Parvalbumin Content Research Articles

3D bio-printing-based vascular-microtissue electrochemical biosensor for fish parvalbumin detection

A novel 3D bio-printing vascular microtissue biosensor was developed to detect fish parvalbumin quickly. The graphite rod electrode was modified with gold and copper organic framework (Cu-MOF) to improve the sensor properties. Polydopamine-modified multi-wall carbon nanotubes (PDA-MWCNT) were mixed with gelatin methacryloyl (GelMA) to prepare a conductive hydrogel. The conductive hydrogel was mixed with mast cells and endothelial cells to produce a bio-ink for 3D bioprinting. High throughput and standardized preparation of vascular microtissue was performed by stereolithography 3D bioprinting. The vascular microtissue was immobilized on the modified electrode to construct the microtissue sensor. The biosensor’s peak current was positively correlated with the fish parvalbumin concentration, and the detection linear concentration range was 0.1 ∼ 2.5 μg/mL. The standard curve equation was IDPV(μA) = 31.30 + 5.46 CPV(μg/mL), the correlation coefficient R2 was 0.990 (n = 5), and the detection limit was 0.065 μg/mL. These indicated a biomimetic microtissue sensor detecting fish parvalbumin has been successfully constructed.

Effect of species, muscle location, food processing and refrigerated storage on the fish allergens, tropomyosin and parvalbumin

Fish tropomyosin is a latest identified fish allergen without full understanding of its biochemical characteristics from the perspective of food allergen. Accordingly, the objective of this study was to investigate the effects of species, muscle location, food processing, and refrigerated storage on fish tropomyosin and compare with main fish allergen, parvalbumin. The result of mass spectrometry analysis revealed tropomyosin as the most abundant thermally stable protein in fish muscle. Fish tropomyosin was ubiquitous among all 28 edible fish species tested, abundant in fish skeletal muscle, resistant to common food processing, and resistant to refrigerated storage up to six days. By contrast, parvalbumin content varied between fish species and was not as thermally stable as tropomyosin under autoclaving. This study demonstrates the intrinsic and processing factors affecting fish allergens and provides valuable information for the presence of major fish allergens and practical consideration of fish allergen detection.

Evaluating the effects of processing on antigenicity and immunochemical detectability of fish proteins by ELISA

The effect of physical (heat treatment at 82 °C and 100 °C), the combination of physical and chemical (buffer pH: 3,5,11 and 12, saccharides: D-glucose and maltose) treatments on the immunoreactive properties of fish proteins and its subset of protein; fish parvalbumins were investigated by two sandwich enzyme-linked immunosorbent assays (sELISA). Calculated immunoreactive protein concentration was inversely proportional to the heat treatment time at 82 °C and 100 °C as assayed by sELISA targeted soluble antigenic proteins. During the first fifteen minutes, the decreased immunoreactive protein concentration was significant (p < 0.0001) for both temperatures. By contrast, immunoreactive parvalbumin concentration in the heated crude extract was significantly increased (p < 0.0001) after 25 min at 82 °C and 15 min at 100 °C to the maximum level as assayed by sELISA targeted parvalbumins (sELISAPV). Combined treatments such as heating the crude extracts altered with pH limited the recognition of immunoreactive parvalbumins by the additional decrease results from the thermally-induced processing step. Solubility drastically decreased near the isoelectric point, correlated with the rapid changes in the immunoreactive parvalbumin concentration. Although low-temperature treatment was favored for glycation of parvalbumins through maillard reaction, faster reactions were observed at high temperatures for both saccharides.

Evaluation of Allergic Cross-Reactivity Among Fishes by Microfluidic Chips and MALDI-TOF MS.

