraham, A., Dickey, R. W., et al. (2008). Monitoring of brevetoxins in the Karenia brevis bloom-exposed Eastern oyster (Crassostrea virginica). Toxicon, 52, 32–38. Poli, M. A., Rein, K. S., & Baden, D. G. (1995). Radioimmunoassay for PbTx-2-type brevetoxins: Epitope specificity of two anti-PbTx sera. Journal of AOAC International, 78, 538–542. Poli, M. A., Rivera, V. R., Neal, D. D., Baden, D. G., Messer, S. A., Plakas, S. M., et al. (2007). An electrochemi-luminescence-based competitive displacement immunoassay for the type-2 brevetoxins in oyster extracts. Journal of AOAC International, 90, 173–178. Trainer, V. L., & Baden, G. D. (1990). An enzyme immunoassay for the detection of Florida red tide brevetoxins. Toxicon, 29, 1387–1394. Trainer, V. L., & Baden, D. G. (1991). An enzyme immunoassay for the detection of Florida red tide brevetoxins. Toxicon, 29, 1387–1394. Twiner, M. J., Bottein Dechraoui, M. Y., Wang, Z. H., Mikulski, C. M., Henry, M. S., Pierce, R. H., et al. (2007). Extraction and analysis of lipophilic brevetoxins from the red tide dinoflagellate Karenia brevis. Analytical Biochemistry, 369, 128–135. Zhou, Y., Li, Y. S., Pan, F. G., Tan, J. H., Liu, Z. S., & Wang, Z. (2006). The study of purified methods of mice ascites IgG McAb. Heilongjiang Animal Science and Veterinary Medicine, 10, 14–17. Zhou, Y., Pan, F. G., Li, Y. S., Zhang, Y. Y., Zhang, J. H., Lu, S. Y., et al. (2009). Colloidal gold probe-based immunochromatographic assay for the rapid detection of brevetoxins in fishery product samples. Biosensors and Bioelectronics, 24, 2744–2747.
PbTx-1
PbTx-3
PbTx-9
Recovery (%) of toxins from Cockle
Oyster
102.1 ± 1.3 99.7 ± 0.8 101.3 ± 0.6 98.4 ± 1.3 101.2 ± 2.1 97.8 ± 0.7 95.4 ± 1.6 99.3 ± 2.5 102.3 ± 0.9 88.4 ± 3.1 98.6 ± 0.4 99.3 ± 1.2
98.7 ± 2.4 104.3 ± 0.3 102.2 ± 2.7 92.9 ± 3.2 99.5 ± 2.2 95.4 ± 3.4 103.2 ± 3.1 101.8 ± 2.3 98.5 ± 4.1 92.3 ± 3.3 89.4 ± 2.9 96.2 ± 0.8
Results are mean ± SD (n = 5). a Assay conditions were described in the text.
0.6 ng well1, close to the sensitivity of immunochromatographic assay (Zhou et al., 2009), higher than electrochemi-luminescence-based competitive displacement immunoassay (Poli et al., 2007) and lower than ELISA (Naar et al., 2001). The assay could be accomplished within 1.5 h, less than that of other immunoassays (Naar et al., 2001; Twiner et al., 2007). 4. Conclusions We produced a monoclonal antibody which shows high-affinity against PbTxs, and developed monoclonal antibody-based competitive ELISA for detection of PbTxs. The IC50 value of the optimised direct competitive ELISA was 5.3 ng mL1 with a detection limit of 0.6 ng well1. The antibody showed high CR with PbTx-1, PbTx-3 and 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 competitive ELISA seems to be a useful method for monitoring PbTxs in mollusk. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 30771657).
