Effect of Maillard reaction conditions on antigenicity of β-lactoglobulin and the properties of glycated whey protein during simulated gastric digestion

Response surface methodology was employed to study the effects of Maillard reaction conditions on the antigenicity of β-lactoglobulin (β-LG) in whey protein isolate (WPI) and to optimise the Maillard reaction conditions of WPI conjugate with oligoisomaltose under which the antigenicity of β-LG reduced to the minimum value. The antigenicity of β-LG and α-lactalbumin (α-LA) in natural and glycated WPI during simulated gastric digestion were investigated. The antigenicity of β-LG was reduced from 272.4 µg mL^?1 to 30.99 µg mL^?1 under the optimal Maillard reaction conditions. After 120 min simulated gastric digestion, the antigenicity of β-LG in natural and glycated WPI were 42.83 µg mL^?1 and 15.66 µg mL^?1, respectively. And the antigenicity of α-LA in natural and glycated WPI were 0.78 µg mL^?1 and 0.03 µg mL^?1, respectively.     

Effects of Maillard reaction conditions on in vitro immunoglobulin G binding capacity of ovalbumin using response surface methodology

This study aims to investigate the effects of temperature, time, pH, ovalbumin/glucose ratio, and ovalbumin concentration on the potential allergenicity of ovalbumin during the Maillard reaction, and to define the key influential factors. Results indicated that immunoglobulin G (IgG) binding of ovalbumin could either be increased or decreased, depending on the reaction temperature, and the glycation parameters could work collectively. Response surface methodology (RSM) was used to optimize conditions under temperature below denaturation of ovalbumin. The key influential factor was temperature, followed by pH and ovalbumin concentrations. Under the optimized condition, a reduction of immunoglobulin E (IgE) binding to 61.86% was observed. RSM was proved to be a good tool in optimizing the Maillard reaction parameters to reduce potential allergenicity of ovalbumin.     

Preparation of anti-melamine antibody and development of an indirect chemiluminescent competitive ELISA for melamine detection in milk

An indirect chemiluminescent competitive ELISA (CL-ELISA) method was developed to detect residues of melamine in milk. A high-quality polyclonal antibody towards melamine cyanurate (MC) was prepared based on synthesis of a novel immunogen. The method is applicable over the range of 0.5–7.0 µg.mL^?1 of MC, with an IC₅₀ value of 1.7 µg.mL^?1. The developed method was used in the detection of melamine residue in milk with the detection limit of 1 ng.mL^?1. There was no cross-reactivity with commonly used veterinary drugs. The CL-ELISA method developed provides an alternative to chromatography spectrometry for regulatory analysis of melamine in milk and could be promisingly used to improve the sensitivity of the available ELISA test kits.     

Development of a fluorescence-linked immunoassay based on quantum dots for fenvalerate

The glutathione-coated CdTe quantum dots (QDs) were synthesized in a paper. After purification, the QDs were coupled with monoclonal antibody to fight against fenvalerate. The conjugates had the same emission wavelength as that of QDs. The excitation wavelength and emission wavelength of the conjugates were determined to obtain the highest signal-to-noise ratio. After the antibody concentration optimization, the fluorescence-linked immunoassay method was developed. The method used the QDs as the signal to quantify the fenvalerate. Compared with the enzyme-linked immunosorbent assay (ELISA), it saved more than 1 h and decreased the false-positive rate using the specified emission wavelength of QDs. The 50% inhibitory concentration (IC₅₀) of the method was 0.28 µg mL^?1. The detection limit was 25 ng mL^?1 and the linear range was 60 ng mL^?1–3.83 µg mL^?1. Via preliminary application, fenvalerate residues in spiked samples were determined. The recovery of fenvalerate in water samples ranged from 84.5% to 96.2% and that in vegetables ranged from 72.5% to 125.7%. It was a rapid detection of the fenvalerate residue in environment and vegetables.     