Fish is one of the most common foods that cause allergic reactions. The study of cross-reactivity among fishes using mass spectrometry (MS) is still limited. We developed a strategy using microfluidic chips coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to evaluate cross-reactivity among fishes. The protocol employed commercial magnetic beads functionalized with anti-human IgE antibodies to carry out the IgEs immunomagnetic separation in blood samples, followed by the capture of allergens from seafood protein extracts in a single-straight microfluidic channel. After elution, the captured allergens were digested and identified by MALDI-TOF MS and high-performance liquid chromatography-tandem mass spectrometry and validated by enzyme-linked immunosorbent assay (ELISA). An investigation of the reproducibility revealed that the protocol can sense well the allergens in a food matrix. Seven fish species were analyzed to evaluate the allergic cross-reactivity among fishes. The commercial ELISA test gave consistent results with the presently developed strategy when the same allergenicity test was performed. Parvalbumins were detected from five of the seven analyzed fishes. The sequence alignment of parvalbumins revealed that the similarity of parvalbumins identified from the analyzed fishes is larger than 64%. Boiling may reduce the allergenicity of fish, as demonstrated by a marginal diminish in the parvalbumin content of crucian carp (Carassius carassius) muscle when boiling with water. The method can potentially be used to predict allergic cross-reactivity among fish species, provide advice and guidance to individuals with a history of seafood allergy, and ensure food safety in the food allergy community.

Targeting parvalbumin promotes M2 macrophage polarization and energy expenditure in mice.

Exercise benefits M2 macrophage polarization, energy homeostasis and protects against obesity partially through exercise-induced circulating factors. Here, by unbiased quantitative proteomics on serum samples from sedentary and exercised mice, we identify parvalbumin as a circulating factor suppressed by exercise. Parvalbumin functions as a non-competitive CSF1R antagonist to inhibit M2 macrophage activation and energy expenditure in adipose tissue. More importantly, serum concentrations of parvalbumin positively correlate with obesity in mouse and human, while treating mice with a recombinant parvalbumin blocker prevents its interaction with CSF1R and promotes M2 macrophage polarization and ameliorates diet-induced obesity. Thus, although further studies are required to assess the significance of parvalbumin in mediating the effects of exercise, our results implicate parvalbumin as a potential therapeutic strategy against obesity in mice.

Novel monoclonal antibody-sandwich immunochromatographic assay based on Fe3O4/Au nanoparticles for rapid detection of fish allergen parvalbumin

In this study, a rapid sandwich immunochromatographic assay (ICA) was developed to detect parvalbumin (PV). Firstly, two optimum primary monoclonal antibody (mAb) against PV had been screened out: mAb1 was used as the capture antibody, and mAb2 conjugated to Fe3O4/Au nanoparticles (Fe3O4/AuNPs) that served as a detection reagent. Using this pair of mAbs, a sandwich ICA strip based on Fe3O4/AuNPs was developed. The results showed that the color intensity of test line positively correlated with the PV concentration in the standard or spiked sample. The limit of detection for qualitative (LOD) and quantitative detection (LOQ) were 2 ng/mL and 0.691 ng/mL, respectively. Besides, the detection time of this ICA strip was within 15 min. The recovery rates ranged from 104.0% to 117.4%, within an acceptable level (80–120%). Moreover, the developed assay also showed high cross reaction in different fish species. These results demonstrated that the established test strip has the potential to be used as a rapid screening tool for large scale determination of PV in foodstuffs.

