| BackgroundImmunospot array assay on a chip (ISAAC) method, from TransChromo(TC) mice, 9 several to extract specific binding human TRAIL-R1 in human immunoglobulin G monoclonal antibody, and classification of these nine kinds of TRAIL-R1 monoclonal cloned antibodies, antibody screening in TRAIL-R1 corresponding to the epitope. Further, screening of the high sensitivity of TRAIL treatment of the tumor cell type and TRAIL treatment of tumor cells more effectively TRAIL-R1 epitope sites. TRAIL and TRAIL-R1 of the epitope conformation and sensitivity of the relationship, to help clarify the mechanism of TRAIL induced apoptosis, the application of TRAIL and TRAIL-R1 signal provides useful information targeted treatment of malignant tumors.MethodELISA competition Category TRAIL-R1 monoclonal antibody and the TRAIL-R1 may be epitope specific antibody. Using flow cytometry, in Colo205, K562, Daudi, KMP4, MCF7 and other 14 kinds of tumor cells to TRAIL-R1 classification and screening of monoclonal antibodies in the TRAIL-R1 on the possibility of antibody epitopes. Further analysis by cell viability, screening TRAIL-R1 antibody epitope (TR1-272-,438-and 419-epitope), and select the most for apoptosis TRAIL-R1 antibody epitope. Then, confocal microscopy analysis and verification, the selection of TRAIL-R1 TRAIL antibody epitope binding may occur after the changes. Further analysis, each cell line the expression of TRAIL-R1 cDNA sequences, detect various cell lines TRAIL-R1 differences expression if in various cell lines is due to TRAIL-R1 base sequence changes. With tunicamycin inhibition of TRAIL-R1 of N-glycosylation in all tumor cellsor benzyl-a-GalNAc inhibition of tumor cell synthesis of O-glycosylation, continue to use the various cell lines by flow cytometry analysis of inhibition of TRAIL-R1 after the glycosylation cells staining. We used Mann-Whitney's U-test P values estimated.ResultsELISA competition speculated 9 type TRAIL-R1 monoclonal antibody binding epitope of TRAIL-R1, from the TR1-272 antibody, TR1-419 antibody, TR1-404 and TR1-416 antibody, TR1-407,417-and 422-antibodies, TR1-412 and 438-among antibodies, completely blocked, partially blocked or hardly the characteristics of blocking analysis, suggesting that the recombinant TRAIL-R1 at least five kinds of antibody epitopes. We do these TRAIL-R1 epitope designated as TR1-272-,419-,416-, 438-or 417-epitope. TR1-272-epitope was almost blocked by the other antibody competition, in addition to TR1-438-antibodies. The TR1-438-epitope can not be TR1-272-blocking antibody competition, but can be TR1-416 and 417-part antibody blocking competition. TR1-416-epitope-based TR1-272-and 419-blocking antibody, but little has been TR1-417-and 438-antibody inhibition. TR1-417 epitope hardly was TR1-272-,416-,419-and 438-antibody inhibition. TR1-419 epitope was almost TR1-272 antibody blocked by TR1-417-and 416-antibody partially blocked, not blocked by TR1-438 antibody. (2) The Next Competitive ELISA analysis TRAIL-R1 monoclonal antibody binding sites and TRAIL of TRAIL-R1 in the relationship between binding sites. The results show that TRAIL blockade TR1-417-and 438-antibody and TRAIL-R1 epitope binding, but almost no blocking TR1-416-,419-and 272-antibody and TRAIL-R1 binding epitope. These results suggest that, TR1-417-and 438-epitope of TRAIL-binding sites located in or near, TR1-272-,416-and 419-epitope may be located outside of TRAIL binding sites. The results from the competition ELISA analysis showed that, TR1-272 and 438-epitope away from each other's. The antibody TR1-416-,417-and 438-epitope, less interference, TR1-417-and 272-epitope and TR1-416-,419-and 272-epitope is close to each other. (3) we used flow cytometry analysis of each antibody in different cell lines combination. Interestingly, some cells were TR1-404-,407-,416-,417-or 422-antibody staining, without being TR1-272-,419-,412-or 438-antibody staining. These results show that classification according to results of competitive ELISA antibody, consistent with cell-binding pattern classification, in each cell line cell surface