| Numerous proteins are abundant in milk and dairy products which are loved by many people, especially for the infants, young children and the elderly. Moreover, they are the main nutrient sources in daily life. However, milk is one of the most important allergen, and survey shows that the incidence of disease in the crowd induced by milk allergy was0.3-7.5%, and among them, the morbidity of infant has amounted to2-3%. Bovine a-lactalbumin (a-La) belongs to the lysozyme superfamily which was considered as one of the major allergens in milk. According to incompletely statistics, more than51%of milk allergy was caused by a-La. The homology of a-La in milk and female was about74%, and another6%amino acid has the similar chemical property. α-lactalbumin possesses high value nutritional and strong allergenicity. Therefore, there is a great theoretical and realistic significance for the detection of a-La in food products.The research was composed of four parts including purification of bovine α-La, preparation and purification of monoclonal antibody against α-La, monoclonal antibodies labelled with quantum dots and the detection of α-La in food products. The research was mainly connected with the following parts.(1) The purification of bovine α-lactalbumin from milk was achieved through DEAE-Sepharose Fast Flow anion exchange chromatography combined with Sephadex G-75gel chromatography. The α-La that obtained from milk was evaluated by SDS-PAGE and gray scan, and its concention was detected with lowry method. The result showed that the purity of α-La was above95%and the yield was about20.5%.(2) Balb/c mice were immuned with the pure α-La, and indirect ELISA was used to. test the titer of antiserum. When the titer was above1:10.0000, the mouse was killed and spleen cells were prepared. The result of sccreeing of hybridoma cell lines showed that the fusion rate was above70%, and more than3%holes were positive. The titer of monoclonal antibody from ascites was about3×105. Ascites monoclonal antibody was purified using caprylic acid-ammonium sulfate precipitation joint, and its purity was detected by SDS-PAGE. Furthermore, the activity and specificity of purified monoclonal antibody were evaluated with indirect competitive ELISA and Western Blotting, respectively. The result showed that the purified monoclonal antibody still has activity and high apecificity, and there is no cross recation with other allergen in milk. (3) The purified monoclonal antibody was coupled with fluorescent quantum dots. The resarch showed a good coupling effect when the reactant ratio of QD, EDC, NHS, monoclonalantibody was1:10:6:4. The purification of coupling product was achieved using Sephadex G-200gel chromatography. The maximum emission peak of fluorospectro spectra for QD has a hypochromatic shift of3nm after coupling with monoclonal antibody. Those results confirmed that the fluorescent qutantum dots have been coupled onto the monoclonal antibody. Moreover, the monoclonal antibodies coupling quantum dots can be combined with a-lactalbumin. It revealed that monoclonal antibody coupled with quantum dot retained strong immune activity.(4) A competitive FLISA was established to detect a-La in milk, and the conditions of which was determined as following:the concentration of coated allergen was1μg/mL, QD-antibody was diluted as1:3200, the ratio of primary antibody and competitive allergen was1:1. The result showed that there was a good linear relation when the concentration of α-La was within from0.1to100ng/mL. The concentration of IC50was0.03μg/mL, and the minimum detection limit was0.1ng/mL. The new established method was used to detect a-lactalbumin in commercial dairy products, and it can provide more sensitive detection results compared with the conventional ELISA method. |
PDF Full Text Request