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Development of a new monoclonal antibody based direct competitive enzyme-linked immunosorbent assay for detection of brevetoxins in 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 Zhang Key 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 2008 Received in revised form 13 April 2009 Accepted 2 May 2009
Keywords: Hybridoma cell line Monoclonal antibody Enzyme-linked immunosorbent assay Brevetoxin Cross-reactivity Recovery
a b s t r a c t Brevetoxin B (PbTx-2) was covalently linked to carrier protein bovine serum albumin and human gamma globulin. 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 monoclonal antibody were studied. An direct competitive enzyme-linked immunosorbent assay (ELISA) for detection of PbTxs was developed, which showed an IC50 value of 5.3 ng mL1 with a detection limit of 0.6 ng well1. The recoveries of PbTxs from cockle (88.4%–102.3%) and oyster (89.4%–104.3%) demonstrated 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 monitoring of PbTxs in mollusk. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Brevetoxins (PbTxs) are potent marine neurotoxins produced by the planktonic red tide dinoflagellate Karenia brevis and are accumulated in filter-feeding molluscan shellfish by dietary transfer. They are lipid-soluble polycyclic polyether compounds and are the only molecules known to activate voltage sensitive sodium channels in mammals through a specific interaction with site 5 of the alpha subunit of the sodium channel (Dechraoui, Naar, Pauillac, & Legrand, 1999). Human ingestion of toxic shellfish causes neurotoxic shellfish poisoning (NSP). Although PbTxs can be used as powerful tools in neuroscience research (Naar, Branaaa, Bottein-Dechraoui, Chinain, & Pauillac, 2001), their detrimental effects on human health and negative impact on seafood industries have mainly raised global awareness. Therefore it is very important to develop a precise, sensitive, reproducible and specific detection method as an alternative to the conventional mouse intraperitoneal (i.p.) bioassay (Lewis, 1995), solid-phase extraction (SPE) assay, receptor binding assay (RBA), radioimmuno-assay (RIA), and liquid chromatography–mass spectrometry (LC–MS) (Twiner et al., 2007), competitive electro-chemiluminescence-based immunoassay (Poli et al., 2007), cell based assay (Fairey, Bottein * Corresponding author. Tel.: +86 0431 87836718. E-mail address: [email protected] (Y. Zhou). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.05.015
Dechraoui, Sheets, & Ramsdell, 2001) and in vitro assays (Plakas et al., 2008). Immunological methods based on antibodies are simple, sensitive, economical and high throughput procedure for quantifying biologically relevant compounds, but require a continuous supply of well-defined specific antibodies. To date, Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins (Naar et al., 2001) and polyclonal antisera to PbTxs have been raised in animals such as 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 high affinity, but usually available in only limited supply and require batch standardisation and continual supply of antigen for booster injections. On the other hand, the antigens (PbTxs) are very expensive, so it is restricted to detect this kind of compounds by polyclonal 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 are small molecules, haptens must be synthesised and coupled to carrier proteins to induce antibody production. In this study, we aimed at obtaining mAbs against PbTxs and developing mAb-based enzyme-linked immunosorbent assay (ELISA) with better performance using a larger number of haptens as competitors.
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(250 mL 95% ethanol + 80 mL acetic acid, distilled water to 1000 mL) with several changes until be satisfied. The pictures of the gels were taken by ultraviolet irradiation (UVI) gel auto imaging system.
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 (the purity P 95% by HPLC) were obtained from Sigma Chemicals Co. (St. Louis, MO, USA). Agarose, complete and incomplete Freund/s adjuvant, polythylene glycol-4000 (PEG), RPMI 1640, foetal bovine serum (FBS), horseradish peroxidase-conjugated goat anti-mice IgG (HRP–IgG), bovine serum albumin (BSA), human gamma globulin (HGG), HT and HAT were purchased from Express China. All other reagents were of analytical grade. The ELISA was carried out in 96-well polystyrene microtiter plates (Stripwell plate 2592, Costar, Changchun, China). Well absorbencies were read with 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, were obtained from Changchun Institute of Biological Products, Jilin province China.