Gold immunochromatographic assay for trimethoprim in milk and honey samples based on a heterogenous monoclonal antibody

Three heterogenous haptens (3,4,5-Trimethoxybenzoic acid [H1], 3,4,5-Trimethoxyphenylacetic acid [H2], 3-(3,4,5-Trimethoxyphenyl) propanoic acid [H3]) were used to synthesize immunogens, with the aim of screening a more sensitive monoclonal antibody (mAb) for trimethoprim (TMP). H2 proved to be the best heterogenous hapten. The mAb was specific with no cross-reactivity with other structurally related chemicals. The 50% maximal inhibitory concentration of the mAb against TMP was 1.98?µg?L^?1, which is more sensitive compared with the mAb reported in our previous work. A mAb-based gold immunochromatographic assay (GICA) was established to analyze TMP residues in milk and honey samples. The concentration of coating antigen in test line and concentration of mAb conjugated with gold nanoparticles was 0.3?µg?mL^?1 and 0.25?µg?mL^?1, respectively. Under optimized conditions, the visual limit of detection concentration of TMP spiked in milk and honey samples was 10?µg?L^?1 and 15?µg?kg^?1, respectively. The proposed GICA provides rapid and simple determination of TMP residues in milk and honey samples.     

Development of an antibody-based colloidal gold immunochromatographic lateral flow strip test for natamycin in milk and yoghurt samples

An immunochromatographic lateral flow strip test was developed for the detection of natamycin (Nata) residues in milk and cheese samples. Monoclonal antibody (mAb) against Nata was produced with a half maximal inhibitory concentration of 1.85?μg?L^?1. MAb conjugated with gold nanoparticles was the detection reagent. Nata conjugated with ovalbumin via the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide method immobilized on a nitrocellulose membrane was the capture reagent. This semi-quantitative method required only 15?min to complete. The optimum concentrations of coating antigen and mAb were 0.15?µg?mL^?1 and 0.2?µg?mL^?1, respectively. Based on an optical density scanner, the visual limit of detection of Nata was 5?μg?L^?1 and 10?μg?kg^?1 in milk and yoghurt samples, respectively.     

Antibacterial and Hemolytic Activities of Quaternary Pyridinium Functionalized Polynorbornenes

In this study, amphiphilic polyoxanorbornene with different quaternary alkyl pyridinium side chains were synthesized. The biological efficiencies of these polymers, with various alkyl substituents, were determined by bacterial growth inhibition assays and hemolytic activity (HC₅₀) against human red blood cells (RBCs) to provide selectivity of these polymers for bacterial over mammalian cells. A series of polymers with different alkyl substituents (ethyl, butyl, hexyl, octyl, decyl and phenylethyl) and two different molecular weights (3 and 10 kDa) were prepared. The impact of alkyl chain length divided the biological activity into two different cases: those with an alkyl substituent containing four or fewer carbons had a minimum inhibitory concentration (MIC) of 200 µg · mL^?1 and a HC₅₀ greater than 1 650 µg · mL^?1, while those with six or more carbons had lower MICs ≤ 12.5 µg · mL^?1 and HC₅₀ ≤ 250 µg · mL^?1. Using MSI‐78, the potent Magainin derivative which has an MIC = 12.0 µg · mL^?1 and HC₅₀ = 120 µg · mL^?1, as a comparison, the polymers with alkyl substituents ≤C₄ (four carbons) were not very potent, but did show selectivity values greater than or equal to MSI‐78. In contrast, those with alkyl substituents ≥C₆ were as potent, or more potent, than MSI‐78 and in three specific cases demonstrated selectivity values similar to, or better than, MSI‐78. To understand if these polymers were membrane active, polymer induced lipid membrane disruption activities were evaluated by dye leakage experiments. Lipid composition and polymer hydrophobicity were found to be important factors for dye release.

