Evaluation of portable vibrational spectroscopy for identifying plasticizers in dairy tubing.

Plasticisers are commonly used to increase the flexibility of a wide variety of food contact materials including the plastic tubing, liners, and gaskets used in the dairy industry. In recent years, some classes of plasticisers have come under scrutiny due to the potential for transfer of these compounds into the milk itself, which can then be further processed into foods such as powdered milks and cheeses, infant formula, and baked goods. One such set of plasticisers that is being evaluated for frequency of use, potential routes of exposure, and risk to consumers is ortho-phthalates, hereafter referred to as phthalates. In order to better understand the actual use of phthalate versus non-phthalate plasticised tubing, a robust, rapid, and portable analytical method is necessary for on-site screening. Laboratory Raman and near-infrared spectrometers have been used extensively for polymer and additive evaluation, and advances in portable/hand-held technology could lead to feasible plasticiser evaluation in the field. This research overviews efforts to evaluate six portable spectroscopy devices for their ability to identify phthalate versus non-phthalate plasticised polyvinyl chloride (PVC) dairy tubing, liners, and gaskets. The most successful method, a hand-held Raman spectrometer along with a plasticiser spectral library or a chemometric model, can rapidly and accurately identify phthalate containing PVC and has the potential to be employed as a future field screening technique for regulators and the dairy industry.

Detection of decomposition in mahi-mahi, croaker, red snapper, and weakfish using an electronic-nose sensor and chemometric modeling.

This study evaluated an electronic-nose (e-nose) sensor in combination with support vector machine (SVM) modeling for predicting the decomposition state of four types of fish fillets: mahi-mahi, croaker, red snapper, and weakfish. The National Seafood Sensory Expert scored fillets were thawed, 10-g portions were weighed into glass jars which were then sealed, and the jars were held at approximately 30°C to allow volatile components to be trapped and available for analysis. The measurement of the sample vial headspace was performed with an e-nose device consisting of nanocomposite, metal oxide semiconductor (MOS), electrochemical, and photoionization sensors. Classification models were then trained based on the sensory grade of each fillet, and the e-nose companion chemometric software identified that eight MOS were the most informative for determining a sensory pass from sensory fail sample. For SVM, the cross-validation (CV) correct classification rates for mahi-mahi, croaker, red snapper, and weakfish were 100%, 100%, 97%, and 97%, respectively. When the SVM prediction performances of the eight MOS were evaluated using a calibration-independent test set of samples, correct classification rates of 93-100% were observed. Based on these results, the e-nose measurements coupled with SVM models were found to be potentially promising for predicting the spoilage of these four fish species. PRACTICAL APPLICATION: This report describes the application of an electronic-nose sensor as a potential rapid and low-cost screening method for fish spoilage. It could provide regulators and stakeholders with a practical tool to rapidly and accurately assess fish decomposition.

Comparison of the Performance of Partial Least Squares and Support Vector Regressions for Predicting Fatty Acids and Fatty Acid Classes in Marine Oil Dietary Supplements by Using Vibrational Spectroscopic Data.

ABSTRACT: Simple, fast, and accurate analytical techniques for verifying the accuracy of label declarations for marine oil dietary supplements containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are required because of the increased consumption of these products. We recently developed broad-based partial least squares regression (PLS-R) models to quantify six fatty acids (FAs) and FA classes by using the spectroscopic data from a portable Fourier transform infrared (FTIR) device and a benchtop Fourier transform near infrared (FT-NIR) spectrometer. We developed an improved quantification method for these FAs and FA classes by incorporating a nonlinear calibration approach based on the machine learning technique support vector machines. For the two spectroscopic methods, high accuracy in prediction was indicated by low root mean square error of prediction and by correlation coefficients (R2) close to 1, indicating excellent model performance. The percent accuracy of the support vector regression (SV-R) model predicted values for EPA and DHA in the reference material was 90 to 110%. In comparison to PLS-R, SV-R accuracy for prediction of FA and FA class concentrations was up to 2.4 times higher for both ATR-FTIR and FT-NIR spectroscopic data. The SV-R models also provided closer agreement with the certified and reference values for the prediction of EPA and DHA in the reference standard. Based on our findings, the SV-R methods had superior accuracy and predictive quality for predicting the FA concentrations in marine oil dietary supplements. The combination of SV-R with ATR-FTIR and/or FT-NIR spectroscopic data can potentially be applied for the rapid screening of marine oil products to verify the accuracy of label declarations.

