Application Of The Immunomagnetic Beads To Capture Exosomes In Serum And The Construction Of Double Antibody Sandwich System In Exosomes

Objective1.Coupling of antibodies associated with exosome membrane surface proteins by application of carboxyl magnetic beads to extract exosomes in serum,Establishing a method for extracting exosomes using immunomagnetic beads.2After the exosomes were captured by immunomagnetic beads prepared,magnetic beads were used as solid-phase carriers,and the exosomes were similar as antigens.A double-antibody sandwich system was constructed to explore whether the method was suitable for preparation of immunomagnetic beads.Methods1.Preparation of Immunomagnetic Beads: Selection of the optimal antibody volume and the immunomagnetic bead preparation process,using enzyme-linked immunosorbent assay detect the efficiency of prepared immunomagnetic beads of carboxyl one-step and carboxyl two-step assays and antibody`s volume of 10 ul,15 ul and 20 ul,The experimental data were repeated three times and the average number was taken.The results were described with (?).The experimental blank control groups was the beads that all subjected to the same experimental operation without adding antibody.Monoclonal mouse anti-mouse CD-63 was used in this experiment.2.Immunomagnetic beads method was used to extract exosomes,and 20 patients with serum from Henan Provincial People's Hospital were screened.Exosomes in serum of patients were extracted with prepared immunomagnetic beads.3.The morphology of exosomes was observed under transmission electron microscope and Western blot was performed on them.Identification of the marker proteins,verifying the success of the extraction;By detecting the change of serum concentration before and after extraction of exosomes.4 The detection of immunomagnetic capture efficiency:By detecting the difference in the concentration of sample protein before and after capture of exosomes in 10 groupsof samples,the capture efficiency was determined.The response ratios of the immunomagnetic beads and serum samples corresponding to group I,II,III were: 1:1,1:2,2:1;the experimental data were described with average,and analyzed by SPSS13.0,the data processing result is statistically significant at P<0.05.5.Evaluate the effect of Encapsulation: By observing the experimental background value,the effect of the prepared sealing solution was evaluated.After the preparation of the immunomagnetic beads,the prepared sealing solution was added and blocked overnight at 4 ° C.The blank control was the magnetic beads stored in the PBS buffer after the preparation was completed.Diluted monoclonal rabbit anti-TSG-101 and HRP-goat anti-rabbit were added sequentially,and their OD values were measured and the test data were described by (?).6.Non-exosomal sample screening: Screening of exosomes after treatment by ultrasonic disruption of exosomes and ultracentrifugation and exclusion of exosomes after two non-exosome treatment methods were performed,and the exosomal markers were identified by enzyme-linked immunosorbent assay,the OD value was measured.The normal sample quality control group and the blank group were set in the experiment.The data was used for(?) description.The experimental design was in accordance with the t-test.The data was analyzed statistically by SPSS 13.0,and the data was P<0.05.Meaning.96-well plate exosomal double-antibody sandwich assays have been validated.Screening was performed in 96-well plates,and the plate was coated with exosome proteins corresponding to the marker protein(monoclonal mouse anti-CD-63)for exosomal capture and extraction.Western blot analysis of exosomal marker proteins verifies whether exosome capture can be performed.7.The double-antibody sandwich method was applied to exosomes.After enrichment of exosomes by immunomagnetic beads method,magnetic beads were used as a solid-phase carrier.An antibody corresponding to another protein on the exosomal membrane surface was added and enzyme-linked immunosorbent assay was used To detects this antibody bound to the surface of the exosomes.The experimental group was a normal exosome extraction group,the two control group one was a blank group,the other was ultrasonically exfoliated exosomes.The experimental data were described with (?) and analyzed by SPSS13.0,The data processing result is statistically significant at P<0.05.Results 1.The prepared immunomagnetic beads were detected by enzyme-linked immunosorbent assay.The preparation process was compared with one step carboxylmethod (?)=174.932,two carboxyl steps (?)=106.032,and the required antibody concentration was 8ul (?)=91.325,15 ul (?)=179.488,and 20 ul (?)=179.702.2.The exosomes were extracted by immunomagnetic beads.The morphology of the exosomes was observed under transmission electron microscope.The diameter of the exosome was about 50-150 nm,which met the requirements of exosomes.The expression of corresponding proteins CD-63 and TSG101 was detected by Western blot.The extracted substance is exosomes and the extraction method is feasible.3.The three sets of data are:18.3%,37.8%,38.0%,Comparison between three sets of data,P=0.502>0.05;?and ?,P=0.00<0.05,?and ?,P=0.00<0.05,?and?,P=0.753>0.05.4.Encapsulation solution data: (?)= 4.588,PBS buffer group data: (?)= 32.630.5.Western blot detection of exosome marker proteins CD-63 and TSG101;normal control group data: 196.37 ± 25.52,ultracentrifuge exclusion exclusion group data:114.83 ± 18.90,ultrasonic fragmentation group data: 117.01 ± 21.40,blank group :(?)= 9.28,between two non-exosome treatment methods: P = 0.44> 0.05.6.The experimental data 190.00 ± 18.81,No exosome group 141.51 ± 22.80,P=0.00<0.05.Conclusion1.The immunomagnetic beads prepared in this experiment can be used for the capture and extraction of exosomes.2The double-antibody sandwich system constructed by using the immunomagnetic beads prepared in the experiment can be used to detect the exosome surface protein,The double antibody sandwich system is suitable for the immunomagnetic beads prepared in this experiment.