TRAIL-R1 antibody epitope expression is different. TR1-416 and 417-epitope in all cell lines expression, the expression of TR1-416 and 417-epitope of the cell lines, not necessarily the expression of TR1-272,419-and 438-epitope. Interesting to note that, Colo205, K562, Daudi and Dul45 cells not express TR1-272 epitopes, and strong expression of other antibody epitopes. (4) Further, we detected different of TRAIL-R1 epitope (TR1-272-, 438-and 419-epitope) expression whether the TRAIL induced apoptosis in tumor cell lines of sensitive related. Among TR1-272-epitope absence Colo205, K562, Daudi and Dul45 tumor cells than TR1-272-epitope positive tumor cells more vulnerable to TRAIL induced apoptosis (p=0.0014). With this contrast, TR1-438-epitope conformation and TRAIL-induced apoptosis to not related (p=0.41). TR1-419 epitope conformation and TRAIL-induced apoptosis also showed no correlation. We observed a number of antibodies, including TR1-416 antibody and TR1-419 antibody, and TRAIL are not competitive binding TRAIL-R1. Here, we again examined TRAIL and TR1-416 antibodies conjunction induced tumor cell apoptosis. The results show that, TR1-416 antibody significantly enhanced the TRAIL to induce Colo205, K562 and Daudi such as tumor cells of apoptosis. Overall, these results show that TR1-272 antibodies can predict the TRAIL in clinical, treatment of tumors with TRAIL sensitivity. TR1-416 antibody can enhance TRAIL induced apoptosis in some tumor cells. (5) In order to understand the different sensitivity adjustment mechanism by TRAIL induced cell death, after treated with TRAIL of TR1-272-epitope-negative or-positive cell lines, using laser scanning confocal microscopy analysis of cell surface TRAIL-R1 situation. Confocal laser microscopy showed that, TRAIL after the role of TR1-272-epitope-negative cells, TRAIL-R1 form a large spotlight, while TR1-272-positive cells can not form a bunch. Show, TRAIL-R1 oligomerization affect and TR1-272-epitope conformation, may affect the tumor cells to TRAIL induced apoptosis of sensitivity. (6) Further analysis of the various cell lines express TRAIL-R1 cDNA sequences found,in various cell lines of TRAIL-R1 is basically the same amino acid sequence, it shows the expression of various cell epitope differences did not occur at the gene level. (7) Next to continue analysis the translation of TRAIL-R1 after the glycosylation is modified. In each cell, with tunicamycin inhibited TRAIL-R1 in the N-glycosylation or Benzyl-a-GalNAc inhibition of synthesis of O-glycosylation, continue to flow cytometry analysis, whether the various cell lines TR1-272,419-or 438-epitope are to be protein post-translational after modification of glycosylations occurrence a negative or positive staining of expression. The results showed that, O-glycosylation or N-glycosylation of inhibition failed to change the negative expression rate. Glycosylation was observed for TRAIL-R1 epitope on the conformation had no effect, indicating glycosylation of conformational epitopes may not play key roles.ConclusionTR1-272-antibody may recognize TRAIL-R1 conformation, which there conformational can release a powerful cell death signal.With this contrast, TR1-438-, TR1-419-, TR1-416 and TR1-417-epitope conformation and not related to TRAIL-induced apoptosis. TR1-416 antibody can significantly enhance the TRAIL to induce Colo205, K562 and Daudi such as tumor cells apoptosis. Various cell lines TRAIL-R1 epitope of express differences did not occur at the gene level, while TRAIL-R1 post-translational modification of glycosylations may be the conformational epitope not afford a key role. Various cell lines TRAIL-R1 epitope of express differences did not occur at the gene level, while TRAIL-R1 post-translational modification of glycosylations may be the conformational epitope not the key role. Further analysis of TRAIL epitopes conformational and sensitivity, can help clarify the mechanism of TRAIL induced apoptosis, and TRAIL and TRAIL-R for the signal to provide useful information to treat cancer.
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