A Rapid, Univariate FT-NIR Procedure to Determine Moisture Concentration in Olive Oil.

We recently observed that the weak near-infrared (NIR) band near 5260 cm-1 was relatively more intense for extra virgin olive oil (EVOO) than for refined olive oil (ROO). We also observed that its intensity was diminished upon heating and erroneously presumed that it may be attributed to volatile carbonyl components in EVOO. In the present study we demonstrate for the first time that this band is primarily attributed to a water O-H combination band. To accurately determine the intensity of this weak band, observed on a shifted and sloping baseline, we measured the peak-to-peak (p-p) height of its first derivative. An exponential calibration curve for p-p height versus gravimetrically-determined concentration of spiked water was satisfactorily generated. The calibration curve was first evaluated by using independent sets of gravimetrically prepared test samples. Subsequently, it was used to determine the moisture content, a quality parameter, for a limited set of authenticated reference olive oils whose quality and purity were confirmed by official methods. These concentrations, 0.098-0.12% H2O (w/w) for EVOO, 0.022-0.030% H2O (w/w) for ROO, and 0.028-0.054% H2O (w/w) for pomace olive oil (POO), were consistent with those reported in the literature. For 88 commercial products investigated, the moisture levels fell in the range from 0.026% to 0.13% (w/w). The correlation between moisture content and other olive oil quality parameters has been reported in the literature and has yet to be further investigated.

Discovery of a Potential Human Serum Biomarker for Chronic Seafood Toxin Exposure Using an SPR Biosensor.

Domoic acid (DA)-producing harmful algal blooms (HABs) have been present at unprecedented geographic extent and duration in recent years causing an increase in contamination of seafood by this common environmental neurotoxin. The toxin is responsible for the neurotoxic illness, amnesic shellfish poisoning (ASP), that is characterized by gastro-intestinal distress, seizures, memory loss, and death. Established seafood safety regulatory limits of 20 µg DA/g shellfish have been relatively successful at protecting human seafood consumers from short-term high-level exposures and episodes of acute ASP. Significant concerns, however, remain regarding the potential impact of repetitive low-level or chronic DA exposure for which there are no protections. Here, we report the novel discovery of a DA-specific antibody in the serum of chronically-exposed tribal shellfish harvesters from a region where DA is commonly detected at low levels in razor clams year-round. The toxin was also detected in tribal shellfish consumers' urine samples confirming systemic DA exposure via consumption of legally-harvested razor clams. The presence of a DA-specific antibody in the serum of human shellfish consumers confirms long-term chronic DA exposure and may be useful as a diagnostic biomarker in a clinical setting. Adverse effects of chronic low-level DA exposure have been previously documented in laboratory animal studies and tribal razor clam consumers, underscoring the potential clinical impact of such a diagnostic biomarker for protecting human health. The discovery of this type of antibody response to chronic DA exposure has broader implications for other environmental neurotoxins of concern.

Non-targeted NIR spectroscopy and SIMCA classification for commercial milk powder authentication: A study using eleven potential adulterants.

A non-targeted detection method using near-infrared (NIR) spectroscopy combined with chemometric modeling was developed for the rapid screening of commercial milk powder (MP) products as authentic or potentially mixed with known and unknown adulterants. Two benchtop FT-NIR spectrometers and a handheld NIR device were evaluated for model development. The performance of SIMCA classification models was then validated using an independent test set of genuine MP samples and a set of gravimetrically prepared mixtures consisting of MPs spiked with each of eleven potential adulterants. Classification models yielded 100% sensitivities for the benchtop spectrometers. Better specificity, which was influenced by the nature of the adulterant, was obtained for the benchtop FT-NIR instruments than for the handheld NIR device, which suffered from lower spectral resolution and a narrower spectral range. FT-NIR spectroscopy and SIMCA classification models show promise for the rapid screening of commercial MPs for the detection of potential adulteration.

Detection of Paralytic Shellfish Toxins in Mussels and Oysters Using the Qualitative Neogen Lateral-Flow Immunoassay: An Interlaboratory Study.

