Exploratory Development Of The New Type Fluorescent Labeling Reagents SPA-EGFP Fusion Protein

The fluorescence immunoassay(FIA)with fluorescent markers as probe founded in the early 1940s,is a historic marker immunoassay.The principle,using the fluorescent materials to labeling monoclonal antibodies,positioning detect the antigenic properties in organization and intracellular,combines the specificity and sensitivity of antigen-antibody reaction with the accuracy of microscopic tracer.The FIA specificity,rapid and accurate at the cellular level of positioning,with various kinds of new fluorescent material found and improved fluorescence labeling method, FIA has been widely used and increasingly played an important role in immunology, microbiology,pathology,and clinical oncology testing,and in so many ways.On the basis of the preliminary studies in our laboratory,The constructed GS115/pPIC9K /spa-egfp engineering bacteria was induced and expressed in the medium BMMY;we add methanol from 24 hours to 12 hours a supplementary added time and do not add histidine and 1%Casamino acid,and the results show no apparent effect on the expression.After supernatant purified by the HisTrap HP column,the outflow was purified once again,the total yield is up to 63.3%,the average yield increased 23.2%.SPA-EGFP fusion protein purity is up to 95%, consistent with the request of fluorescence immunoassay.In this study,purified SPA-EGFP fusion protein as a fluorescent labeling reagents,by indirect fluorescence immunoassay(IFIA),hepatitis B surface antibody (HBsAb)as the test object,research detection conditions of the SPA-EGFP fusion protein.The results showed that pH 7.2,100 mM PBS buffer(plus 0.1%between 20), as washing liquid,the effect of washing is better;it is more appropriate to use washing liquid.The results showed that the antigen-antibody complex by the SPA-EGFP fusion protein marked in the inverted fluorescence microscope excited by the Blu-ray,showing the bright green fluorescence spots,and the background was more uniform bleak black,identified at a high level.Prolonged observation,the fluorescence intensity is essentially invariably,no obvious quenching phenomenon.We used the detection condition to detect the hepatitis B surface antibody(HBsAb) of 283 medical conditions of serum by IFIA and synchronous detected by ELISA.The results showed that the positive rate for the FIA is 72.08%,73.85%for the ELISA, with higher consistency the two detection methods,their test results consistency is up to 98.23%.So using the definite detection condition,detection of HBsAb by SPA-EGFP-FIA is practicable and with good effect.This study used SPA-EGFP fusion protein to substitute the FITC labeled goat anti-human IgG antibodies in EUROIMMUN anti-nuclear antibody detection kit, detected the patients serum diagnosed with systemic lupus erythematosus(SLE) throughâ…¡FA.Studies show that all the 25 samples showed positive serum, fluorescence pattern is the same SPA-EGFP fusion protein and FITC,and, SPA-EGFP fusion protein is staining background light,fluorescence stability,and low non-specific staining;the identified PBS eluant is to achieve the best results detection requirements.SPA-EGFP fusion protein as a new type of fluorescent labeling reagents,its application in fluorescence immunoassay will become wider and wider.With further research,it perhaps can be applied in immunology array and flow cytometry chip technology.