| Glucoamylase can convert starch into low molecular number sugars and has been used widely in the sugar industry. Generally there are the residues of glucoamylase in many plant sugar products.The contents of glucose and fructose in honey are about65%which leads to mix surgar into the honey easily. For the past few years, quality and safety issues of the Chinese honey constantly have been emerged. In2002, European Union and some countries took the trade barriers of banning inlet and stopping sale for the Chinese honey, which brought a great loss to the Chinese beekeeping. Therefore, it has great practical importance to establish a method that can quickly check out external glucoamylase mixed into the honey for detection of mixing surgar into the bee productions.There are several methods to detect the plant sugars in honey, including stable carbon isotope ratio analysis (SIRA), fourier transform infrared spectroscopy and high performance liquid chromatography method. SIRA analysis on the identification of honey mixed with C4plant sugars have a good effect, but the incorporation of C3plants as raw materials starch sugar can not be identified, which bring some difficulties to detect honey mixed with plant sugars; Fourier transform infrared spectroscopy method is convenient, fast, accurate and simple, however, the equipment is expensive and the requirements is complicated; High performance liquid chromatography is sensitive and accurate, but the equipment is expensive and pre-treatment is complicated. Compared with the three methods introduced before, the immunological methods, especially the enzyme-linked immunosorbent assay(ELISA) methods have been Large-scale extension, because of its advantages of sensitive, speed, and special simple and easy and practical. This paper aims to study a fast and accurate enzyme-linked immunological methods to detective the residues of glucoamylase in honey.In this study, using the glucoamylase immunized new Zealand rabbits to obtain the polyclonal antibodies(PcAbs), western-blot analysis porved that the PcAbs had good immunoeraetivity against GA. Glucoamylase was used to immunize BALB/c mouse, after screening,12hybridoma cells in all which could stability excrete antibodies against GA by hybridoma technology and monoclonal antibody technology. Western-blot analysis showed that the12McAbs specifically recognized Glucoamylase. Hybridoma2H4F9,6H9D8,8F2F11,8F2E9,1A8G6and1C4D5excreting anti-Glucoamylase McAbs were used to inject into mice(ICR) abdominal cavity. The titers of the McAbs were all above1:1×104.Six strains of monoclonal antibodies(McAbs) were confirmed to be specific for four different kinds epitopes on glucoamylase competitive binding assay. Of them, McAb-6H9D8and McAb-8F2F11might direct to the same antigenic determinant on the GA, McAb-1A8G6and McAb-1C4D5to another antigenic site, McAb-8F2E9to the third antigenic site, McAb-2H4F9to the fourth antigenic site. To obtain enzyme-marked special monoclonal antibody-6H9D8which marked by horseradish peroxidase (HRP) for a double-antibody sandwich ELISA method.Establish the method of double-antibody sandwich ELISA by using polyclonal antibody or monoclonal antibody. The results showed that the test method based on monoclonal antibody has more specificity and higher sensitivity. In the test, we established the double-antibody sandwich ELISA method by using special monoclonal antibody and enzyme-marked special monoclonal antibody together.The best concentration of antibody (McAb-2H4F9) was12mg/L, the best dilution of GA solution was1:100and the optimal dilution of antibody labeled by Horseradish peroxidase was1:1000. The results showed that double-antibody sandwich ELISA method had good specificity, the ascites had no cross-reactivity with a-amlase (alpha-G) and beta-fructofuranosidase ((3-F). The method was initially applied to the detection of honey samples. The intervention disappeared when the sample was diluted8times. In the range from5to80ng/mL, the standard curve equation was Y=-0.4555x+1.2409, R2=0.9914. |
PDF Full Text Request