     

Development of a monoclonal antibody-based immunochromatographic strip for cephalexin

An anti-cephalexin (CEX) monoclonal antibody was prepared by the immunogen of CEX-keyhole limpet hemocyanin connected by disuccinimidyl suberate, which was used for the establishment of a rapid immunochromatographic test strip. With this semi-quantitative detection method, a low limit of deduction of 10 ng mL^?1 was obtained with the naked eyes and 1.3 ± 0.1 ng mL^?1 by a strip reader in unprocessed milk within 5 min, which was much lower than the European Union Maximum Residue Limit of 100 µg kg^?1 in milk. The recovery in the range of 87–120% was measured in another milk sample. In conclusion, this method with high sensitivity and satisfactory recovery shows significant potential for CEX residue detection in milk.     

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide EPN

An immunochromatographic assay (ICA) based on competitive antigen-coated format and colloidal gold label was developed for the detection of the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with EPN hapten-OVA conjugate (test line) and anti-mouse IgG (control line). As the competition is between the migrating analyte and the immobilised analyte hapten for the binding sites of the migrating antibody–colloidal gold (Ab–CG) conjugate, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. Based on this criterion, a nitrocellulose (NC) membrane was chosen as the most suitable for EPN ICA. The detection limit of the ICA for EPN standard and EPN spiked into agricultural samples were 10^?2 and 5×10^?2 µg mL^?1, respectively.     

Chemiluminescence microfluidic system of gold nanoparticles enhanced luminol-silver nitrate for the determination of vitamin B12

A rapid and sensitive chemiluminescence (CL) system coupled with a microfluidic chip has been presented to determine vitamin B12 (VB12) based on the reaction of luminol and silver nitrate (AgNO₃) in the presence of gold nanoparticles (AuNPs). A microfluidic chip was fabricated by a soft-lithographic procedure using polydimethyl siloxane (PDMS) having four inlets and one outlet with a 200 μm wide, 250 μm deep, and 100 mm long microchannel. Ag⁺ was used as a chemiluminogenic oxidant in this CL reaction which oxidized luminol to produce strong CL signal in the presence of AuNPs. Luminol reacted with AgNO₃ under the catalysis of AuNPs to produce luminol radicals which reacted with dissolved oxygen and emitted CL light. The proposed CL system was applied to determine the amount of VB12 in VB12 tablets and multivitamin. Under the optimum conditions, the CL intensity of the system was increased with the concentration of VB12 in the range of 0.25–100 ng mL^?1 with the correlation coefficient of 0.9982. The limit of detection was found to be 0.04 ng mL^?1 with the relative standard deviation of 1.56 % for five replicate determinations of 25 ng mL^?1 of VB12. The CL reaction mechanism was demonstrated by UV-visible spectra and CL emission spectra.     

IgE antibodies of fish allergic patients cross-react with frog parvalbumin

BACKGROUND: The major allergens in fish are parvalbumins. Important immunoglobulin (Ig)E cross-recognition of parvalbumins from different fish species has been shown. Recently frog parvalbumin alpha has been found to be responsible for a case of IgE-mediated anaphylaxis triggered by the ingestion of frog meat. The aim of this study was to investigate whether IgE antibodies of fish allergic persons cross-react with frog parvalbumin and to appreciate its clinical relevance. METHODS: The sera of 15 fish allergic patients and one fish and frog allergic patient were tested by IgE-immunoblotting against frog muscle extract. Sera were tested against recombinant parvalbumin alpha and beta from Rana esculenta. Skin prick tests were performed in selected patients with recombinant frog parvalbumin. Ca(2+) depletion experiments and inhibition studies with purified cod and frog recombinant parvalbumin were done to characterize the cross-reactive pattern. RESULTS: Fourteen of the sera tested had IgE antibodies recognizing low molecular weight components in frog muscle extract. Calcium depletion experiments or inhibition of patient sera with purified cod parvalbumin led to a significant or complete decrease in IgE binding. When tested against recombinant parvalbumins, three of 13 sera reacted with alpha parvalbumin and 11 of 12 reacted with beta parvalbumin from R. esculenta. Skin prick tests performed with recombinant frog parvalbumin were positive in fish allergic patients. Inhibition studies showed that a fish and frog allergic patient was primarily sensitized to fish parvalbumin. CONCLUSION: Cod parvalbumin, a major cross-reactive allergen among different fish species, shares IgE binding epitopes with frog parvalbumin. This in vitro cross-reactivity seems to be also clinically relevant. Parvalbumins probably represent a new family of cross-reactive allergens.     