2.6. Immunization Immunogen (PbTx–HGG 100 lg, in 0.1 mL PBS, pH 7.4) and freunds complete adjuvant (0.1 mL) was injected (female Balb/C mice, 6- to 7-week old) at multiple subcutaneously (s.c.) sites as the first immunization. The subsequent injections were intraperitoneally (i.p.) with 50 lg of immunogen (0.1 mL) and the same volume Freunds incomplete adjuvant at the 3rd, 5th, 7th, and 9th week, respectively. After the third immunization, at the 10th day of each injection, the animals were bled by tail–tip cut method, and the serum was tested for its ability to bind to PbTx–BSA or the carrier protein HGG and BSA. The animals, whose serum titres (serum antibody titres) bind to PbTx–BSA were 4 103 or higher were selected to be spleen donors for hybridoma production, and received intravenous injection (i.v.) boosts of 20 lg immunogen conjugate in PBS (pH 7.0). Four days after the final boost, spleens were removed from immunized mice. 2.7. Hybridoma screening and generation
2.3. Buffers and solutions 1
The buffers used regularly were coating buffer, 50 mmol L carbonate buffer (pH 9.5); phosphate buffered saline (PBS), 10 mmol L1 sodium phosphate buffer (pH 7.4) containing 140 mmol L1 NaCl; dilution buffer, PBS containing 0.1% (w/v) gelatin; washing buffer (PBST), 10 mmol L1 sodium phosphate buffer (pH 7.4) containing 140 mmol L1 NaCl and 0.05% (v/v) Tween 20; and TMB solution, 70 lL of 0.65% H2O2, 250 lL of 10 mg mL1 3,3/ 5,5/-tetramethylbenzidine (TMB) in dimethylsulfoxide (DMSO) per 25 mL of phosphate citrate buffer, pH 5.4. 2.4. Preparation of PbTx–protein conjugates Conjugates were prepared according to a modified method of Naar et al. (2001). Briefly, A 10-fold molar excess of succinic anhydride solubilised in 10 mL of anhydrous pyridine was added to 8 mg of crystalline PbTx-2. After incubation (6 h at 65 °C) the solvent was evaporated under a stream of nitrogen and the residue was reacted with a 10-fold molar excess of tributylamine and isobutyl chlorocarbonate as 1/10th dilutions in dry peroxide-free dioxan for 30 min at room temperature. Then the carrier protein HGG and BSA (the same hapten/carrier molar ratio) were added at room temperature and incubated for more than 30 min, that were used for immunisation (PbTx–HGG) and antibody screening (PbTx–BSA) respectively. Conjugates were recovered by acetone precipitation, resuspended in 1 mL distilled water, filter sterilised (0.22 lm), dispensed into sterile tubes then freeze-dried overnight and stored at 20 °C until use. 2.5. Conjugate analysis The conjugates were analysed using a modification of the methods of Kamps, Carlin, and Shefield (1993). Briefly, TAE was employed for electrophoresis buffer and the mixture solution of 0.04% bromophenoland blue and 6.67% sucrose was used as loading buffer. Each sample (5 lg lL1) 1 lL mixed with an equal volume of loading buffer was applied to the gel and samples were separated at 230 V for 30–40 min. The gel was fixed with 20% trichloroacetic acid for 30 min, stained with Coomassie blue R-250 for more than 2 h and destained with ethanol–acetic acid
The immunized mice spleen cells were mixed with myeloma cells (SP2/0) at a 5–10:1 (spleen: myeloma) ratio in the presence of polyethylene glycol (PEG), mol. wt 4000, and plated into 96-well tissue culture plates filled with RPMI + 20% FBS/HAT medium at 37 °C in an atmosphere of 5% CO2. Specific antibody-secreting hybridoma were screened by ELISA employing PbTx–BSA as target antigen, BSA and HGG as non-relevant control antigens. Hybridoma showing significant PbTx-specific inhibition were cloned three times by limiting dilution. One clone was chosen for further study. 2.8. Ascites produce and mAb affinity determination Hybridoma (2 106 cells for each mice) were injected into abdomens of the 9- to 10-week old Balb/C mice for seven days after liquid olefin was injected. The ascites were obtained through the needle of a 20 mL injector about seven days later. The mAb was purified from ascites using the method of Zhou et al. (2006). MAb subtyping was performed by an ELISA commercial kit ‘‘mouse monoclonal antibody isotyping reagents” (Sigma), and the operational procedure based on its manufacturer’s instructions. The affinity of mAb for PbTxs was measured by a competitive enzyme immunoassay according to the method of Dong and Wang (2002). 