Linear epitopes of bony fish ß-parvalbumins.

Introduction: Fish ß-parvalbumins are common targets of allergy-causing immunity. The nature of antibody responses to such allergens determines the biological outcome following exposure to fish. Specific epitopes on these allergens recognised by antibodies are incompletely characterised. Methods: High-content peptide microarrays offer a solution to the identification of linear epitopes recognised by antibodies. We characterized IgG and IgG4 recognition of linear epitopes of fish ß-parvalbumins defined in the WHO/IUIS allergen database as such responses hold the potential to counter an allergic reaction to these allergens. Peripheral blood samples, collected over three years, of 15 atopic but not fish-allergic subjects were investigated using a microarray platform that carried every possible 16-mer peptide of known isoforms and isoallergens of these and other allergens. Results: Interindividual differences in epitope recognition patterns were observed. In contrast, reactivity patterns in a given individual were by comparison more stable during the 3 years-course of the study. Nevertheless, evidence of the induction of novel specificities over time was identified across multiple regions of the allergens. Particularly reactive epitopes were identified in the D helix of Cyp c 1 and in the C-terminus of Gad c 1 and Gad m 1.02. Residues important for the recognition of certain linear epitopes were identified. Patterns of differential recognition of isoallergens were observed in some subjects. Conclusions: Altogether, comprehensive analysis of antibody recognition of linear epitopes of multiple allergens enables characterisation of the nature of the antibody responses targeting this important set of food allergens.

Insight into the allergenicity and structure changes of parvalbumin from Trachinotus ovatus induced by dense-phase carbon dioxide.

Parvalbumin (PV) is a major allergen in fish, and traditional treatments cannot reduce its sensitization. The effects of dense-phase carbon dioxide (DPCD) treatment on the sensitization and spatial structure of PV in Trachinotus ovatus were evaluated in this study. Western blotting and indirect ELISA were used to determine the allergenicity changes and spatial conformations of PV treated by DPCD. Tris-tricine-SDS-PAGE, circular dichroism, surface hydrophobicity, endogenous fluorescence, UV spectrophotometry, free amino group, total sulfhydryl group and SEM analyses were applied to characterize PV structure. The results showed that DPCD treatment (15 MPa, 30 min, 50 °C) could reduce PV-induced allergic reactions by 39-41 %, which destroyed the normal conformational epitopes and reduced the risk of PV-induced allergy. The secondary structure changed from ordered to disordered with a decreased content of α-helical groups, while the internal hydrophobic groups were exposed. The total sulfhydryl group content decreased significantly (P < 0.05). The surface hydrophobicity and ultraviolet absorption spectrum were enhanced, and the endogenous fluorescence peak shifted to a long wavelength. Meanwhile, the content of free amino groups increased significantly (P < 0.05). This study could provide a theoretical basis and a promising technical approach for reduction of PV allergenicities.

Comparative analysis of allergenicity and predicted linear epitopes in α and ß parvalbumin from turbot (Scophthalmus maximus).

BACKGROUND: Parvalbumin (PV) can be subdivided into two phylogenetic lineages, αPV and ßPV. The bony fish ßPV is considered a major fish allergen. However, there is no available report on the immunological property and epitope mapping of bony fish αPV. RESULTS: To characterize the allergenic property of bony fish αPV and investigate the difference in allergenic property of bony fish αPV and ßPV, turbot (Scophthalmus maximus) αPV and ßPV were identified by mass spectrometry and were expressed in Escherichia coli system in this study. Spectra analysis and three-dimensional (3D) modeling showed the similar structure between αPV and ßPV. However, αPV exhibited lower immunoglobulin E/immunoglobulin G (IgE/IgG) binding capacity than ßPV. Three identified ßPV epitopes possessed higher IgE reactivity and more hydrophobic residues than three identified αPV epitopes. In addition, less similarity in sequence homology of αPV epitopes was observed with allergen sequences in database. CONCLUSION: These finding expanded information on fish PV epitopes and substantiated the difference in allergenicity and epitope mapping between fish αPV and ßPV, which will improve the epitope-based detection tools of PV and diagnostic of PV induced fish allergy. © 2023 Society of Chemical Industry.

Kinetics of Ca2+ Dissociation from Cod Parvalbumin Studied by Fluorescent Stopped-flow Method.

