ABSTRACT
Objective To reduce immunogenicity of porcine skin by removingα-Gal epitopes expressed in cell surface and extracellular matrix using recombinant α-galactosidase produced by Bacteroides fragilis.Methods The porcine skin was harvested from healthy 2-month-old pigs without any skin disorders before being sterilized by iodine and 75%alcohol, respectively.Enzymatic removal of α-Gal antigen was followed by washing with PBS.The α-Gal antigen in the prepared porcine skin was measured with immunofluorostaining of cryosections and the residual enzyme was measured with a double-antibody sandwich ELISA method.Enzymatic removal procedures were optimized by detecting residual enzyme and the effi-cacy ofα-Gal removal under different enzymatic and washing conditions.Results Efficient enzymatic and washing methods were established to removeα-Gal antigen.Theα-Gal removal efficacy was above 90% and residual enzyme was undetect-able (αprescribed minimum ofα-galactosidase detection with indirect ELISA was 1 ng/ml) .Conclusion It is feasible to efficiently removeα-Gal antigen under these enzymatic and washing conditions, and a method of producing low-immunoge-nicity pig skin dressing for burn is established.
ABSTRACT
Objective To establish a method of quantiying trace α-galactosidase from Bacteroides fragilis in enzymatic conversion of blood group B to O red blood cells ( B-ECO RBCs) .Methods BALB/c mice were immunized with purified recombinant B.fragilisα-galactosidase ( the purity>90%) to prepare monoclonal antibodies.The ascites were prepared using hybridoma cell lines stably secreting antibody and purified by HiTrap rProtein A column.The antibody titer and spe-cificity were detected by ELISA and Western blotting, respectively.Purified monoclonal antibody and rabbit polyclonal an-tibody were applied to detect residual enzyme in B-ECO RBCs and the washing solution was analyzed by indirect ELISA. Results A high titer and purity antibody was obtained.Western blotting showed that the antibody specifically reacted with B.fragilisα-galactosidase.Moreover, indirect ELISA was sensitive enough to detect the minimal amount of residualα-gal-actosidase at the concentration of 1 ng/ml.After four repeat washing cycles with 1∶4 ( v/v) phosphate-buffered saline, the amount of residual enzyme in B-ECO RBCs was less than 10 ng/ml.Conclusion An effective method of detecting the min-imal amount of residual α-galactosidase in blood conversion is established for safety evaluation of universal RBCs prepara-tion by enzymatic treatment.
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<p><b>OBJECTIVE</b>To explore the strategies which reduce the amount of xenoantigen Galalpha1,3Gal.</p><p><b>METHODS</b>Human alpha-galactosidase gene and alpha1,2-fucosyltransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV.15 and human alpha-galactosidase transgenic mice were produced. The Galalpha1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed.</p><p><b>RESULTS</b>Human alpha-galactosidase gene alone reduced 78% of Galalpha1,3Gal on PEDSV.15 cell surface while human alpha-galactosidase combined with alpha1,2-fucosyltransferase genes removed Galalpha1,3Gal completely. Decrease of Galalpha1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of alpha-galactosidase gene and alpha1,2-fucosyltransferase gene. RT-PCR indicated positive human alpha-galactosidase gene expression in all organs of positive human alpha-galactosidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galalpha1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice.</p><p><b>CONCLUSIONS</b>Human alpha-galactosidase gene and alpha1,2-fucosyltransferase gene effectively reduce the expression of Galalpha1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human alpha-galactosidase in mice can also eliminate the Galalpha1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.</p>
Subject(s)
Animals , Humans , Mice , Antigens, Heterophile , Metabolism , Cell Death , Cells, Cultured , Disaccharides , Metabolism , Endothelial Cells , Metabolism , Fucosyltransferases , Genetics , Metabolism , Graft Rejection , Genetics , Mice, Transgenic , Spleen , Cell Biology , Swine , Transfection , alpha-Galactosidase , Genetics , MetabolismABSTRACT
Objective: Soy products may cause excessive intestinal gas because of soybean oligosaccharides . The effect of recombinant ?-galactosidase on eliminating mouse flatus was observed. Methods:The mouse model of flatulence was set up by ig raffinose and stachyose and the flatulence was investigated by measuring intestinal flatulent volume. Oligosaccharides were examined by TLC test and soybean protein was examined by SDS-PAGE. Results: Raffinose and stachyose can result in mouse flatus and recombinant ?-galactosidase can eliminate it without any effect on soybean protein. Conclusion: Recombinant ?-galactosidase can eliminate flatus, and be used as food additive.
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Objectives:To establish hybridomas secreting monoclonal antibodies(McAbs) against O6-methylguanine-DNA methyltransferase(MGMT) and to observe the relationship between MGMT expression and clinical responses to ACNU and BCNU in human brain tumors.Methods:The hybridomas were established by cell fusion.MGMT expression in 60 glioma specimens was detected by means of immunohistochemical assay.Results: Seven hybridomas secreting McAbs against MGMT were obtained.Thirty tumor specimens had no detectable or low level of MGMT expression(Mer-), while 30 specimens had high level of MGMT expression(Mer+). The Mer- patients showed more sensitive to ACNU and BCNU than the Mer+ patients.Conclusions: The high specific hybridomas secreting monoclonal antibodies(McAbs) against MGMT were established.The preliminary study indicated that MGMT negative tumors were sensitive to ACNU and BCNU, whereas MGMT positive ones were more resistant to nitrosourea drugs.