Convenient large-scale purification of yessotoxin from Protoceratium reticulatum culture and isolation of a novel furanoyessotoxin

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.     

检测多杀性巴氏杆菌毒素抗体的单抗竞争ELISA方法的建立

本研究以纯化的多杀性巴氏杆菌毒素基因片段的原核表达产物作为抗原免疫小鼠制备单抗,并利用表达蛋白和多杀性巴氏杆菌毒素单抗酶结合物建立了竞争ELISA方法检测多杀性巴氏杆菌毒素抗体。经过研究确定抗原包被浓度为223ng／mL,待检血清最佳稀释度为1：2,酶标单抗工作浓度为1：3200,血清抑制率大于50%为阳性。应用单抗竞争ELISA和细胞毒性中和试验同时对82份血清进行猪多杀性巴氏杆菌毒素抗体检测,竞争ELISA的检出率为40.2％,细胞毒性中和试验检出率为36.6％,两者符合率达91.5％。试验结果表明,该ELISA方法特异性强,敏感性高,稳定性和重复性好,操作简便。本方法的建立在实验室诊断的标准化、猪群萎缩性鼻炎疫苗免疫效果的评价及流行病学调查方面具有应用价值。     

Validation of the tetrasensor honey test kit for the screening of tetracyclines in honey

Regarding anti-infectious agents, no maximum residue limits are fixed for honey in the European legislation. Discussions are being conducted in order to set working limits at the European level; for example, for tetracyclines, 20 microg/kg was proposed. The Tetrasensor Honey test kit is a receptor-based assay using dipsticks for a rapid screening (30 min) of honey on the presence of tetracyclines. The test was validated according to Commission Decision 2002/657/EC. The test detects tetracycline, oxytetracycline, chlortetracycline, and doxycycline in honey in a specific and sensitive way. Depending on the type of tetracycline, detection capabilities (CCbeta) between 6 and 12 microg/kg were obtained (4-7 microg/kg for dried dipsticks). The test is rugged and participation with the test in an international ring trial gave compliant results. It can be concluded that the Tetrasensor Honey test kit is a simple and reliable test that can even be used at the production site.     

Development of a monoclonal antibody against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal gold immunostrip

A monoclonal antibody (mAb) specific to domoic acid was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with domoic acid--keyhole limpet hemocyanin. The 9F1F11 mAb belongs to the immunoglobulin G1 (kappa-chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. In the cdELISA, the concentration causing 50% inhibition (IC50) of binding of domoic acid-horseradish peroxidase to the antibody by domoic acid was found to be 0.58 ng/mL. A sensitive and rapid mAb-based colloidal gold immunostrip was also developed. The immunostrip assay, which has a detection limit of 5 ng/mL for domoic acid, can be completed in 10 min. Analysis of domoic acid in blue mussel samples revealed that data obtained from immunostrip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunostrip assay established in this study were sensitive and accurate for rapid screening of domoic acid in shellfish samples.     

Development of an enzyme-linked immunosorbent assay for bisphenol a using chicken immunoglobulins

Bisphenol A was coupled, after derivatization into a suitable hapten, to bovine serum albumin and ovalbumin in order to produce immunizing and coating antigens. The immunizing antigens were injected into chickens, which allowed the isolation of specific bisphenol A immunoglobulins from the egg yolk. These antibodies were used in an indirect competitive enzyme-linked immunosorbent assay for the determination of bisphenol A in aqueous solutions. Various parameters, influencing the assay sensitivity, were evaluated. The applicability of the assay for the determination of bisphenol A in milk was also studied. The assay was not as sensitive as other analytical techniques used in bisphenol A analysis, since typical I(50) levels of 2.5 microM were reached in aqueous solutions. This study nevertheless illustrates the usefulness and the potency of chicken antibodies in the analysis of migration residues from packaging materials using immunochemical techniques. In addition, the assay showed to be quite specific for bisphenol A as well. Only for bisphenol A analogues, cross reactivities of about 40% were reached, enabling the use of the antibodies for the screening of bisphenol A and alike compounds.     