Paralytic shellfish toxins (PSTs) in bivalve molluscs represent a public health risk and are controlled via compliance with a regulatory limit of 0.8 mg saxitoxin (STX)⋅2HCl equivalents per kilogram of shellfish meat (eq/kg). Shellfish industries would benefit from the use of rapid immunological screening tests for PSTs to be used for regulation, but to date none have been fully validated. An interlaboratory study involving 16 laboratories was performed to determine the suitability of the Neogen test to detect PSTs in mussels and oysters. Participants performed the standard protocol recommended by the manufacturer and a modified protocol with a conversion step to improve detection of gonyautoxin 1&4. The statistical analysis showed that the protocols had good homogeneity across all laboratories, with satisfactory repeatability, laboratory, and reproducibility variation near the regulatory level. The mean probability of detection (POD) at 0.8 mg STX⋅2HCl eq/kg using the standard protocol in mussels and oysters was 0.966 and 0.997, respectively, and 0.968 and 0.966 using the modified protocol. The estimated LOD in mussels was 0.316 mg STX⋅2HCl eq/kg with the standard and 0.682 mg STX⋅2HCl eq/kg with the modified protocol, and 0.710 and 0.734 mg STX⋅2HCl eq/kg for oysters, respectively. The Neogen test may be acceptable for regulatory purposes for oysters in accordance with European Commission directives in which the standard protocol provides, at the regulatory level, a probability of a negative response of 0.033 on 95% of occasions. Its use for mussels is less consistent at the regulatory level due to the wide prediction interval around the POD.

Pathology and Epidemiology of Oxalate Nephrosis in Cheetahs.

To investigate cases of acute oxalate nephrosis without evidence of ethylene glycol exposure, archived data and tissues from cheetahs ( Acinonyx jubatus) from North America ( n = 297), southern Africa ( n = 257), and France ( n = 40) were evaluated. Renal and gastrointestinal tract lesions were characterized in a subset of animals with ( n = 100) and without ( n = 165) oxalate crystals at death. Crystals were confirmed as calcium oxalate by Raman spectroscopy in 45 of 47 cheetahs tested. Crystals were present in cheetahs from 3.7 months to 15.9 years old. Cheetahs younger than 1.5 years were less likely to have oxalates than older cheetahs ( P = .034), but young cheetahs with oxalates had more oxalate crystals than older cheetahs ( P < .001). Cheetahs with oxalate crystals were more likely to have renal amyloidosis, interstitial nephritis, or colitis and less likely to have glomerular loop thickening or gastritis than those without oxalates. Crystal number was positively associated with renal tubular necrosis ( P ≤ .001), regeneration ( P = .015), and casts ( P ≤ .001) but inversely associated with glomerulosclerosis, renal amyloidosis, and interstitial nephritis. Crystal number was unrelated to the presence or absence of colitis and was lower in southern African than American and European animals ( P = .01). This study found no evidence that coexisting chronic renal disease (amyloidosis, interstitial nephritis, or glomerulosclerosis), veno-occlusive disease, gastritis, or enterocolitis contributed significantly to oxalate nephrosis. Oxalate-related renal disease should be considered as a potential cause of acute renal failure, especially in young captive cheetahs. The role of location, diet, stress, and genetic predisposition in the pathogenesis of oxalate nephrosis in cheetahs warrants further study.

Effects of the Adulteration Technique on the Near-Infrared Detection of Melamine in Milk Powder.

The United States Pharmacopeial Convention has led an international collaborative project to develop a toolbox of screening methods and reference standards for the detection of milk powder adulteration. During the development of adulterated milk powder reference standards, blending methods used to combine melamine and milk had unanticipated strong effects on the near-infrared (NIR) spectrum of melamine. The prominent absorbance band at 1468 nm of melamine was retained when it was dry-blended with skim milk powder but disappeared in wet-blended mixtures, where spray-dried milk powder samples were prepared from solution. Analyses using polarized light microscopy, Raman spectroscopy, dielectric relaxation spectroscopy, X-ray diffraction, and mass spectrometry indicated that wet blending promoted reversible and early Maillard reactions with lactose that are responsible for differences in melamine NIR spectra between wet- and dry-blended samples. Targeted detection estimates based solely on dry-blended reference standards are likely to overestimate NIR detection capabilities in wet-blended samples as a result of previously overlooked matrix effects arising from changes in melamine hydrogen-bonding status, covalent complexation with lactose, and the lower but more homogeneous melamine local concentration distribution produced in wet-blended samples. Techniques used to incorporate potential adulterants can determine the suitability of milk reference standards for use with rapid detection methods.

Effects of Wet-Blending on Detection of Melamine in Spray-Dried Lactose.