Development of a highly sensitive icELISA to detect semicarbazide based on a monoclonal antibody

A highly sensitive and specific monoclonal antibody was prepared to detect semicarbazide (SEM). Hapten 4-{[(aminocarbonyl)hydrazono]methyl}benzoic acid (4-CPSEM) was synthesised through the condensation reactions of SEM and 4-carboxybenzaldehyde (4-CBA). The active ester method was employed to couple the hapten to bovine serum albumin, keyhole limpet hemocyanin and ovalbumin (OVA) to obtain conjugates of immunogens and coating antigens. A novel hapten 2-[(aminocarbonyl)hydrazono]acetic acid (SEM-A) was also synthesised from SEM and oxoacetic acid to prepare a heterologous coating antigen (SEM-A-OVA). The linear ranges of inhibition curves in the optimised indirect competitive enzyme-linked immunosorbent assay (icELISA) method were in the range of 0.0071–0.056 ng mL^?1 and 0.018–0.209 ng mL^?1 for coating antigens SEM-A-OVA and 4-CPSEM-OVA, respectively. The IC₅₀ was 0.019 ng mL^?1 for SEM (SEM in the form of 4-nitrobenzaldehyde semicarbazone) and 0.13 ng mL^?1 for 2-nitrobenzaldehyde semicarbazone, both of which were well below the minimum required performance limit of 1 µg·kg^?1 set by the European Commission.     

Effects of pH,temperature, enzyme-to-substrate ratio and reaction time on the antigenicity of casein hydrolysates prepared by papain

The effects of pH, temperature, enzyme-to-substrate ratio and reaction time on the antigenicity of casein hydrolysates were investigated. Response surface methodology (RSM) was employed to optimise the reaction conditions. Enzymatic hydrolysis with papain could reduce the antigenicity of α-casein and β-casein effectively and the reduction of antigenicity could be controlled by regulation of the reaction conditions. The model for optimal reaction conditions of a lower antigenicity of α-casein and β-casein was established. Under the range of conditions studied, enzyme-to-substrate ratio had the most significant effects on the antigenicity of α-casein and β-casein. The anti-α-casein IgG binding inhibition and anti-β-casein binding inhibition were both significantly negatively related with the degree of hydrolysis (DH).     

Effect of instant controlled pressure drop (DIC) treatment on milk protein's immunoreactivity

In this work, we tried to investigate the impact of treatment with instant controlled pressure drop technology (DIC) on the allergenicity of milk proteins. In DIC treatment, milk proteins are exposed to a high-pressure saturated steam (0.4 MPa, 144 °C) during 25 s and ended with an abrupt pressure drop towards a vacuum (5 kPa, 32 °C). Immunoglobulin E (IgE)-binding ability is determined with indirect enzyme linked immuno spectrophotometric analysis and western blot test. Results indicate significant changes in the IgE binding of treated proteins with DIC. Treated caseins showed increase in the IgE-binding ability; this indicates that DIC enhance the antigenicity of treated caseins. In the opposite, treated whey proteins (ß-lactoglobulin and α-lactalbumin) showed decrease in the IgE binding of these proteins. DIC treatment at 0.4 MPa during 25 s causes a greater reduction of the allergenicity of whey proteins than treatment with 0.6 MPa for 25 s.     

Substitution of antibody with molecularly imprinted 96-well plate in chemiluminescence enzyme immunoassay for the determination of chloramphenicol residues

A direct competitive chemiluminescence enzyme immunoassay was developed by using the molecularly imprinted 96-well plate as an artificial antibody to detect chloramphenicol (CAP). The artificial antibody was synthesised on the well surface of MaxiSorp polystyrene 96-well plate and CAP was conjugated with the horseradish peroxidase. The results showed that the imprinted plate exhibited antibody-like binding ability. The plate showed fast adsorption rate, 66% adsorption was finished within 20 min. The cross-reactivity for CAP, florfenicol and thiamphenicol were 100%, 1.25% and 2.08%, respectively. And the imprinted plate could be reused for many times without loss of sensitivity. The IC₅₀ and the detection limit values under optimum conditions were 30±2 µg·L^?1 and 0.9±0.01 µg·L^?1, respectively. The plate was used to detect CAP in sea cucumber, which showed excellent recoveries ranging from 89% to 98.7%. And the result correlated well with that obtained by the CAP enzyme-linked immunosorbent assay (ELISA) kit.     