BACKGROUND: Small Ca2+-binding protein parvalbumin possesses two strong Ca2+/Mg2+- binding sites located within two EF-hand domains. Most parvalbumins have no tryptophan residues, while cod protein contains a single tryptophan residue, which fluorescence (spectrum maximum position and fluorescence quantum yield) is highly sensitive to the Ca2+ association/dissociation. OBJECTIVE: Intrinsic protein fluorescence of cod parvalbumin can be used for elucidating the mechanism of Ca2+ binding to this protein. Fluorescence of the single tryptophan residue of cod parvalbumin has been used to monitor Ca2+-induced changes in the protein, both in steady-state and kinetic mode. METHODS: Steady-state fluorescence spectra of cod parvalbumin were measured using Cary Eclipse spectrofluorimeter. Stopped-flow accessories in combination with a novel high-speed spectrofluorimeter were used for measurements of kinetics of Ca2+ dissociation from cod parvalbumin after fast mixing of Ca2+-loaded protein with a chelator of divalent metal cations ethylenediaminetetraacetic acid (EDTA). RESULTS: The fluorescent phase plots (fluorescence intensity at a fixed wavelength plotted against a fluorescence intensity at another fixed wavelength), constructed from steady state and kinetical data, shows a break at [Ca2+]/[parvalbumin] ratio close to 1. This means that the transition passes through an intermediate state, which is a protein with one bound calcium ion. These observations indicate that the binding of Ca2+ to cod parvalbumin is sequential. CONCLUSION: The results of the present spectral study showed that the binding of Ca2+ to cod parvalbumin is a sequential process. Calcium dissociation rate constants for the two binding sites of cod parvalbumin evaluated from the kinetic data are koff1 = 1.0 s-1 and koff2 = 1.5 s-1.

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.

Strontium Binding to α-Parvalbumin, a Canonical Calcium-Binding Protein of the "EF-Hand" Family.

Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr2+ interference with Ca2+ binding to proteins of the EF-hand family, we studied Sr2+/Ca2+ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human α-PA ('CD' and 'EF' sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 109 M-1 and 4 × 109 M-1 for Ca2+, and 2 × 107 M-1 and 2 × 106 M-1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of α-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded α-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 × 105 M-1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of α-PA exhibit similar states of their COO- groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of α-PA, compared to the Ca2+-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of α-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of α-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.

Recruitment of parvalbumin and somatostatin interneuron inputs to adult born dentate granule neurons.

GABA is a key regulator of adult-born dentate granule cell (abDGC) maturation so mapping the functional connectivity between abDGCs and local interneurons is required to understand their development and integration into the hippocampal circuit. We recorded from birthdated abDGCs in mice and photoactivated parvalbumin (PV) and somatostatin (SST) interneurons to map the timing and strength of inputs to abDGCs during the first 4 weeks after differentiation. abDGCs received input from PV interneurons in the first week, but SST inputs were not detected until the second week. Analysis of desynchronized quantal events established that the number of GABAergic synapses onto abDGCs increased with maturation, whereas individual synaptic strength was constant. Voluntary wheel running in mice scaled the GABAergic input to abDGCs by increasing the number of synaptic contacts from both interneuron types. This demonstrates that GABAergic innervation to abDGCs develops during a prolonged post-mitotic period and running scales both SST and PV synaptic afferents.

Identification and characterization of parvalbumin-like protein in Trichophyton violaceum.

Parvalbumins play crucial physiological roles in neuromuscular systems of vertebrates, such as cell-cycle, development of neurons, contraction of muscles, and regulation of intracellular calcium. To perform these neuromuscular functions, parvalbumin may be in associated with other proteins including calbindin, carbonic anhydrase, and cytochrome oxidase. Humans may show an IgE-specific hypersensitivity to parvalbumins after consumption of some distinct fish species. While this protein is abundant in fish muscles, literature review of publications related to fish parvalbumins, do not point to the presence of parvalbumins in eukaryotic microbes. In this study, we propose that distantly related parvalbumins may be found in some non-fish species. Bioinformatics studies such as multiple sequence alignment (MSA), phylogenetic analysis as well as molecular-based experiments indicate that, at least two parvalbumins sequences (UniProt IDs: A0A178F775 and A0A178F7E4) with EF-hand domains and Ca2+-binding sites could be identified in Trichophyton violaceum, a pathogenic fungal species. It was determined that both genes consisted of a single exon and encoded for parvalbumin proteins possessing conserved amino acid motifs. Antigenicity prediction revealed antigenic sites located in both sides of the Ca2+-binding site of the first EF-hand domain. Our phylogenetic analysis revealed that one of parvalbumins (UniProt ID: 0A178F775) can be evolved to other parvalbumins in T. violaceum (UniProt ID: A0A178F7E4) and fish species through evolutionary phenomenon. To confirm our in-silico findings, we designed three primer pairs to detect one of the T. violaceum parvalbumins (UniProt ID: A0A178F7E4) by polymerase chain reaction (PCR); one primer pair showed a strong and specific band in agarose gel electrophoresis. To evaluate the specificity of the method, the primers were tested on extracted DNA from Trichophyton rubrum and T. mentagrophytes. The results demonstrated that the evaluated parvalbumin gene (UniProt ID: A0A178F7E4) was T. violaceum-specific and this pathogenic fungus can be differentiated from T. rubrum and T. mentagrophytes through identification of parvalbumin genes. Further studies are necessary to unravel the biochemical and physiological functions of parvalbumins in T. violaceum.