Basis for a new procedure to eliminate diarrheic shellfish toxins from a contaminated matrix.

The natural contamination of shellfish with diarrheic shellfish toxins (DSP) has important public health implications. To avoid the economic effects of toxic episodes on shellfish farmers and the related industry, research on artificial methods alternative to the natural detoxification of shellfish is needed. Because the usual thermal processes are not efficient, alternative technologies have to be studied. Here preliminary results are presented about the lability of the DSP toxin okadaic acid in a supercritical atmosphere of carbon dioxide with acetic acid. Most of the toxin is eliminated (up to 90%), and the biological activity against its target enzyme is also severely affected (up to 70% reduction). Detoxification of contaminated shellfish requires a partial dehydration, and the detoxification yield is lower than that obtained with free toxin. Mass spectrometry experiments suggest that acetylation of the toxin molecule is not the basis of the inactivating mechanism, but a conformational change is suggested. This is the first report of the use of supercritical fluids to inactivate toxins.     

Enzyme-linked immunosorbent assay-based detection of free trenbolone in bovine bile

A rapid antibody-based detection system has been developed for the presence of free trenbolone in bovine samples. Polyclonal antibodies were produced that showed specificity toward epitopes located around the steroidal A-ring of the trenbolone molecule. These antibodies were shown to have little or no recognition for many closely related compounds. The antibodies were utilized as the specific biorecognition molecules in competitive and inhibitive enzyme-linked immunosorbent assay systems. While both assays were able to detect low nanogram concentrations of trenbolone in bovine bile, the competitive format was more sensitive (2.41 vs 17.15 ng/mL for TRAb2 and 3.31 vs 30.73 ng/mL for TRAb1). This format was also more accurate and the data produced by this assay fitted more closely to the four parameter equation used to calculate the standard curve. This was a common finding with both of the polyclonal antibodies, suggesting that this was a characteristic of the format used.     

Development of immunoassays for tyramine and tryptamine toxins of Phalaris aquatica L

The leaves of the perennial pasture grass Phalaris aquatica L. (phalaris) contain two groups of known toxins, indole alkaloids, primarily dimethyltryptamines and N-methyltyramines, which cause illnesses in grazing animals, especially sheep. Using amino-reactive and phenolic hydroxyl-reactive homobifunctional reagents, simple methods were devised for coupling toxins representative of those in phalaris to carrier proteins and enzymes for ELISA development. ELISAs were produced for both groups of toxins. Dimethyltryptamines were most sensitively detected [lower limit of detection (LLD) of 1 microg/L for bufotenine] using rabbit anti-bufotenine antibodies, coupled to ovalbumin using divinyl sulfone, with detection using a peroxidase conjugate prepared using the same hapten coupled with 1, 4-butanediol diglycidyl ether. The assay cross-reacted with other toxins of the same class (N,N-dimethyltryptamine and N, N-dimethyl-5-methoxytryptamine) but not with the structurally related amino acids histidine and tryptophan. The most sensitive N-methyltyramine assay (LLD of 1 microg/mL for N-methyltyramine) utilized antisera to tyramine with N-methyltyramine coupled to peroxidase. Significant cross-reaction was seen with the low-grade toxin hordenine, but detection of tyramine was poorer, whereas the amino acid tyrosine was not detected. These assays could be applied to the analysis of simple extracts of Phalaris leaves with minimal interference. A good correspondence was observed between toxin levels by ELISA and estimates from a more tedious thin-layer chromatography method. The method has now been incorporated in a Phalaris breeding program.     