Soluble polystyrene derivatives of metal bis(3,4-dicarboxybenzoyl)phthalocyaninates: Synthesis,thermal analysis,and sensitivity towards toxic gases

Polystyrene-bound metal [2,9 or 2,10 (or 2,16 or 2,17) bis(3,4-dicarboxybenzoyl)]phthalocyaninates were synthesized by Friedel-Crafts reaction of polystyrene with the corresponding metal phthalocyaninates. Co(II) and Cu(II) [2,9 or 2,10 (or 2,16 or 2,17) bis(3,4-dicarboxybenzoyl)]-phthalocyaninate (PS-CodaPc and PS-CudaPc) contained 0,13 mmol · g^?1 (12,4 wt.-%) and 0,13 mmol · g^?1 (12,8 wt.-%) of CodaPc and CudaPc, respectively. They were soluble in N,N'-dimethylformamide, but only partially soluble in chloroform, tetrahydrofuran (THF), dimethyl sulfoxide, N-methyl-2-pyrrolidone, and pyridine. The THF extracts contained 0,12 mmol · g^?1 (11,4 wt.-%) and 0,18 mmol ? g^?1 (17,2 wt.-%) of PS-CodaPc and PS-CudaPc, respectively. The thermal stability of the polymers was studied using thermogravimetric and differential thermal analysis in nitrogen and synthetic air atmosphere. The contents of MdaPc(M: metal) in THF-extracted polymers calculated from the data of residue in thermogravimetric analysis are 0,12 mmol · g^?1 for PS-CodaPc and 0,19 mmol · g^?1 for PS-CudaPc. In addition, the sensitive properties of the polymers towards toxic gases were also investigated by quartz microbalance transducers. The results show that the quartz microbalance sensors coated with both polymers were sensitive to NO₂ and chlorinated hydrocarbons, i.e. chloroform and perchloroethylene. The sensitivity to NO₂ was 6,53 · 10^?7 m³ · mL^?1 · s^?1 for PS-CodaPc and 1,90 · 10^?6 m³ · mL^?1 · s^?1 for PS-CudaPc, and that to chloroform and perchloroethylene was 2,33 · 10^?8 and 4,60 · 10^?8 m³ · mL^?1 · s^?1, respectively, for PS-CodaPc and 4,79 · 10^?8 and 9,51 · 10^?7 m³ · mL^?1 · s^?1 for PS-CudaPc.     

The polymerization of isobutyl vinyl ether by trifluoroacetic acid

The polymerization of isobutyl vinyl ether (IBVE) initiated by trifluoroacetic acid (TFA) in 1,2-dichloroethane and in 1,2-dichloroethane/carbon tetrachloride mixtures has been studied over the temperature range ?2,5°C to 35°C. Provided that the ratio [IBVE]/[TFA] did not exceed 88 and that 2·10^?4 mol·1^?1 < [TFA] < 4·10^?3 mol·1^?1 polymerization was the only detectable reaction, and the initial rate of reaction R₀ was equal to k[IBVE]²[TFA]. The activation energy of the rate = (34 ± 2) kJ · mol^?1 and the activation energy of the molecular weight = ? (11,9 ± 0,5) kJ · mol^?1. When [TFA] < 1·10^?4 mol/l, no reaction was detectable. The polymers produced had molecular weights less than 4400; the only transfer is by monomer and this is the main chain-breaking step. The reaction was unaffected by the addition of water for [H₂O] < [TFA]. The rate depended on the solvent dielectric constant, but in a manner suggestive of a dipole-dipole reaction. A mechanism is proposed for the reaction in which the reactive intermediates are trifluoroacetate esters solvated by monomer.     