Allergenicity and Stability of 6 New Korean Bony Fish Extracts

PurposeDiagnostic tests for allergen sensitization should reflect real exposure. We made 6 new bony fish extracts, which are consumed popularly in Korea, and evaluated their allergenicity and stability.MethodsWe manufactured fish extracts from codfish, mackerel, common eel, flounder, cutlass, and catfish. Protein and parvalbumin (PV) were evaluated by Bradford assay, 2-site enzyme-linked immunosorbent assay, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and anti-PV immunoblotting. The immunoglobulin E (IgE) reactivities of the extracts were evaluated with ImmunoCAP and IgE immunoblotting using sera from 24 Korean fish allergy patients, 5 asymptomatic sensitizers, and 11 non-atopic subjects. Stability of the extracts stored in 4 different buffers were evaluated for up to a year.ResultsThe protein concentrations of commercial SPT fish extracts varied with up to a 7.5-fold difference. SDS-PAGE showed marked differences in the PV concentrations of commercial SPT reagents. Specific IgE measurements for the following investigatory fish extracts—iCodfish, iMackerel, and iEel—were concordant with that of their corresponding Phadia ImmunoCAP measurements. ImmunoCAP results showed marked IgE cross-reactivity among the fish species, and the overall sensitivity of ImmunoCAP with the investigatory fish extracts for identification of culprit fish species was 85.7%. The protein and PV concentrations in the investigatory extracts were highly stable in saline with 0.3% phenol–50% glycerol at 4°C for up to a year.ConclusionsThe commercial SPT fish extracts exhibited considerable variation in terms of allergenicity, which may impact on diagnostic accuracy. Our new fish extracts have sufficient allergenicity and stability and may be adequate to various clinical applications.

A colorimetric and fluorescent gold nanoparticle-based dual-mode aptasensor for parvalbumin detection

Food allergy has risen as a worldwide food safety concern due to the increasing prevalence over the last decades. It’s in urgent need to develop straight yet sensitive detection methods for food allergens to help people avoid the potential risks imposed by food allergy. Here, we developed a dual-mode aptasensor that gave both colorimetric and fluorescent readouts in response to parvalbumin (PV), a major allergen in fish. The aptasensor was constructed by hybridizing the aptamer modified gold nanoparticles (AuNP-APT), the complementary short chain modified gold nanoparticles (AuNP-CS1) and the fluorescent dye-labeled complementary short chain (FAM-CS2). The presence of PV led to disassembly of the aptasensor through competitive interaction between APT and PV, which resulted in a change in the solution color of the AuNPs and a recovery of the once quenched fluorescence signals of FAM-CS2. The aptasensor exhibited a colorimetric response in the PV concentration range of 2.5–20 μg/mL and a linear fluorescent response in the PV concentration range of 2.38–40 μg/mL. Moreover, the aptasensor showed encouraging specificity, precision, accuracy as well as efficiency in real fish sample detection, which held great potential in recognition and analysis of food allergens.

Absence of parvalbumin increases mitochondria volume and branching of dendrites in inhibitory Pvalb neurons in vivo: a point of convergence of autism spectrum disorder (ASD) risk gene phenotypes

BackgroundIn fast firing, parvalbumin (PV)-expressing (Pvalb) interneurons, PV acts as an intracellular Ca2+ signal modulator with slow-onset kinetics. In Purkinje cells of PV−/− mice, adaptive/homeostatic mechanisms lead to an increase in mitochondria, organelles equally capable of delayed Ca2+ sequestering/buffering. An inverse regulation of PV and mitochondria likewise operates in cell model systems in vitro including myotubes, epithelial cells, and oligodendrocyte-like cells overexpressing PV. Whether such opposite regulation pertains to all Pvalb neurons is currently unknown. In oligodendrocyte-like cells, PV additionally decreases growth and branching of processes in a cell-autonomous manner.MethodsThe in vivo effects of absence of PV were investigated in inhibitory Pvalb neurons expressing EGFP, present in the somatosensory and medial prefrontal cortex, striatum, thalamic reticular nucleus, hippocampal regions DG, CA3, and CA1 and cerebellum of mice either wild-type or knockout (PV−/−) for the Pvalb gene. Changes in Pvalb neuron morphology and PV concentrations were determined using immunofluorescence, followed by 3D-reconstruction and quantitative image analyses.ResultsPV deficiency led to an increase in mitochondria volume and density in the soma; the magnitude of the effect was positively correlated with the estimated PV concentrations in the various Pvalb neuron subpopulations in wild-type neurons. The increase in dendrite length and branching, as well as thickness of proximal dendrites of selected PV−/− Pvalb neurons is likely the result of the observed increased density and length of mitochondria in these PV−/− Pvalb neuron dendrites. The increased branching and soma size directly linked to the absence of PV is assumed to contribute to the increased volume of the neocortex present in juvenile PV−/− mice. The extended dendritic branching is in line with the hypothesis of local hyperconnectivity in autism spectrum disorder (ASD) and ASD mouse models including PV−/− mice, which display all ASD core symptoms and several comorbidities including cortical macrocephaly at juvenile age.ConclusionPV is involved in most proposed mechanisms implicated in ASD etiology: alterations in Ca2+ signaling affecting E/I balance, changes in mitochondria structure/function, and increased dendritic length and branching, possibly resulting in local hyperconnectivity, all in a likely cell autonomous way.