2.9. Protocol of direct competitive ELISA A direct competition ELISA format was utilised to measure PbTx-2 binding and cross-reactivity (CR) to related compounds PbTx-1, PbTx-3, PbTx-9, ciguatoxin-1, okadaic acid, microcystin LR and dinophysistoxin. Indirect ELISA was carried out as follows: (1) 200 lL of coating antigen PbTx–BSA diluted with the coating buffer at 0.5 lg mL1 was added into a microtiter plate and incubated overnight at 4 °C. (2) Plates were washed three times using 300 lL well1 of PBST (10 mM PBS containing 0.05% Tween 20, pH 7.4) and then followed by incubation with 50 lL well1 of standard analyte (for inhibition assay) in 0.01 M PBS at different dilutions or sample solution or blank together with the mAb (1:8000 with the dilution buffer solution, 50 lL well1) for 30 min at 37 °C. (3) After washing three times, 100 lL of HRP–IgG (working concentration recommend 1:4000) was then added to each well and incubated for 30 min at 37 °C. (4) The plates were washed
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again, and 100 lL well1 of TMB solution was added. (5) After incubation for 15 min at 37 °C, the reaction was stopped by adding 50 lL of 2 M H2SO4 well1. (6) The absorbance was measured at 450 nm and recorded. Standard curves were obtained by plotting absorbance against the logarithm of analyte concentration. 2.10. Spiking of mollusk extracts Non-toxic cockle Austrovenus stuchibuli and oyster Crassostrea virginica were purchased from a local supermarket. The tissue was thoroughly disrupted in dimethyl sulfoxide (50% w/v) and then centrifuged at 1500 g for 15 min. The supernatant was passed sequentially through a 100 mm nylon mesh, Whatman No. 1 filter and then finally through a Whatman GF/B filter yielding a slightly opalescent yellowcolored extract. Standard analyte of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 was incorporated respectively at the required concentrations using vortexing. Recoveries were determined at concentrations of 4, 40 and 400 ng/g of whole shellfish meat (n = 5) by the use of non-toxic cockle or oyster homogenates spiked with standard analytes. In order to see if the under- or overestimation in recoveries can be rationalised by the matrix effect of the extract of the mollusks, unspiked cockles or oysters were extracted with 10 mL of dimethyl sulfoxide as described above and standard analyte of PbTx-2, PbTx1, PbTx-3 and PbTx-9 was spiked respectively into the extract to obtain standard curves. The standard curve in the buffer was also obtained. 3. Results and discussion 3.1. Analysis of the conjugates Conjugates were prepared according to the procedure described in Section 2. In nondenaturing agarose gel electrophoresis, the more negative charged protein migrates further in the gel towards the anode (+) than the more positive charged protein. The coupling of protein with protein modifier and haptens will induce the changes of the protein charge, therefore the migration of carrier protein, treated protein and hapten-carrier protein will be different. Fig. 1 illustrates the results of nondenaturing agarose gel electrophoresis for conjugates. The net charge of PbTx–HGG (lane 3) becomes more negative than that of treated HGG (lane 2) and untreated HGG (line 1), so it migrates to a greater extent towards the anode (+), and the migration of PbTx–HGG and treated HGG