Molecular Dynamics Simulation and Kinetic Study of Fluoride Binding to V21C/V66C Myoglobin with a Cytoglobin-like Disulfide Bond.

Protein design is able to create artificial proteins with advanced functions, and computer simulation plays a key role in guiding the rational design. In the absence of structural evidence for cytoglobin (Cgb) with an intramolecular disulfide bond, we recently designed a de novo disulfide bond in myoglobin (Mb) based on structural alignment (i.e., V21C/V66C Mb double mutant). To provide deep insight into the regulation role of the Cys21-Cys66 disulfide bond, we herein perform molecular dynamics (MD) simulation of the fluoride-protein complex by using a fluoride ion as a probe, which reveals detailed interactions of the fluoride ion in the heme distal pocket, involving both the distal His64 and water molecules. Moreover, we determined the kinetic parameters of fluoride binding to the double mutant. The results agree with the MD simulation and show that the formation of the Cys21-Cys66 disulfide bond facilitates both fluoride binding to and dissociating from the heme iron. Therefore, the combination of theoretical and experimental studies provides valuable information for understanding the structure and function of heme proteins, as regulated by a disulfide bond. This study is thus able to guide the rational design of artificial proteins with tunable functions in the future.

Reconstruction of fish allergenicity from the content and structural traits of the component ß-parvalbumin isoforms.

Most fish-allergic patients have anti-ß-parvalbumin (ß-PV) immunoglobulin E (IgE), which cross-reacts among fish species with variable clinical effects. Although the ß-PV load is considered a determinant for allergenicity, fish species express distinct ß-PV isoforms with unknown pathogenic contributions. To identify the role various parameters play in allergenicity, we have taken Gadus morhua and Scomber japonicus models, determined their ß-PV isoform composition and analyzed the interaction of the IgE from fish-allergic patient sera with these different conformations. We found that each fish species contains a major and a minor isoform, with the total PV content four times higher in Gadus morhua than in Scomber japonicus. The isoforms showing the best IgE recognition displayed protease-sensitive globular folds, and if forming amyloids, they were not immunoreactive. Of the isoforms displaying stable globular folds, one was not recognized by IgE under any of the conditions, and the other formed highly immunoreactive amyloids. The results showed that Gadus morhua muscles are equipped with an isoform combination and content that ensures the IgE recognition of all PV folds, whereas the allergenic load of Scomber japonicus is under the control of proteolysis. We conclude that the consideration of isoform properties and content may improve the explanation of fish species allergenicity differences.

Identification and comparison of allergenicity of native and recombinant fish major allergen parvalbumins from Japanese flounder (Paralichthys olivaceus).

Parvalbumin is the major fish allergen that can trigger anaphylactic reactions in predisposed individuals. We extracted and purified native parvalbumins from Japanese flounder (Paralichthys olivaceus) with gradient ammonium sulfate fractionation, and cloned DNAs into the expression vector pET-28a (+) to produce highly purified recombinant parvalbumin in Escherichia coli. The identification of native and recombinant parvalbumins was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and mass spectrometry. The IgG-binding capacity was examined by indirect and inhibition ELISA. The immuno-reactivity was further evaluated by the release assay of ß-hexosaminidase in an RBL-2H3 cell model. Three parvalbumin isoforms were purified, namely PVI, PVII and PVIII with a purity of over 90%. Mass data showed that PVII and PVIII matched with XP_019958408.1 and XP_019938975.1 in the NCBI database, respectively. The recombinant parvalbumin was successfully expressed with high purity and matched with PVIII, which has been proved as the major parvalbumin isoform. Data from an ELISA assay revealed that the recombinant PVIII contains most of the IG-binding epitopes of the native PVIII. Meanwhile, the recombinant PVIII showed a lower immuno-reactivity in the RBL-2H3 cell model. The results suggest that recombinant PVIII could be a useful tool for the confirmation of fish allergens and diagnosis of fish allergies.