Determination of a major allergen in fish samples by simple and effective label-free capillary electrophoretic analysis after background suppression in ion-exchange chromatography

A simple analytical method using ion-exchange chromatography (IEC) coupled with capillary zone electrophoresis (CZE) was newly developed for detection and quantification of parvalbumin (PV), a major allergen, in fish. The procedure of IEC for simple enrichment of PV crude extract was optimized. By using 25 mmol/L borate-borax buffer with 15 kV separation voltage at pH 9.2 for CZE separation, the migration time, separation efficiency and electrophoretic resolution greatly improved. Under the optimal conditions, PV was determined in 2.8 min, with the limit of detection (LOD) at 0.71 μg/mL (S/N = 3) and the recoveries at 89.6%–104.7%. We also found that only when the concentration of PV was above LOD reported here was the protein capable of stimulating human mast cell degranulation, indicating the biological significance of the LOD. Finally, the use of this method to analyze fish samples with simple sample preparation highlights the applicability for detection of allergens in seafood matrices.

The opposite effects of nandrolone decanoate and exercise on anxiety levels in rats may involve alterations in hippocampal parvalbumin-positive interneurons.

The aim of this study was to evaluate the behavioral effects of chronic (six weeks) nandrolone decanoate (ND, 20 mg/kg, s.c., weekly in single dose) administration (in order to mimic heavy human abuse), and exercise (swimming protocol of 60 minutes a day, five days in a row/two days break), applied alone and simultaneously with ND, in male rats (n = 40). Also, we evaluated the effects of those protocols on hippocampal parvalbumin (PV) content and the possible connection between the alterations in certain parts of hippocampal GABAergic system and behavioral patterns. Both ND and exercise protocols induced increase in testosterone, dihydrotestosterone and estradiol blood levels. Our results confirmed anxiogenic effects of ND observed in open field (OF) test (decrease in the locomotor activity, as well as in frequency and cumulative duration in the centre zone) and in elevated plus maze (EPM) test (decrease in frequency and cumulative duration in open arms, and total exploratory activity), that were accompanied with a mild decrease in the number of PV interneurons in hippocampus. Chronic exercise protocol induced significant increase in hippocampal PV neurons (dentate gyrus and CA1 region), followed by anxiolytic-like behavioral changes, observed in both OF and EPM (increase in all estimated parameters), and in evoked beam-walking test (increase in time to cross the beam), compared to ND treated animals. The applied dose of ND was sufficient to attenuate beneficial effects of exercise in rats by means of decreased exercise-induced anxiolytic effect, as well as to reverse exercise-induced augmentation in number of PV immunoreactive neurons in hippocampus. Our results implicate the possibility that alterations in hippocampal PV interneurons (i.e. GABAergic system) may be involved in modulation of anxiety level induced by ND abuse and/or extended exercise protocols.

Protein and DNA-based assays as complementary tools for fish allergen detection.