became decentralized. The conjugate band migrations were different from those of treated proteins and carrier proteins alone. The net charge of PbTx–BSA (lane 6) becomes more negative, so PbTx–BSA migrates to a greater extent towards the anode (+) than that of treated BSA and untreated BSA, and the migration of PbTx– BSA and treated BSA became decentralized. The results indicated that 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 immunogenicity and must be conjugated to a protein carrier to add immunogenicity. A previous study indicated that the titres of produced antibody for the hapten changed according to the carrier protein used in the conjugation and different mice (Kentaro, Yonekazu, & Tamao, 1999). In the present study, six mice were immunized by PbTx–HGG. Table 1 shows the results of serum titres of immunized mice. There are four mice whose serum titres were 4 103 higher. No. 6 mouse was selected to be spleen donors for hybridoma production. Hybridoma supernatants were screened by ELISA for binding to target and control antigens in parallel, then positive clones were checked for PbTx-2 reactivity by competitive inhibition ELISA. A screened hybridoma cell line designated 2C4, was established after being subcloned for 3 cycles by ‘‘limiting dilution”. 3.3. The characteristics of mAb The hybridoma cells were expanded and injected into Balb/C mouse abdomen. The mAb was obtained from ascites and purified by the caprylic/ammonium sulphate precipitation (CA–AS) method. The protein concentration of ascites was 24.5 mg mL1, calculated as following equation (Liu, Cai, Wang, & Li, 1999): Here Concentration (mg mL1) = 1.45 OD280–0.74 OD260. OD280 is the absorption value of ascites at 280 nm, OD260 is the absorption value of ascites at 260 nm. The ELISA titres of ascites and purified mAb were 3.2 105 and 6.4 105, respectively. The mAb secreted by 2C4 belongs to the IgG1 class. The average affinity of mAb was 0.82 109 M1 calculated as following equation (Dong & Wang, 2002):
Ka ¼
n1 0 2ðn½Ab ½AbÞ
ð1Þ
Here n is the concentration time of two different concentration plate coating antigens in one group, [Ab0 ] and [Ab] are the mAb concentrations (ng L1) correspond to 50% of maximum absorption values of two different concentration plate coating antigens. The CR was calculated according to the following equation:
CR% ¼
standardIC50 cross-reactantIC50
ð2Þ
Table 1 Titres of antisera from the immunized mice by indirect ELISAa.
Fig. 1. Analysis of PbTx–HGG and PbTx–BSA conjugations by non-denaturing agarose gel electrophoresis. Lane 1. HGG, lane 2: treated HGG (the procedure was performed according to Section 2, only PbTxs was not added), lane 3: PbTx–HGG conjugates, lane 4: BSA, lane 5: treated BSA (the procedure was performed according to Section 2, only PbTxs was not added), lane 6. PbTx–BSA conjugates.
Mouse no.
Titers of antiserum
PbTx–HGGb
PbTx–BSAc
BSAc
HGGc
1 2 3 4 5 6
1:8000 1:400 1:16000 1:8000 1:4000 1:32000
Negative 1:200 Negative Negative 1:400 Negative
1:16000 1:8000 1:64000 1:32000 1:1600 1:64000
a The titrations by indirect ELISA were performed for the serum of mice after the fourth booster. b As immunogen. c As plate-coating antigen.
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IC50 is PbTx concentration reducing the ELISA maximum response to 50%. CR (%) of PbTx-2 was defined as 100%. The CR of the mAb with some other marine toxins is shown in Table 2. The mAb showed high CR values with PbTx-1, PbTx-3 and PbTx-9, low CR values with ciguatoxin-1 and okadaic acid, and no significant CR values with microcystin LR and dinophysistoxin in the direct competitive ELISA. Therefore the mAb secreted by 2C4 is classselective and has potential to be used for broad spectrum assay of PbTx-1, PbTx-2, PbTx-3 and PbTx-9. It is expensive and time consuming to produce the brevetoxin antiserum, which depends on
Table 2 Specificity of the mAb to different marine toxins in the competitive ELISAa using PbTx–BSA as coating antigen. Compound
IC50 (ng mL1)b
CR (%)
PbTx-2 PbTx-1 PbTx-3 PbTx-9 Ciguatoxin-1 Okadaic acid Microcystin LR Dinophysistoxin
6.4 6.57 5.31 7.15 175.82 216.22 #c #c
100.00 97.45 120.63 89.56 3.64 2.96 <0.1 <0.1
a Antigen coating solution was made in PBS at 500 ng mL1 and final ascites dilution was 1:64,000 in PBS-T. b Data represent the means of five experiments. c The IC50 cannot be evaluated.