Evaluation of the IgE reactivity of common pandora parvalbumin in a Moroccan population and action of heating and enzymatic treatments

The aim of this study was to evaluate the immunoglobulin E (IgE) sensitivity to common pandora (Pagellus erythrinus) parvalbumin (CPP) in a Moroccan population from the Fez region, and then to study the effect of temperature and enzymatic digestion on the allergenicity of CPP. This work was conducted with a questionnaire completed by a sera-bank, obtained from 500 patients recruited from Fez hospitals. Their sera were analyzed for specific IgE against CPP. Evaluation of specific IgE showed that 11.8% of patients present higher values (>150?IU/ml). Further indirect ELISA and dot-blot results indicated that CPP showed a decrease in the binding of anti-IgE under heating with an average diminution of 41.9%, while pepsin hydrolysis reduced IgE recognition by 22.9%. These results demonstrate that this population was sensitive to CPP and the sensitivity could be reduced by heating and pepsin hydrolysis with an action higher with temperature than enzymatic digestion processing.     

Expression of recombinant parvalbumin from wolf-herring fish and determination of its IgE-binding capability

In this study, we produced the recombinant form of parvalbumin from wolf-herring fish and determined its IgE reactivity. Parvalbumin cDNA was sub-cloned into pET28 and expressed in Escherichia coli BL-21. The immunoreactivities of the recombinant and native parvalbumins were compared, and the effect of calcium binding was determined by sera from 25 fish-allergic patients. ELISA and Western blotting confirmed similar IgE-reactivities of the recombinant and native proteins and confirmed that this phenomenon is highly dependent on calcium binding. The recombinant protein was 94.5% similar to carp parvalbumin (Cyp c1). Approximately 72% of patients reacted strongly with recombinant parvalbumin, 80% of them reacted with the native form and only 56% showed IgE reactivity with crude extract. Because the IgE-binding capacity of recombinant wolf-herring parvalbumin is retained and is highly similar to Cyp c1, the wild and hypoallergenic forms of this allergen could be used for diagnosis and immunotherapy of fish allergy, respectively.     

Molecular size and configuration of poly(3-vinylpyrene)

Solution properties of poly(3-vinylpyrene) (PVπ) have been studied to determine the degree of hindrance to the flexibility of the vinyl chain by the pyrene pendant group. Fractionation of this polymer prepared by anionic polymerization, yielded nine narrow distribution fractions in the M_w range of 35 000–490 000 and the fractions were characterized by osmometry, light-scattering and viscometry. Intrinsic viscosities of the fractions were obtained in chloroform and tetrahydrofuran at 25°C and in o-dichlorobenzene at different temperatures from 25–80°C. From the KUHN-MARK-HOUWINK relationship, [η] = KM^a, K and a values were determined and the exponent varied in the order, chloroform (a = 0.500) < tetrahydrofuran (a = 0.547) < o-dichlorobenzene (a = 0.655) suggesting that chloroform at 25°C is a theta solvent for PVπ. Cloud point titration indicated that a mixture of THF/methanol in the ratio of 92:8 (V/V) is also a theta solvent for PVπ at 25°C. A K_Θ value of 5.2·10^?4 determined for PVπ at 25°C gave a value for the unperturbed dimension (R /M)^1/2 of 5.77·10^?9 cm. The conformational parameter, σ was computed as 2.84 for PVπ which suggests that the hindrance to rotation of the vinyl chain by the pyrene group is approximately the same as in the case of carbazole in poly(n-vinylcarbazole). Values of [η] in o-dichlorobenzene decreased with the increase of temperature yielding an average negative d log [η]/dT value of 1.1·10^?3. The expansion factor decreased in all cases with the rise of temperature. A decrease of unperturbed dimension with the increase of temperature was reflected in the d log K_Θ/dT value of ?2.0·10^?3 from which (R /M)^1/2 at 80°C was computed as 5.35·10^?9 cm.