During the development of rapid screening methods to detect economic adulteration, spray-dried milk powders prepared by dissolving melamine in liquid milk exhibited an unexpected loss of characteristic melamine features in the near-infrared (NIR) and Raman spectra. To further characterize this "wet-blending" phenomenon, spray-dried melamine and lactose samples were produced as a simplified model and investigated by NIR spectroscopy, Raman spectroscopy, proton nuclear magnetic resonance (1H NMR), and direct analysis in real time Fourier transform mass spectrometry (DART-FTMS). In contrast to dry-blended samples, characteristic melamine bands in NIR and Raman spectra disappeared or shifted in wet-blended lactose-melamine samples. Subtle shifts in melamine 1H NMR spectra between wet- and dry-blended samples indicated differences in melamine hydrogen-bonding status. Qualitative DART-FTMS analysis of powders detected a greater relative abundance of lactose-melamine condensation product ions in the wet-blended samples, which supported a hypothesis that wet-blending facilitates early Maillard reactions in spray-dried samples. Collectively, these data indicated that the formation of weak, H bonded complexes and labile, early Maillard reaction products between lactose and melamine contribute to spectral differences observed between wet- and dry-blended milk powder samples. These results have implications for future evaluations of adulterated powders and emphasize the important role of sample preparation methods on adulterant detection.

Non-targeted detection of milk powder adulteration using Raman spectroscopy and chemometrics: melamine case study.

Raman spectroscopy in combination with chemometrics was explored as a rapid, non-targeted screening method for the detection of milk powder (MP) adulteration using melamine as an example contaminant. Raman spectroscopy and an unsupervised pattern-recognition method, principal component analysis (PCA), allowed for the differentiation of authentic MPs from adulterated ones at concentrations > 1.0% for dry-blended (DB) samples and > 0.30% for wet-blended (WB) ones. Soft independent modelling of class analogy (SIMCA), a supervised pattern-recognition method, was also used to classify test samples as adulterated or authentic. Combined statistics at a 97% confidence level from the SIMCA models correctly classified adulteration of MP with melamine at concentrations ≥ 0.5% for DB samples and ≥ 0.30% for WB ones, while no false-positives from authentic MPs were found when the spectra in the 600-700 cm-1 range were pre-processed using standard normal variate (SNV) followed by a gap-segment derivatisation. The combined technique of Raman spectroscopy and chemometrics proved to be a useful tool for the rapid and cost-efficient non-targeted detection of adulteration in MP at per cent spiking levels.

Characterising variances of milk powder and instrumentation for the development of a non-targeted, Raman spectroscopy and chemometrics detection method for the evaluation of authenticity.

There is a need to develop rapid tools to screen milk products for economically motivated adulteration. An understanding of the physiochemical variability within skim milk powder (SMP) and non-fat dry milk (NFDM) is the key to establishing the natural differences of these commodities prior to the development of non-targeted detection methods. This study explored the sources of variance in 71 commercial SMP and NFDM samples using Raman spectroscopy and principal component analysis (PCA) and characterised the largest number of commercial milk powders acquired from a broad number of international manufacturers. Spectral pre-processing using a gap-segment derivative transformation (gap size = 5, segment width = 9, fourth derivative) in combination with sample normalisation was necessary to reduce the fluorescence background of the milk powder samples. PC scores plots revealed no clear trends for various parameters, including day of analysis, powder type, supplier and processing temperatures, while the largest variance was due to irreproducibility in sample positioning. Significant chemical sources of variances were explained by using the spectral features in the PC loadings plots where four samples from the same manufacturer were determined to likely contain an additional component or lactose anomers, and one additional sample was identified as an outlier and likely containing an adulterant or differing quality components. The variance study discussed herein with this large, diverse set of milk powders holds promise for future use as a non-targeted screening method that could be applied to commercial milk powders.

Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay.

Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV-Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.

Microarray chip development using infrared imaging for the identification of catfish species.

Several families of catfish species are extensively aquacultured around the world; however, only those from the family Ictaluridae can be labeled as catfish in the United States. Non-Ictalurid catfish species that are marketed as "catfish" in the USA are considered misbranded. Misbranding in general has led to an increased interest in developing deoxyribonucleic acid (DNA)-based methods such as DNA barcoding, polymerase chain reaction restriction fragment length polymorphism, and DNA microarrays with fluorescence detection for the identification of fish species. In this proof-of-concept study, DNA microarrays coupled with a newly developed mid-infrared imaging detection method were applied to the identification of seven species of catfish for the first time. Species-specific DNA probes targeting three regions per species of the cytochrome c oxidase 1 (barcoding) gene were developed and printed as microarrays on glass slides. Deoxyribonucleic acid targets labeled with biotin were hybridized to their complementary probes using a strategy that allowed the selective formation of a silver layer on hybridized spots needed for detection. Using this three-probe format, the seven species were all identified correctly, even when a limited number of false positive spots were observed. Raman spectroscopy was employed to further characterize the arrays.

