| Exosomes are membranous vesicles with particle size ranging from 30 to 150 nm secreted by living cells into the extracellular environment.Exosomes are important mediators of intercellular information transmission and play an important role in the process of tumor genesis,development and metastasis.Exosomes are widely distributed in blood,saliva,urine and other body fluids,and are an important part of tumor"liquid biopsy",which have become one of the most promising tumor markers.The tetraspanins are specifically expressed on the surface of exosome bilayer membranes,which can be used as markers for their sorting and identification.The number of exosomes secreted by tumor cells is closely related to the occurrence and development of tumors.The precise quantification of exosomes derived from tumor cells can establish a basis for exploring their correlation with cancer progression.Traditional exosome quantification techniques are complicated in operation and expensive in equipment,which limit their clinical application.Based on the magnetic quantitative immunochromatography technology of protein molecules established in the previous stage,this project intends to complete the recognition and capture of exosomes through tetrameric transmembrane proteins specifically expressed on the surface of exosomes,thereby establishing a magnetic quantitative detection method for exosomes.In order to provide a new possibility of technological expansion for exosome research.The specific experimental design is as follows:Carboxyl magnetic beads were labeled with one of the tetrameric transmembrane protein antibody specifically expressed on the surface of the exosome membrane,and the labeled magnetic beads were co-incubated with the exosome samples.Then,the composite product was loaded on the chromatographic test strip.The"carboxyl magnetic bead-exosome"complex will be captured by another exosome membrane surface-specific tetrameric transmembrane protein antibody embedded on the chromatographic test strip,so that it will accumulate at the test line to form color band.The quantification of exosomes is accomplished by detecting the output intensity of the magnetic signal contributed by the carboxyl magnetic beads at the band.In this study,we first carried out laboratory verification on isolation and identification of cell,plasma and urine exosomes,and studied in detail the optimal conditions for short-term storage of exosomes.Secondly,the exosome magnetic quantitative immunochromatography method was comprehensively and systematically constructed using standard HEK 293 cell-derived exosomes.Which included the pairing study of antibodies,preparation and screening of immune probes,optimization of pre-incubation system,optimization of the conditions of the chromatography system and determination of quantitative relationship.Finally,we detected exosome content in the culture medium of cell lines representing different grades of bladder cancer by the constructed method,and compared the results with commercial ELISA kits.The results show that the successfully constructed exosome magnetic quantitative immunochromatography system can present a good quantitative relationship in the range of(10-160)x10~6exosomes/μL exosomes,with the lowest detection limit of 2.1x10~6exosomes/μL,and the detection results were comparable with ELISA results.Although we do not have the opportunity to apply the constructed detection system in the blood and urine of real bladder cancer patients due to the difficulty in obtaining cancer samples,the work of this project will provide an important basis for the subsequent detection of tumor-derived specific exosomes and revealing the correlation between the number of exosomes and bladder cancer. |
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