the continual supply of animals and brevetoxins, and it is difficult to get the uniform antiserum due to the individual difference of animals. The uniform mAb can be readily obtained which dose not need brevetoxins. 3.4. Extraction recovery In order to understand the under- and overestimation in recovery values, unspiked cockles or oysters were extracted with 10 mL of dimethyl sulfoxide and standard analyte of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 was spiked respectively into the extract to obtain standard curves. The standard curves obtained in the matrix extract are presented in Fig. 2. No remarkable matrix extract intersecting appears in standard curves of buffer, cockle and oyster matrix extract. Recoveries of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 extracted from tissue homogenates spiked at three dose levels are shown in Table 3. Recoveries of PbTx-2, PbTx-1, PbTx-3 and PbTx-9 were 98.7%–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 consistent with the corresponding standard curves in the matrix extract intersecting the standard curve in the buffer (Fig. 2). The results demonstrated that the matrices of cockle and oyster where PbTxs are found do not interfere remarkably with the assay. The optimised ELISA detection limit was calculated by taking the mean absorbance value of 10 blank wells plus 2 times standard deviations of the mean. The detection limit was found to be
Fig. 2. Direct competition ELISA curves for PbTxs in matrix extract of cockle and oyster. Standard curve (a) PbTx-2 in extract by direct competition ELISA, (b) PbTx-1 in extract by direct competition ELISA, (c) PbTx-3 in extract by direct competition ELISA, (d) PbTx-9 in extract by direct competition ELISA. The details of assay protocol were described in text.
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Table 3 Rcoveries of toxins from spiked homogenates of whole meat of shellfisha.
References
Toxin
Concentration (ng/g)
PbTx-2
4 40 400 4 40 400 4 40 400 4 40 400
Dechraoui, M. Y., Naar, J., Pauillac, S. A., & Legrand, M. (1999). Ciguatoxins and brevetoxins, neurotoxic polyether compounds active on sodium channels. Toxicon, 37, 125–143. Dong, Z. W., & Wang, Y. (2002). Antibody engineering. Beijing: Beijing Medical University Press. p. 281. Fairey, E. R., Bottein Dechraoui, M. Y., Sheets, M. F., & Ramsdell, J. S. (2001). Modification of the cell based assay for brevetoxins using human cardiac voltage dependent sodium channels expressed in HEK-293 cells. Biosensors & Bioelectronics, 16, 579–586. Kamps, C., Carlin, R. G., & Shefield, L. (1993). Analysis of hapten-carrier protein conjugates by nondenaturing gel electrophoresis. Journal of Immunological Methods, 164, 245–253. Kentaro, K., Yonekazu, H., & Tamao, N. (1999). Production and characterization of a monoclonal antibody against domoic acid and its application to enzyme immunoassay. Toxicon, 37, 1579–1589. Levine, L., & Shimizu, Y. (1992). Antibodies to brevetoxin B: Serologic differentiation of brevetoxin B and brevetoxin A. Toxicon, 30, 411–418. Lewis, R. J. (1995). Detection of ciguatoxins and related benthic dinoagellate toxins: In vivo and in vitro methods. Manual on Harmful Marine Microalgae, 923, 135–161. Liu, X. B., Cai, M. Y., Wang, X., & Li, X. R. (1999). One simple and efficient method for purification of IgG McAb from mice ascites: Caprylic acid/ammonium sulfate precipitation. Journal of Sichuan University (Medical Science Edition), 30, 455–456. Melinek, R., Rein, K. S., Schultz, D. R., & Baden, D. G. (1994). Brevetoxin PbTx-2 immunology: differential epitope recognition by antibodies from two goats. Toxicon, 32, 883–890. Naar, J., Branaaa, P., Bottein-Dechraoui, M. Y., Chinain, M., & Pauillac, S. (2001). Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins using minute amount of hapten-protein conjugates obtained in a reversed micellar medium. Toxicon, 39, 869–878. Plakas, S. M., Jester, E. L. E., El Said, K. R., Granade, H. R., Abraham, A., Dickey, R. W., et al. (2008). Monitoring of brevetoxins in the Karenia brevis bloom-exposed Eastern oyster (Crassostrea virginica). Toxicon, 52, 32–38. Poli, M. A., Rein, K. S., & Baden, D. G. (1995). Radioimmunoassay for PbTx-2-type brevetoxins: Epitope specificity of two anti-PbTx sera. Journal of AOAC International, 78, 538–542. Poli, M. A., Rivera, V. R., Neal, D. D., Baden, D. G., Messer, S. A., Plakas, S. M., et al. (2007). An electrochemi-luminescence-based competitive displacement immunoassay for the type-2 brevetoxins in oyster extracts. Journal of AOAC International, 90, 173–178. Trainer, V. L., & Baden, G. D. (1990). An enzyme immunoassay for the detection of Florida red tide brevetoxins. Toxicon, 29, 1387–1394. Trainer, V. L., & Baden, D. G. (1991). An enzyme immunoassay for the detection of Florida red tide brevetoxins. Toxicon, 29, 1387–1394. Twiner, M. J., Bottein Dechraoui, M. Y., Wang, Z. H., Mikulski, C. M., Henry, M. S., Pierce, R. H., et al. (2007). Extraction and analysis of lipophilic brevetoxins from the red tide dinoflagellate Karenia brevis. Analytical Biochemistry, 369, 128–135. Zhou, Y., Li, Y. S., Pan, F. G., Tan, J. H., Liu, Z. S., & Wang, Z. (2006). The study of purified methods of mice ascites IgG McAb. Heilongjiang Animal Science and Veterinary Medicine, 10, 14–17. Zhou, Y., Pan, F. G., Li, Y. S., Zhang, Y. Y., Zhang, J. H., Lu, S. Y., et al. (2009). Colloidal gold probe-based immunochromatographic assay for the rapid detection of brevetoxins in fishery product samples. Biosensors and Bioelectronics, 24, 2744–2747.
PbTx-1
PbTx-3
PbTx-9
Recovery (%) of toxins from Cockle
Oyster
102.1 ± 1.3 99.7 ± 0.8 101.3 ± 0.6 98.4 ± 1.3 101.2 ± 2.1 97.8 ± 0.7 95.4 ± 1.6 99.3 ± 2.5 102.3 ± 0.9 88.4 ± 3.1 98.6 ± 0.4 99.3 ± 1.2
98.7 ± 2.4 104.3 ± 0.3 102.2 ± 2.7 92.9 ± 3.2 99.5 ± 2.2 95.4 ± 3.4 103.2 ± 3.1 101.8 ± 2.3 98.5 ± 4.1 92.3 ± 3.3 89.4 ± 2.9 96.2 ± 0.8
Results are mean ± SD (n = 5). a Assay conditions were described in the text.
0.6 ng well1, close to the sensitivity of immunochromatographic assay (Zhou et al., 2009), higher than electrochemi-luminescence-based competitive displacement immunoassay (Poli et al., 2007) and lower than ELISA (Naar et al., 2001). The assay could be accomplished within 1.5 h, less than that of other immunoassays (Naar et al., 2001; Twiner et al., 2007). 4. Conclusions We produced a monoclonal antibody which shows high-affinity against PbTxs, and developed monoclonal antibody-based competitive ELISA for detection of PbTxs. The IC50 value of the optimised direct competitive ELISA was 5.3 ng mL1 with a detection limit of 0.6 ng well1. The antibody showed high CR with PbTx-1, PbTx-3 and 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 competitive ELISA seems to be a useful method for monitoring PbTxs in mollusk. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 30771657).