Background: Fish is one of the most important, allergenic foods worldwide. Parvalbumin is the well characterized, major allergen in fish muscle. In this study, we developed a protein- and a DNA-based method for the sensitive detection and authentication of eight commonly consumed fishes in food and compared their applicability. Methods: Fish parvalbumins were purified. Polyclonal, anti-parvalbumin antibodies were raised in rabbits and mice. Protein extracts from food were analyzed by quantitative ELISA. Parvalbumin genes were cloned and sequenced for the design of parvalbumin gene-specific PCR-primers. DNA extracted from food was subjected to specific PCR. Results: Increasing parvalbumin contents were quantified by ELISA in fresh fish, in the order of tuna < mackerel < cod < salmon/trout < redfish < carp < herring. The parvalbumin content of processed fish was up to 67% lower than in fresh fish. In spiked food samples, 1 to 15 ppm fresh fish and 30 to 170 ppm processed fish were still detectable by ELISA. The eight fishes were identified by specific PCR using 0.2 to 10 ng fish DNA. PCRs detected still 3 ppm fresh fish and 30 to 150 ppm processed fish in spiked samples. Conclusions: Both the protein- and the DNA-based method have sufficient sensitivity to protect fish-allergic consumers. The ELISA allows allergen quantification, while the PCR identifies the fish present in the food. The detection limits of both methods vary depending on different factors. Both methods need to be carefully validated for each fish and fish product when used in detection assays.

Quantitative analysis of species specificity of two anti-parvalbumin antibodies for detecting southern hemisphere fish species demonstrating strong phylogenetic association

This study aimed to develop a novel approach to determine the correlation between the parvalbumin (PAV) contents and their corresponding immunoreactivity (detectability) in southern hemisphere fish species. The immuno-detected PAV contents of the test fish species were estimated by a quantitative SDS-PAGE. A quantitative Enzyme-Linked ImmunoSorbent Assay (ELISA) was formatted to assess relative immunoreactivity of PAV. Sixteen species (forty-three percent) displayed a positive correlation with the anti-cod PAV polyclonal antibody, but no correlation with the anti-carp PAV monoclonal antibody. There was a strong phylogenetic association of the PAV immunoreactivity. Species from the order of Perciformes showed strong binding with both antibodies; whereas species from Salmoniformes, Ophidiiformes, Scombriformes, Scorpaeniformes, and Tetraodontiformes showed weak or no binding. This approach showed for the first time a statistical correlation between the PAV content and the immunoreactivity and allowed to rank the relative species/order specificity of the two antibodies for the southern hemisphere fish PAV.

Axotomy Leads to Reduced Calcium Increase and Earlier Termination of CCL2 Release in Spinal Motoneurons with Upregulated Parvalbumin Followed by Decreased Neighboring Microglial Activation.

Motoneurons with naturally elevated calcium binding protein content, such as parvalbumin, are more resistant against injury. Furthermore, increase of intracellular calcium, which plays a pivotal role in injury of neurons, could be moderated by elevating their calcium binding proteins. To test whether by elevating parvalbumin content of motoneurons, activation of neighboring microglial cells, a robust component of the inflammatory reaction after injury, could be influenced. Mice overexpressing neuronal parvalbumin were derived and the spinal motoneurons were challenged by cutting the sciatic nerve. At postoperative days 1, 4, 7, 14 and 21 the change of the chemokine ligand 2 immunostaining in the motoneurons and the activation of microglial cells, measured as alterations in CD11b immunostaining were determined. Calcium level of motoneurons was tested electron microscopically at postoperative day 7. After axotomy, increased level of chemokine ligand 2 was detected in the lumbar motoneurons. The staining intensity reached its maximum at day 7 and decayed faster in transgenic mice compared to controls. Microglial activation around motoneurons attenuated faster in parvalbumin overexpressing mice, too, but the decrease of microglial activation was delayed compared to the decline of the chemokine ligand 2 signal. At the time when the microglial reaction peaked, no intracellular calcium increase was detected in the motoneurons of transgenic mice, in contrast to the twofold increase in wild type animals. Increased calcium buffering capacity, which augments resistance of motoneurons against calcium-mediated injury, leads to earlier termination of motoneuronal emission of CCL2 followed by a reduction of neighboring microglial activation after axotomy.