Development of a sensitive enzyme-linked immunosorbent assay for the determination of domoic Acid in shellfish

Polyclonal antibodies for domoic acid were generated from rabbits after the animals had been immunized with either domoic acid-keyhole limpet hemocyanin (KLH) or domoic acid-bovine serum albumin (BSA). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of domoic acid in blue mussels and clams. The antibody titers in the serum of rabbits immunized with domoic acid-KLH were considerably higher than those in rabbits immunized with domoic acid-BSA. The antibodies from the rabbits immunized with domoic acid-KLH were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of domoic acid-horseradish peroxidase to the antibodies by domoic acid and a domoic acid analogue, kainic acid, were found to be 0.75 and 200 ng/mL, respectively. In the presence of blue mussel matrix, the detection limit of domoic acid was <25 ng/g. The overall analytical recovery of domoic acid (25-500 ng/g) added to the blue mussels and then extracted with 50% aqueous methanol in the cdELISA was found to be 81.1%. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method. Analysis of domoic acid in shellfish samples showed that 10 of the 15 shellfish examined were contaminated with domoic acid at levels of <50 ng/g.     

Cell bioassay for paralytic shellfish poisoning (PSP): comparison with postcolumn derivatization liquid chromatographic analysis and application to the monitoring of PSP in shellfish

We performed a neuroblastoma cell (Neuro2a) culture assay modified slightly from a method reported previously to provide a simple and sensitive evaluation of paralytic shellfish poisoning (PSP) toxicity in shellfish. The cell bioassay was just as sensitive for C-toxins as for gonyautoxins. The sensitivity of our cell bioassay was 4 times that of the current standard mouse bioassay. Using the cell bioassay, we evaluated PSP toxicity in 361 shellfish samples collected from Mikawa Bay and Ise Bay, Aichi Prefecture, Japan, from April 1999-March 2002. The results were compared with those obtained in a postcolumn derivatization liquid chromatographic analysis. PSP toxins were detected in 236/361 samples by both assays, and there was a fairly good correlation (r = 0.9001, n = 236, p < 0.001) between the results from the two assays. We applied this cell bioassay when short-necked clams in the bay turned poisonous in 2001. The chronological changes in PSP toxicity in the short-necked clams were analyzed and compared with those of the cell density of poisonous plankton (Alexandrium tamarense) occurring in the bay. The PSP toxicity in shellfish peaked 2 weeks after the cell density reached a maximum. We recommend using the cell bioassay for routine monitoring of PSP toxicity in shellfish living in natural marine environments.     

Removal of lipids and diarrhetic shellfish poisoning toxins from blue mussels (Mytilus edulis) during acid and alkaline isolation of proteins

Diarrhetic shellfish poisoning (DSP) toxins pose a serious health risk for consumers of bivalves and other shellfish, as well as a huge economic burden for the bivalve-producing farmers. In this work, the aim was to utilize a solubilization-based protein-isolation method to produce a low-DSP toxin protein isolate from toxic blue mussels that are unsuitable for the whole shellfish market. A homogenate of whole mussel meat was solubilized at low pH (2.8) or high pH (11.1), followed by centrifugation and reprecipitation of the solubilized mussel proteins at the isoelectric pH. In a second centrifugation, precipitated proteins were collected. These processes resulted in 81 (acid solubilization) and 72% (alkaline solubilization) reduction in the initial DTX-1 toxin content of the mussel meat. No other DSP toxins were found in the protein isolates. Acid processing of mussel meat resulted in 50% reduction in the total lipid content, while alkaline treatment did not significantly affect the lipid content. The effect of citric acid and calcium chloride addition to the mussel meat-water homogenate on lipid and toxin content was also investigated. A poor correlation factor was surprisingly obtained between reductions in DTX-1 toxin and lipids in protein isolates from processed toxic mussels. Results from an analytical mass balance of the DTX-1 toxin during acid processing showed that 61% of this toxin ended up in the aqueous supernatant after the second centrifugation. The present study presents a promising alternative way of utilizing mussels for food production in periods when they are toxic.     

