Determination Of Royal Jelly Freshness And Detection Of Adulterated Honey By ELISA With A Highly Specific Anti-apalbumin 1, Major Royal Jelly Protein 1 Antibody

Royal jelly (RJ) is secreted by the nutritional glands (including back of the head glands, glands and mandibular gland swallow, etc.) of worker bee head. The milky substance is rich in biologically active ingredients, and has a variety of physiological activities. Major royal jelly protein 1 (MRJP1), the most abundant protein component, is a key functional component of RJ. Honey is a kind of natural sweet substances brewed with honeydew of plants collect by honeybees, and bound with the secretions from honeybees. MRJP1 exists also in honey. This study established a detection method of MRJP1 in RJ and honey by double-antibody sandwich ELISA. The main results are as follows:1. Establishment of a sandwich ELISA detecting MRJP1 contentA specific polyclone antibody of MRJP1 (SP-2) was prepared with an announced chemically synthesized polypeptide, and labeled with horseradish peroxides (HRP-). A double-antibody sandwich ELISA was established with another polyclone antibody SP-1 (established previously) as capture antibody and HRP-SP-2 as detecting antibody. The best conditions for each step were:coating SP-1 at 37℃ for 2 h, blocking with 5% skim milk overnight at 4℃, coating antigen overnight at 4℃, coating HRP-SP-2 at 37℃ for 2 h.2. Comparison of indirect ELISA and the double-antibody sandwich ELISAThe content of MRJP1 was measured by indirect ELISA method and sandwich ELISA method, and the detecting range, repeatability, accuracy of the two methods were then compared. The results were as follows:the detecting range of each methods were 1-5.5μg/mL and 2.5-10μg/mL; the recovery rate were 77.96%-178.97% and 92.74%-100.72%; coefficient of variation were 2.22%-19.39% and 2.75%-12.4%. The results showed a higher accuracy and repeatability of sandwich ELISA method and a faster detection of indirect ELISA method.3. Determination of freshness of royal jelly by sandwich ELISAA sandwich ELISA to detect the freshness of RJ was established. The fresh samples were stored 0,7,14,21,28 and 35 days under 40℃, respectively. The degradation rate of MRJP1 of the 6 RJ samples was detected. Then the determined results with sandwich ELISA, indirect ELISA and SDS-PAGE analysis were compared and validated, respectively. It was proved that MRJP1 degrade during storage, and degradation rates of RJ was positively associeated with the storage days.4. Detection of the MRJP1 content in honey with SDS-PAGEThe MRJP1 content in honey was mesured by SDS-PAGE and the gray scale values were analyzed by ImageJ program. Using the purified MRJP1 as the standard or protein marker, MRJP1 content in honey was estimated. Corn syrup was mixed with acacia honey in proportion. SDS-PAGE electrophoresis analysis of showed that the gray scale values were consisted with the theoretical value of the MRJP1 content, which showed this method to identify authenticity of honey known nectar sources is feasible. Analysis resuls of 10 commercial honey samples showed that 5 sampls are real honey,2 samples are fake and 3 samples might be fake.5. Detection of MRJP1 content in honey with ELISAThe contents of MRJP1 in 6 honey samples were detected with both indirect ELISA and sandwich ELISA. In order to verify the impact of color, the decolorized honey sample were also detected. The results showed that light-colore honey should not, dark-colored honey should be decolorized for indirect ELISA. However, sandwich ELISA was lightly infected by honey color and showed a higher accuracy.6. Authenticity identification of honey with ELISACorn syrup was added proportionly to acacia honey, and the contents of MRJP1 in honey were then detected with sandwich ELISA. The results showed that the MRJP1 content in honry was negatively associated with the proportion of corn syrup in honey, which indicated the ELISA to identify adulterated honey is feasible. The analysis of MRJP1 contents on 10 commercial honey samples showed the determined result form SDS-PAGE is similar to that from sandwich ELISA.