Fish allergy in patients with parvalbumin-specific immunoglobulin E depends on parvalbumin content rather than molecular differences in the protein among fish species.

Allergenic characteristics of purified parvalbumins from different fish species have not been thoroughly investigated. We revealed that purified parvalbumins from nine different fish species have identical IgE-reactivities and high cross-reactivities. We also showed that fish allergenicity is associated with the parvalbumin content of the fish species, rather than species-specific differences in the molecular characteristics of the individual parvalbumin proteins.

PECULIARITIES OF THE STRUCTURAL ORGANIZATION OF VENTRAL POSTEROMEDIAL, VENTRAL POSTEROLATERAL, AND RETICULAR NUCLEI OF RAT THALAMUS (AN IMMUNOHISTOCHEMICAL STUDY)

The aim of this work was an immunohistochemical study of the expression of neuronal and glial proteins, and of gap junctions proteins (connexin 36, connexin 43) in ventral posteromedial (VPMN), ventral posterolateral (VPLN) and reticular (RТN) nuclei of the thalamus in rats. It was found that VPMN and VPLN of the thalamus were characterized by a homogeneous distribution of synaptophysin, grouped arrangement of astrocytes, horizontal orientation of somatostatin-containing myelinated and unmyelinated nerve fibers, forming the bundles, and running through the barreloid septum, expression of connexin 36 and 43 as well as of parvalbumin revealing barreloids in 4 μm-thick sections. In RTN the content of myelin basic protein, neurofilaments, parvalbumin, and somatostatin was increased, while the amount of glial fibrillary acidic protein and connexin 43 was moderate, and synaptophysin and connexin 36 were absent.

Quantification of major allergen parvalbumin in 22 species of fish by SDS–PAGE

Fish is an important causative material of food allergy. Although the allergenicity of fish is considered to correlate with the content of parvalbumin, the major fish allergen, available information about the parvalbumin content in fish is limited. In this study, a simple and reliable quantification method for fish parvalbumin by SDS–PAGE was first established. Application of the SDS–PAGE method to 22 species of fish revealed a marked variation in parvalbumin content among fish. Furthermore, the parvalbumin content was found to be higher in dorsal white muscle than in ventral white muscle, in rostral part of white muscle than in caudal part of white muscle and in white muscle than in dark muscle. IgE reactivity of fish was roughly proportional to parvalbumin content. Interestingly, large-sized migratory fish, such as salmon, swordfish and tuna, were commonly very low in both parvalbumin content and IgE reactivity.

Creatine loading elevates the intracellular phosphorylation potential and alters adaptive responses of rat fast-twitch muscle to chronic low-frequency stimulation.