Effect of tyrosinase and caffeic acid crosslinking of turbot parvalbumin on the digestibility, and release of mediators and cytokines from activated RBL-2H3 cells.

Turbot can induce allergy in susceptible individuals due to the presence of parvalbumin (PV), a major fish allergen. This study aimed at evaluating the digestibility and the ability of PV to elicit the release of cellular degranulation, following treatment with tyrosinase (PV-Tyr), caffeic acid (PV-CA) and in combination (PV-Tyr/CA), using in vitro digestion and RBL-2H3 (passive rat basophil leukemia) cell line. The digestion assay products revealed that the stability of PV in simulated gastric fluid (SGF) was stronger, while in simulated intestinal fluid (SIF) was rather weak. Western blot analysis revealed that the IgG-binding abilities of the cross-linked PV were markedly reduced. Moreover, crosslinking hampered the release of cellular degranulation process in RBL-2H3 cell lines. PV-Tyr/CA showed highly significant reduction in the release rate of ß-hexosaminidase (66.02%), histamine (35.01%), tryptase (29.25%), cysteinyl leukotrienes (29.72%), prostaglandin D2 (34.96%), IL-4 (43.99%) and IL-13 (38.93%) and shown potential in developing hypoallergenic fish products.

Feasibility of trifluoromethyl TROSY NMR at high magnetic fields.

Insights into the structure and dynamics of large biological systems has been greatly improved by two concurrent NMR approaches: the application of transverse relaxation-optimized spectroscopy (TROSY) techniques in multi-dimensional NMR, especially the methyl-TROSY, and the resurgence of 19F NMR using trifluoromethyl (CF3) probes. Herein we investigate the feasibility of combining these approaches into a trifluoromethyl-TROSY experiment. Using a CF3-labelled parvalbumin, we have evaluated the natural abundance 13C-19F correlation spectra and find no indication of a CF3 TROSY at high magnetic fields.

Effect of laccase-catalyzed cross-linking on the structure and allergenicity of Paralichthys olivaceus parvalbumin mediated by propyl gallate.

The aim of the present study was to evaluate Paralichthys olivaceus parvalbumin (PV) following treatment by laccase (LAC) in the presence of propyl gallate (PG) on the structure and potential allergenicity. The structure of LAC + PG treated PV was analyzed through SDS-PAGE, CD, fluorescence, and allergenicity was analyzed by immunological and cell model. Our results showed that LAC + PG treatment can induce structural changes through PV cross-linking. Western blotting and indirect ELISA analysis revealed the decrease in IgG binding capacity of PV, corresponding with the structural changes. The results of in vitro digestion illustrate that LAC + PG treated PV showed more resistance to gastrointestinal digestion compared to untreated PV. The release rate of ß-hexosaminidase and histamine decreased by 35.6% and 66.9%, respectively, with LAC + PG treatment by RBL-2H3 cell assay. Considering the wide utilization of LAC in food industry, our treatment reveals its potential for creation of hypoallergenic fish products under mild reaction conditions.

Designing Calcium-Binding Proteins for Molecular MR Imaging.

Early diagnosis, noninvasive detection, and staging of various diseases, remain one of the major clinical barriers to effective medical treatment and prevention of disease progression toward major clinical consequences. Molecular imaging technologies play an indispensable role in the clinical field in overcoming these major barriers. The increasing application of imaging techniques and agents in early detection of different diseases such as cancer has resulted in improved treatment response and clinical patient management. In this chapter we will first introduce criteria for the design and engineering of calcium-binding protein (CaBP) parvalbumin as a protein Gd-MRI contrast agent (ProCA) with unprecedented metal selectivity for Gd3+ over physiological metal ions. We will then discuss the further development of targeted MRI contrast agent for molecular imaging of PSMA biomarker for early detection of prostate cancer.

Ca2+-Binding Proteins of the EF-Hand Superfamily: Diagnostic and Prognostic Biomarkers and Novel Therapeutic Targets.

A multitude of Ca2+-sensor proteins containing the specific Ca2+-binding motif (helix-loop-helix, called EF-hand) are of major clinical relevance in a many human diseases. Measurements of troponin, the first intracellular Ca-sensor protein to be discovered, is nowadays the "gold standard" in the diagnosis of patients with acute coronary syndrome (ACS). Mutations have been identified in calmodulin and linked to inherited ventricular tachycardia and in patients affected by severe cardiac arrhythmias. Parvalbumin, when introduced into the diseased heart by gene therapy to increase contraction and relaxation speed, is considered to be a novel therapeutic strategy to combat heart failure. S100 proteins, the largest subgroup with the EF-hand protein family, are closely associated with cardiovascular diseases, various types of cancer, inflammation, and autoimmune pathologies. The intention of this review is to summarize the clinical importance of this protein family and their use as biomarkers and potential drug targets, which could help to improve the diagnosis of human diseases and identification of more selective therapeutic interventions.