Monoclonal-based ELISA for the identification of herbicidal cyclohexanedione analogues that inhibit graminaceous acetyl coenzyme-A carboxylase

Cyclohexanediones are one of four known structural classes of herbicides that inhibit graminaceous acetyl coenzyme-A carboxylase (ACCase; EC 6.4.1.2). Five monoclonal antibodies were raised against cyclohexanediones conjugated to bovine serum albumin. Cross-reactivity studies using a homologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) against 24 cyclohexanedione analogues revealed that two monoclonal antibodies (mAb A and mAb B) could segregate the analogues into active and inactive ACCase inhibitors on the basis of the analogue concentration required to inhibit 50% of antibody binding to the coating conjugate (IC(50)). Both mAb A and mAb B were also found to cross-react with various members of the indolizidinedione structural class of ACCase inhibitors in ciELISA, suggesting that both cyclohexanediones and indolizidinediones possess features recognized by monoclonal antibodies important for the inhibition of ACCase activity. In conclusion, pharmacophore-specific antibodies may be potentially valuable screening tools for the identification of new lead chemistries in a pesticide discovery program.     

Characterization of phospholipid molecular species in the edible parts of bony fish and shellfish

The phospholipid molecular species of freshwater (pangasius, Nile perch, trout), marine fish fillets (horse mackerel, European hake, common sole, European anchovy, European pilchard, Atlantic mackerel) and the edible muscle foot of bivalves (clam, mussel, oyster) commonly available in the Italian market during spring and summer were characterized by means of normal-phase high performance liquid chromatography coupled online with positive electrospray ionization ion-trap tandem mass spectrometry. From principal component analysis (PCA), it was observed that the total fatty acid profile was not suitable to differentiate among the shellfish genera. The fatty acid molecular combinations of phosphatidylcholine, the main phospholipid class, as well as phosphatidylinositol and phosphatidylethanolamine allowed for the differentiation of shellfish from the bony fishes. Phosphatidylserine and phosphatidylethanolamine plasmalogen profile allowed for the discrimination of each bony fish or shellfish genus since PS and pPE classes included a large number of fatty acid combinations that were specific for a fish genus or group.     

Simultaneous analysis of T-2 toxin and HT-2 toxin by an indirect enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of HT-2 toxin in the presence or absence of T-2 toxin is described. In the indirect ELISA, the relative cross-reactivities of antibodies against T-2 toxin (anti-T-2) with T-2 toxin and HT-2 toxin were 1 and 0.1, whereas anti-HT-2 cross-reactivities with T-2 toxin and HT-2 toxin were 0.33 and 1, respectively. Using such relationships, a formula was established that could be used to calculate the individual toxin concentration in a mixed sample after experimentally analyzing for T-2 and HT-2 toxins in the 2 indirect ELISAs. This method was tested by analyzing urine samples spiked with HT-2 toxin alone and samples spiked with both T-2 toxin and HT-2 toxin. A cleanup protocol for treatment of urine samples before ELISA was also established. The overall analytical recovery of HT-2 toxin when it was added at concentrations of 0.1-10 parts per billion (ppb) to the urine samples was ca 89%. When both T-2 and HT-2 toxins were added to the urine samples at equal concentrations of 0.5 to 5.0 ppb, their recoveries were 112 and 109%, respectively.     

Evaluation of laboratory performance of the AOAC method for PSP toxin in shellfish

Laboratory performance of the official AOAC method for paralytic shellfish poison (PSP) toxin in shellfish was evaluated. Two series of naturally toxic shellfish split samples were distributed (15 in 1979 and 19 in 1982) to state shellfish-monitoring laboratories which participate in the National Shellfish Sanitation Program. The laboratories performed bioassays on duplicate 100 g portions of each 220 g split sample. Bioassays were consistent among the laboratories and compared favorably with those of previous studies.     

Development of an immunoassay for the pyrethroid insecticide esfenvalerate.

A competitive enzyme-linked immunosorbent assay was developed for the detection of the pyrethroid insecticide esfenvalerate. Two haptens containing amine or propanoic acid groups on the terminal aromatic ring of the fenvalerate molecule were synthesized and coupled to carrier proteins as immunogens. Five antisera were produced and screened against eight different coating antigens. The assay that had the least interference and was the most sensitive for esfenvalerate was optimized and characterized. The I(50) for esfenvalerate was 30 +/- 6.2 microg/L, and the lower detection limit (LDL) was 3.0 +/- 1.8 microg/L. The assay was very selective. Other pyrethroid analogues and esfenvalerate metabolites tested did not cross-react significantly in this assay. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction (SPE) was used for water matrix. With this SPE step, the LDL of the overall method for esfenvalerate was 0.1 microg/L in water samples.     