First report of a direct surface plasmon resonance immunosensor for a small molecule seafood toxin.

Tetrodotoxin (TTX), a small molecular weight neurotoxin, is responsible for poisoning events that traditionally occur from consumption of contaminated puffer fish. Recent studies have shown a growing number of foods contaminated with TTX and a larger number of waters and associated countries where the toxin may occur. The apparent expanding prevalence of TTX supports a growing need for screening assays that can be used to detect potentially harmful food. In the past few years, surface plasmon resonance (SPR) biosensors have been developed for rapid, robust detection of TTX; however, these assays focus on detection of unbound antibody from an inhibition reaction with the toxin. This manuscript introduces the first direct immunoassay for a seafood toxin, specifically TTX. Major advantages of this assay compared to indirect assays include increased speed of analysis, decreased use of biological reagents, and improved confidence in the detection of the toxin, along with the ability to characterize the antibody/toxin interaction. The analytical method introduced in this paper could be applied to other seafood toxins, as well as to a wide range of low molecular weight targets.

Surface plasmon resonance biosensor for detection of feline calicivirus, a surrogate for norovirus.

The human noroviruses are the most common non-bacterial cause of gastroenteritis and are responsible for as much as 50% of all gastroenteritis outbreaks worldwide. Norovirus (NoV), a single stranded RNA virus, is highly contagious with an infectious dose of less than 100 viral particles. While techniques exist for the identification of NoV, the lack of a reliable cell culture system, NoV genetic variability, and time-consuming sample preparation steps required to isolate the virus (or its genome) prior to molecular based methods has hindered rapid virus detection. To better protect the public from virus-contaminated food and enable better detection in clinical and environmental samples, sensitive and selective methods with simple sample preparation are needed. Surface plasmon resonance (SPR) biosensors represent an emerging detection platform, and this approach has been applied to the rapid detection of foodborne small molecule toxins, protein toxins, and bacteria. This analytical technique, however, has yet to be fully investigated for rapid virus detection, especially for intact viral particles extracted from food matrices. For this study, the culturable, non-human pathogen feline calicivirus (FCV), which has similar morphology and is genetically related to NoV, was chosen as a surrogate virus for designing and evaluating an SPR assay. An antibody-based assay was performed by first immobilizing anti-FCV to an SPR chip surface and then directly measuring virus binding and subsequent secondary antibody binding. The resulting biosensor directly detected intact FCV particles with limits of detection of approximately 10(4)TCID50FCV/mL from purified cell culture lysates. In addition, intact virus detection in FCV-spiked oyster matrix was possible when using a simple extraction procedure and employing a secondary antibody to FCV for quantitation. The results from these preliminary studies show promise for the development of a rapid assay for detecting intact viruses, such as NoV, using an SPR biosensor. While the current level of sensitivity achieved with this SPR biosensor may be more applicable to virus detection in clinical specimens, broader application and increased sensitivity of this method for foodborne viruses may be achieved when performed in conjunction with efficient virus extraction and concentration methods.

First report of the use of a saxitoxin-protein conjugate to develop a DNA aptamer to a small molecule toxin.

Saxitoxin (STX) is a low molecular weight neurotoxin mainly produced by certain marine dinoflagellates that, along with its family of similarly related paralytic shellfish toxins, may cause the potentially fatal intoxication known as paralytic shellfish poisoning. Illness and fatality rates are low due to the effective monitoring programs that determine when toxins exceed the established regulatory action level and effectuate shellfish harvesting closures accordingly. Such monitoring programs rely on the ability to rapidly screen large volumes of samples. Many of the screening assays currently available employ antibodies or live animals. This research focused on developing an analytical recognition element that would eliminate the challenges associated with the limited availability of antibodies and the use of animals. Here we report the discovery of a DNA aptamer that targets STX. Concentration-dependent and selective binding of the aptamer to STX was determined using a surface plasmon resonance sensor. Not only does this work represent the first reported aptamer to STX, but also the first aptamer to any marine biotoxin. A novel strategy of using a toxin-protein conjugate for DNA aptamer selection was successfully implemented to overcome the challenges associated with aptamer selection to small molecules. Taking advantage of such an approach could lead to increased diversity and accessibility of aptamers to low molecular weight toxins, which could then be incorporated as analytical recognition elements in diagnostic assays for foodborne toxin detection. The selected STX aptamer sequence is provided here, making it available to any investigator for use in assay development for the detection of STX.