Effect of tyrosinase-catalyzed crosslinking on the structure and allergenicity of turbot parvalbumin mediated by caffeic acid.

BACKGROUND: Enzymatic treatment of allergenic protein can alter their functional properties under a mild reaction condition due to specificity of enzymes. Phenolic compounds act as mediators and enhance the crosslinking reactions. The study aimed to assess the changes in the structure and immunoglobulin G (IgG) binding capacity of turbot parvalbumin (PV) upon crosslinking with tyrosinase (Tyr) in the absence and presence of caffeic acid. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the appearance of higher molecular weight bands (24, 36 kDa) in the crosslinked PV. The secondary structure of crosslinked PV became loosened and disordered. The results of intrinsic fluorescence and ultraviolet absorption spectral analyses, as well as surface hydrophobicity and free amino group analyses also revealed structural changes. As observed by western blot analysis, the intensity of the PV bands reduced upon Tyr treatment, indicating reduced binding of specific IgG to PV. Moreover, the indirect ELISA (enzyme-linked immunosorbent assay) analysis confirmed that the IgG binding ability of crosslinked PV was reduced 34.94%. CONCLUSION: Enzymatic treatment mitigated the allergenicity of fish PV, which was closely related to the alterations in the conformational structure. This treatment showed potential for developing hypoallergenic fish products under mild reaction conditions. © 2019 Society of Chemical Industry.

The binding of monomeric amyloid ß peptide to serum albumin is affected by major plasma unsaturated fatty acids.

Human serum albumin (HSA) serves as a natural depot of amyloid ß peptide (Aß). Improvement of Aß binding to HSA should impede Alzheimer's disease (AD). We developed a method for quantitation of the interaction between monomeric Aß40/42 and HSA using surface plasmon resonance spectroscopy. The dissociation constant of HSA complex with recombinant Aß40/42 is 0.2-0.3 µM. Flemish variant of Aß40 has 2.5-10-fold higher affinity to HSA. The parameters of the HSA-Aß interaction are selectively sensitive to HSA binding of major plasma unsaturated fatty acids and Cu2+. Linoleic and arachidonic acids promote the HSA-Aß42 interaction. The developed methodology for quantitation of HSA-Aß interaction may serve as a tool for search of compounds favoring HSA-Aß interaction, thereby preventing AD progression.

Effects of his-tags on physical properties of parvalbumins.

A comparative study of His-tagged and non-tagged rat ß-parvalbumin (rWT ß-PA), calcium binding protein with the EF-hand calcium binding domains, has been carried out. The attachment of His-tag increases α-helical content and decreases ß-sheets and ß-turns content of the metal free form (apo-state) of ß-PA. In contrast to this, the attachment of His-tag decreases α-helical content by more than 10% and increases contents of ß-sheets and ß-turns of the Ca2+-loaded state. According to the dynamic light scattering analysis, apo-state of His-tagged rat ß-PA seems to be less compact compared with the apo-state of non-tagged rat ß-PA. Surprisingly, the attachment of His-tag practically does not change mean hydrodynamic radius of Ca2+-loaded rat ß-PA. The attachment of His-tag shifts thermal denaturation peaks of both apo- and Ca2+-loaded states of rat ß-PA towards higher temperatures by 3-4 °C and slightly decreases its Ca2+ affinity. These results should be taken into consideration in the use of His-tagged parvalbumins.

Matrix effect on food allergen detection - A case study of fish parvalbumin.

Two fish parvalbumin models were established to study relationships among matrix effect, extractability, and thermostability during in vitro immunodetection using two parvalbumin-specific monoclonal antibodies (3E1 and PARV19). Our results illustrated that matrix-induced thermal instability of parvalbumin was due mainly to physical (hydrophobic effect) and chemical (thiol-disulfide interchange) interactions. The addition of sodium dodecyl sulfate (SDS, surfactant), ß-mercaptoethanol (reducing agent) or ethylenediaminetetraacetic acid (EDTA, metal chelator) during sample preparation could not only increase the extractability of parvalbumin but also enhanced its immunodetection. Our findings demonstrated excess EDTA completely chelated Ca2+ in parvalbumin and rendered it undetectable using PARV19 (a Ca2+-dependent antibody). Overall, our resulted showed that matrix effect on in vitro analyte quantification cannot be underestimated. Any false negative or positive results could lead to severe or life-threatening allergic reactions.