| Recently,Food allergies have become a major public health concern.In order to protect public health and safety,food allergies problem must be discussed always.So far,there is no special treatment to food allergy,and an important method to prevent people jfrom food allergy is decreasing exposure to allergens.At present,the research is focused on the development of low sensitivity food and food allergen detection and analysis.In this paper,Bovine serum albumin(BSA)was applied as the experiment material to combine high-Hydrostatic pressure(HHP)and low temperature treatment.The effects of HHP synergistic temperature which were bond to kinetics and thermodynamics on the BSA molecular structure were studied and discussed in this study.Meanwhile,the effects of HHP on BSA structure and the change of antigenicity were discussed.1.The application of indirect competitive ELISA method for the detection of animal allergens BSAIn this paper,a sensitive,specific indirect competitive immunoassay method was established.Monoclonal antibody against BSA was made by immunizing BALB/c mice with allergenic BSA.The purified monoclonal antibody against BSA was made by using BSA as the coating antigen and homemade antibody as the primary antibody.Horseradish peroxidase labeled goat anti-mouse IgG as enzyme secondary antibody and tetramethyl benzidine(TMB)as the substrate.The optimal antigenic coating concentration for indirect competitive enzyme-linked immunoassay was 0.25,g/mL.The optimal concentration of monoclonal antibody was 1:32000 and the optimal working concentration was 1:10000.The minimum detection limit was 6.71 ng/mL while the linear detection range was 3.5938-460 ng/mL.The curve regression equation is y =-47.4489x + 135.8663 and the correlation coefficient is 0.9973.The method has no cross reaction with horse serum,porcine serum,rabbit serum,sheep serum,dog serum,chicken serum and mouse serum.The method is specific,sensitive,reproducible and it can be applied for rapid detection of allergen BSA.2.The application of Surface Plasmon Resonance method for combination ability detection of animal allergen BSA and antibody.The CM5 chip was used to optimize the experimental conditions of the antigen and antibody combination.We aimed to establish a method to get the change of BSA and antibody combining dynamic and thermodynamic constants.The best antibody coupling concentration was 100 p-g/mL.The minimum detection limit was 1.6 nmol/L while the curve regression equation is y=0.10462x-+8.1897 and the correlation coefficient is 0.9975.The concentration range of the BSA dynamic detection was 156 nmol/L,312 nmol/L,625 nmol/L,1.25 ?mol/L,2.5 ?mol/L and 5 ?mol/L.The regenerative liquid was glycine-hydroelectric acid and the PH=2.0.3.The effect of ultrahigh pressure treatment on BSA structure and allergenicityUltra-high pressure treatment couldn't change the primary structure of BSA.Otherwise,HHP made different effects on the secondary structure and tertiary structure.Changes of the secondary structure showed that the a-helix content decreases,and the ?-fold structure content,?-turn and random coil is increasing.And the structure of the BSA protein increases with the increasing heat effect resulted from the high-pressure when causing the hydrogen bond structure disorder in the natural conformation.In the tertiary structure,the results of hydrophobicity,free thiol content and endogenous fluorescence intensity of the protein showed a tendency to increase and then decrease.The decrease of endogenous fluorescence intensity indicated that HHP effect makes BSA molecules stretch and make the hydrophobicity of the surroundings of tryptophan residues increase.Furthermore.some aromatic amino acid residues are embedded in the molecule.At the same time,some of the original embedded in the BSA protein molecules within the amino acid residues were exposed to the molecular surface.And protein hydrophobicity increased disulfide bond breakage.However,with the treatment of pressure and temperature improving,protein molecules aggregate.Some of the aggregates might also result in precipitation,which caused original exposed hydrophobic groups re-embedded in the molecule.And the hydrophobic decreased,and the bonds between free thiol were re-Associated to form disulfide bonds.The results showed that ultra-high pressure synergistic temperature treatment could effectively reduce the antigenicity of allergen BSA by using the established ELISA method.In this study,the correlation between BSA antigenicity and ?-helix structure was also analyzed by linear fitting analysis,and both of them showed significant positive correlation(P<0.05)which further confirmed that the antigenicity of the allergen BSA is closely related to the ?-helix.Moreover,the change of the ?-helix content will result in the change of the conformation of the protein,and then the antigenicity would be reduced.The kinetic and thermodynamic data show that the dissociation of BSA-IgG is accelerated and the stability decreases with the increase of KD,and the binding affinity of BSA is decreased.The reaction is driven by the entropy of the natural state into enthalpy.The main force of the combination is changed from the typical hydrophobic effect of the natural state to the hydrogen bond van der Waals force. |
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