This study tested the hypothesis that elevating the intracellular phosphorylation potential (IPP = [ATP]/[ADP]free) within rat fast-twitch tibialis anterior muscles by creatine (Cr) loading would prevent fast-to-slow fibre transitions induced by chronic low-frequency electrical stimulation (CLFS, 10 Hz, 12 h/day). Creatine-control and creatine-CLFS groups drank a solution of 1% Cr + 5% dextrose, ad libitum, for 10 days before and during 10 days of CLFS; dextrose-control and dextrose-CLFS groups drank 5% dextrose. Cr loading increased total Cr (P < 0.025), phosphocreatine (PCr) (P < 0.003), and the IPP (P < 0.0008) by 34%, 45%, and 64%, respectively. PCr and IPP were 46% (P < 0.002) and 76% (P < 0.02) greater in creatine-CLFS than in dextrose-CLFS. Higher IPP was confirmed by a 58% reduction in phospho-AMP-activated protein kinase α (Thr172) (P < 0.006). In dextrose-CLFS, myosin heavy chain (MyHC) I and IIa transcripts increased 32- and 38-fold (P < 0.006), respectively, whereas MyHC-IIb mRNA decreased by 75% (P < 0.03); the corresponding MyHC-I and MyHC-IIa protein contents increased by 2.0- (P < 0.03) and 2.7-fold (P < 0.05), respectively, and MyHC-IIb decreased by 30% (P < 0.03). In contrast, within creatine-CLFS, MyHC-I and MyHC-IIa mRNA were unchanged and MyHC-IIb mRNA decreased by 75% (P < 0.003); the corresponding MyHC isoform contents were not altered. Oxidative reference enzymes were similarly elevated (P < 0.01) in dextrose-CLFS and creatine-CLFS, but reciprocal reductions in glycolytic reference enzymes occurred only in dextrose-CLFS (P < 0.02). Preservation of the glycolytic potential and greater SERCA2 and parvalbumin contents in creatine-CLFS coincided with prolonged time to peak tension and half-rise time (P < 0.01). These results highlight the IPP as an important physiological regulator of muscle fibre plasticity and demonstrate that training-induced changes typically associated with improvements in muscular endurance or increased power output are not mutually exclusive in Cr-loaded muscles.

Nano-gold capillary immunochromatographic assay for parvalbumin.

A novel non-instrumental bioanalysis based on colloidal-gold immunochromatography in a modified glass capillary was developed and named capillary immunochromatographic assay (CICA). In this report, glass capillary was proposed as a support in immunochromatographic assay because of its excellent characteristics. Goat anti-rabbit IgG and parvalbumin (PV) were immobilized on the inner wall of the glass capillary as control zone and test zone, respectively. The CICA was constructed, and main variables for the performance were optimized. Using an important allergen of fish products (parvalbumin, PV) as the target, the analytical efficiency of the developed technique was investigated and the visual detection limit (VDL) and semi-quantitative limit of detection (LOD) were estimated to be 70 ng mL(-1) and 40 ng mL(-1), respectively. The coefficient of variation (CV) for the intra-assay and inter-assay was calculated for the PV concentration of 50 ng mL(-1), and the entire operation, including sample preparation, was consistently performed in 30 min. The developed technique was implemented and validated with different foodstuffs, including Scophthalmus maximus (Linnaeus), surimi products, and livestock, confirming sufficient accuracy and precision of results and verifying the method to be efficacious. These results enabled us to propose CICA as a new and promising technique for simple, rapid, and on-site screening of PV in biological samples.

Parvalbumin in fish skin-derived gelatin: is there a risk for fish allergic consumers?

The major allergen parvalbumin was purified from cod muscle tissues, and polyclonal antibodies were raised towards it. The antibodies were tested for specificity and an enzyme-linked immunosorbent assay (ELISA) was developed using these antibodies. The ELISA was applied to measure parvalbumin in cod skin, the starting material for fish gelatin made from deep sea, wild fish. The ELISA was sufficiently sensitive (LLOQ = 0.8 ng ml−1 in extracts, corresponding to 0.02 µg of parvalbumin per g of tissue), and did not cross-react with common food constituents. Fish gelatin, wine and beer, matrices for the potential use of this ELISA, did not cause disturbance of the assay performance. The data show that the parvalbumin content in cod muscle tissue is 6.25 mg g−1, while the skins contained considerably less, 0.4 mg g−1. Washing of the skins, a common industrial procedure during the manufacturing of fish gelatin, reduced the level of parvalbumin about 1000-fold to 0.5 µg g−1, or 0.5 ppm. From 95 commercial lots of fish gelatin it is shown that 73 are below 0.02 µg g−1 parvalbumin. From the other 22 lots, the one with the highest concentration contained 0.15 µg g−1 of parvalbumin. These levels are generally assumed to be safe for fish-allergic individuals.