The Application Of Immunochromatographic Test Strip In The Detection Of Glycoprotein From Edible Bird’s Nest

Edible bird’s nest is formed by the mixture of saliva and velvet feathers of swiftlet bird which belongs to the Apodidae family.The edible bird’s nest is mainly produced in some coastal countries of Southeast Asian and China(including Fujian,Hainan and Guangdong).Edible bird’s nest has a wealth of nutritional and medicinal value.It has been knowing as nourishing treasures since ancient times.However,the adulterant of the edible bird’s nest was emerged and found on the markets,and resulted in huge economic losses and physical and mental harm to the customers.Currently,the edible bird’s nest detection analysis andauthenticity identification methods mainly include physical and chemical properties testing,instrumental analysis and enzyme-linked immunoassay,etc.However,most of the detection methods require sophisticated instruments,complicated procedures and long analysis time,the detection of the edible bird’s nest is limited to the well-equipped laboratories and places.It is necessary to develop a highly sensitive,fast and convenient detection assay for on-site analysis of edible bird’s nest.This thesis was aimed to extract and purify glycoprotein from edible bird’s nest to prepare polyclonal antibody which was capable of specifically recognizing glycoprotein.The immunochromatographic test strip based on sandwich detection assay was established using the glycoprotein antibody as the recognition element to achieve the qualitative and quantitative detection of glycoprotein in edible bird’s nest.The sandwich assay based immunochromatographic test strip could provide a theoretical basis for the authenticity of edible bird’s nest.The main contents and conclusions were summarized as follows:(1)The optimal extraction conditions of glycoprotein from edible bird’s nest were determined by the single factor experiment and the response surface analysis.The optimal conditions of glycoprotein extraction were as follows:2.6 hours of extraction time,1:47 of material to liquid ratio and extracting for 2 times.The extraction rate of protein was 3.96%,polysaccharide extraction rate was 1.54%.(2)The glycoprotein extracted from edible bird’s nest was directly isolated and purified by mono-Q column.SDS-PAGE and HPLC were used to determine the purity of glycoprotein as a single component with a molecular weight of about 75 kDa,and the purity was 90%.DSC results demonstrated that the thermal denaturation temperature of glycoprotein was 119.9℃,and the IR absorption spectrum indicated that the sugar chain of glycoprotein was the β-type glycosidic bond structure of pyranose.The compositional assay indicated that the glycoprotein was a characteristic component of edible bird’s nest-sialoglycoprotein.The purified glycoprotein was used as antigen to prepare the polyclonal antibody.The indirect ELISA method was carried out to determine the antiserum titer which was in accordance with the immune requirements.The polyclonal antibody was purified by affinity chromatography column with the concentration of 0.6 mg/mL.(3)The purified polyclonal antibody was used as an immunological recognition element to construct nanocomposite(GNP-SiNR)based immunochromatographic test strip for the detection of glycoprotein from edible bird’s nest.The optimization of the working conditions and the analytical performance of the immunochromatographic test strip were investigated and determined.The optimum pH for the conjugation of GNP-SiNR with the detection antibody was 9;the dispensing times of capture antibody on the test line was measured as 1 time;theconcentration of detection antibody in GNP-SiNR-Ab conjugate was optimized as 0.72 μg/mL;the optimum loading amount of the GNP-SiNR-Ab complex on the conjugate pad was 8μL;the best running buffer was determined as PBST with 0.25%of Tween-20.Under the optimum working conditions,the test strip showed a good linear relationship to the glycoprotein from edible bird’s nest in the concentration range of 10-1200 ng/mL,with the detection limit of 7.02 ng/mL.The immunochromatographic test strip showed good reproducibility and specificity without any non-specific target cross-reactions,and presented a good stability under room temperature for 1 month.