Production and characterization of monoclonal and polyclonal antibodies to forchlorfenuron

The development of immunoassays for the detection of the plant growth regulator forchlorfenuron (CPPU) is described. To achieve that purpose, a set of CPPU derivatives has been obtained by the previous synthesis of the adequate p-aminophenyl alkanoic acid. Protein conjugates of these compounds have been used as immunogens to produce rabbit polyclonal antibodies and a collection of mouse monoclonal antibodies. Additionally, a battery of structural analogues of the target analyte has been synthesized and used for the characterization of antibody binding. This strategy has demonstrated that most antibodies followed Landsteiner's principle, although some monoclonal antibodies showing important deviations from this behavior have also been found. Finally, different assay formats have been developed with a variety of antibodies and conjugates, and a rapid procedure has been optimized for the indirect ELISA format using monoclonal and polyclonal antibodies. In the indirect competitive ELISA, assay IC50 values for CPPU below 0.5 nM were found with LODs as low as 0.013 nM.     

Development of a monoclonal antibody-based enzyme-linked immuosorbent assay for the beta-adrenergic agonist zilpaterol

Zilpaterol is a beta-adrenergic agonist approved for use as a growth promoter in cattle in South Africa and Mexico but not in the European Union, United States, or Asia. Here, we report the development of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for zilpaterol. Mice immunized with zilpaterol-butyrate-keyhole limpet hemocyanin were utilized for monoclonal antibody generation whereas zilpaterol-butyrate-bovine serum albumin was used as a coating antigen for ELISA. Thirteen clones were isolated, and after the initial sensitivity and isotyping experiments, three clones were selected for further ELISA optimization. Studies indicated that the optimum pH was near 7.4. Clone 3H5 had the highest sensitivity to zilpaterol and some interaction with clenbuterol and terbutaline at high concentrations but not other N-alkyl [bamethane, (-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (fenoterol, isoxsuprine, ractopamine, or salmeterol) beta-agonists tested. However, clone 3H5 was not functional at high salt concentrations, which precluded further development for urine analysis. Clone 2E10 showed increased sensitivity as salt concentrations were increased and did not cross-react with any of the structural analogues tested. However, its sensitivity to salt and urine concentration changes could cause high variability. Clone 7A8 showed good sensitivity and only a modest change with the salt concentration changes. Clone 7A8 also demonstrated smaller changes in IC(50) and B(0) with increasing sheep urine or cattle urine concentrations as compared to clones 2E10 or 3H5 and, thus, was selected for further development. The IC(50) for all of the antibodies showed exponential increases with increasing organic solvents concentrations, making it desirable to minimize solvent levels. In conclusion, a sensitive, specific zilpaterol monoclonal antibody-based ELISA has been developed that can serve as a rapid screening assay.     

Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting 19-nortestosterone residue in animal tissues

This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and β-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 μg/kg in beef, and 2 μg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.     

Identification of pinnatoxins and discovery of their fatty acid ester metabolites in mussels ( Mytilus edulis ) from eastern Canada

Pinnatoxins are a group of fast-acting cyclic imine toxins previously identified in shellfish from Asia, the southern Pacific, and northern Europe. In this work pinnatoxins were detected in mussels from locations across the eastern coast of Canada. Pinnatoxin G (6) was the major structural variant present, sometimes at levels >80 μg/kg, whereas much lower levels of pinnatoxin A (1) were detected in some samples. Increased concentrations were observed following base hydrolysis of extracts, leading to the discovery by LC-MS of a range of fatty acid esters of 6. Information on the structures of these acylated derivatives was provided through a series of mass spectrometric experiments, supported by partial synthesis, and it is proposed that the compounds are 28-O-acyl esters of 6. Although acyl esters of a range of other phycotoxins are known to form as metabolites in shellfish, this is the first report of their existence for this particular toxin class. The occurrence of pinnatoxins in North American shellfish further highlights the international distribution of these toxins.