Developing improved immunoassays for paralytic shellfish toxins: the need for multiple, superior antibodies.

Paralytic shellfish toxins (PSTs) are a risk to humans upon consumption of contaminated seafood. The PST family is comprised of more than twenty congeners, with each form having a different potency. In order to adequately protect consumers yet reduce unnecessary closures of non-contaminated harvesting areas, a rapid method that allows for analysis of sample toxicity is needed. While a number of PST immunoassays exist, the outstanding challenge is linking quantitative response to sample toxicity, as no single antibody reacts to the PST congeners in a manner that correlates with potency. A novel approach, then, is to combine multiple antibodies of varying reactivity to create a screening assay. This research details our investigation of three currently available antibodies for their reactivity profiles determined using a surface plasmon resonance biosensor assay. While our study shows challenges with detection of the R1-hydroxylated PSTs, results indicate that using multiple antibodies may provide more confidence in determining overall toxicity and the toxin profile. A multiplexed approach would not only improve biosensor assays but could also be applied to lateral flow immuno-chromatographic platforms, and such a theoretical device incorporating the three antibodies is presented. These improved assays could reduce the number of animal bioassays and confirmatory analyses (e.g., LC/MS), thereby improving food safety and economic use of shellfish resources.

Surface plasmon resonance biosensor for determination of tetrodotoxin: prevalidation study.

A label-free surface plasmon resonance biosensor method was applied to determine tetrodotoxin (TTX) in pufferfish matrixes using an antibody inhibition assay format. A prevalidation study was conducted to demonstrate the assay performance characteristics, such as selectivity, LOD, LOQ, repeatability, reproducibility, and accuracy. Three participating laboratories reported standard curves in buffer and pufferfish matrix. A set of blind samples with TTX spiked into buffer as well as in 10% pufferfish extract were analyzed. Additionally, three blind naturally contaminated samples were analyzed, and the results were compared to those obtained using a reference method (HPLC/electrospray ionization-selected reaction monitoring-MS). The developed method was demonstrated to be capable of detecting TTX in pufferfish matrix standard samples in a broad concentration range (2-9000 ng/mL) with an LOD of 1.5 ng/mL. Between-laboratory recovery values were in the range of 51-190% with a mean of 107%, and 64-180% with a mean of 103% for TTX-spiked samples in buffer and pufferfish matrix, respectively. Between-laboratory recoveries were in the satisfactory range of 101-119% for naturally contaminated samples. This robust, rapid, and noninvasive method may serve as an attractive alternative to established methods for detection of TTX in pufferfish extracts.

Antibody characterization and immunoassays for palytoxin using an SPR biosensor.

Palytoxin (PLTX), a polyether marine toxin originally isolated from the zoanthid Palythoa toxica, is one of the most toxic non-protein substances known. Fatal poisonings have been linked to ingestion of PLTX-contaminated seafood, and effects in humans have been associated with dermal and inhalational exposure to PLTX containing organisms and waters. Additionally, PLTX co-occurrence with other well-characterized seafood toxins (e.g., ciguatoxins, saxitoxins, tetrodotoxin) has hindered direct associations of PLTX to seafood-borne illnesses. There are currently no validated methods for the quantitative detection of PLTX(s). As such, a well-characterized, robust, specific analytical technique is needed for the detection of PLTX(s) in source organisms, surrounding waters, and clinical samples. Surface plasmon resonance (SPR) biosensors are ideally suited for antibody characterization and quantitative immunoassay detection. Herein, we describe a newly developed SPR assay for PLTX. An anti-mouse substrate was used to characterize the kinetic values for a previously developed monoclonal anti-PLTX. The characterized antibody was then incorporated into a sensitive, rapid, and selective PLTX assay. Buffer type, flow rate, analyte-binding time, and regeneration conditions were optimized for the antibody-PLTX system. Cross-reactivity to potentially co-occurring seafood toxins was also evaluated. We show that this optimized assay is capable of measuring low- to sub-ng/mL PLTX levels in buffer and two seafood matrices (grouper and clam). Preliminary results indicate that this SPR biosensor assay allows for (1) rapid characterization of antibodies and (2) rapid, sensitive PLTX concentration determination in seafood matrices. Method development information contained herein may be broadly applied to future PLTX detection and/or antibody characterization efforts.