In vivo and in vitro optimization of depletion of IgM and IgG xenoantibodies by immunoadsorption using cell membrane proteins.
A system of immunoadsorption was developed for in vitro depletion of xenoreactive natural antibodies of classes IgG and IgM from monkey and human plasma. Porcine endothelial cell membrane proteins, platelet membrane proteins, and endothelial cells were used as affinity ligands, and cyanogen bromide-activated Sepharose 6 Fast Flow and Sepharose CL-4B gels were used for chromatography. Adsorption capacity was evaluated by means of ELISA, immunonephelometry, and cytotoxicity testing. Several consecutive adsorption-desorption cycles were performed. Different parameters influencing immunoadsorption were examined: ligand density on the column gel, adsorbent-plasma contact time, ratio of plasma volume to immunoadsorbent volume, desorption conditions, and temperature. After 2 adsorption-desorption cycles, 99% and 82 to 85% of IgG and IgM antipig antibodies were adsorbed, respectively. Furthermore, there was a 74 to 77% decrease in cytotoxicity. In vivo, we observed that after one adsorption-desorption cycle, 97% of antipig IgG antibodies and 96% of antipig IgM antibodies were adsorbed, and there was an 85% decrease in cytotoxicity. The immunoadsorption method studied and optimized in vitro and in vivo therefore efficiently depleted xenoantibodies and reduced the cytotoxicity. Thus, it can be used in xenotransplantation experiments without eliminating non-specific antibodies.
Lucchiari
N
N
Departamento Clínica Cirúrgica, Universidade Federal de Santa Catarina, Florianópolis-SC, Brazil.
Azimzadeh
A
A
Wolf
P
P
Regnault
V
V
Cinqualbre
J
J
D000327:Adsorption; D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003488:Cyanogen Bromide / Q000737:chemistry; D003601:