Application Of Monoclonal Antibody ELISA To Determination Of Haze-Active Protein In Beer

The haze-active protein (HAP) is one of the major factors which result in the unstability downtrend. Therefore, the establishment of reliable, rapid, efficient and specific method has great significance. Firstly, the HAP was isolated by the adsorption characteristics of silica gel, and purified by Gel filtration column (Sephadex G-75) secondly, removing the protein fragment of 10-25kDa. Identifying by SDS-PAGE and the high puritying protein (about 40kDa MW) was obtained finaly. The concentration of HAP was 1.616mg·mL^-1 evaluated by Bradford method.Western Blot indicated that the antibody against silica gel elution protein has high specification. Compared the content of amino acid between the protein in beer and in HAP by HPLC, the HAP was rich in proline about 44% which was higher than in beer apparently. Meanwhile, the content of silica gel and the silica aperture size was also compared against adsorption effects, the results showed that the silica content reached equilibrium at 1.5g·L^-1 basically, and small aperture silica was beneficial to HAP adsorption, which has far-reaching significance to improving beer colloid stability.In this study, the BALB/c mice were immunized by purified protein, and the 3 monoclonal antibodies (Mcbs) were obtained successfully, named HP1, HP2 and HP3 respectively. Results of the identification for the Mcbs was that the HP1 is IgG2b subclass, the HP2 and HP3 was IgG2a subclass, HP1 and HP2 had different antigen epitopes, but HP2 and HP3 had the same antigen epitope. Specific results proved three Mcbs had better specificity reaction, and did not cross-react with LTP1, protein Z4 and Z7.This paper established DAS-ELISA method determining the haze protein in beer. After repeated screening, the optimum antigen coated buffer was pH9.6 carbonate buffer, the optimum antibody coated concentration was 5.5μg·mL^-1, blocking by 0.5% BSA with better effects, incubating antigen concentration was 4.04μg·mL^-1, HP2 was diluted 1:1000, the optimum concentration of HRP-goat antimouse IgG was 1:3000, the optimum reaction time of HP2 was 60min, and the Substrate was 20min.DAS-ELISA for detecting HAP in beer had high specificity, appearing no cross-reaction with the Z4, Z7 and LTP1, and the detection limits for HAP with DAS-ELISA was 2.52μg·mL^-1 (sensitivity 252ng), and the method has good repeatability and stability.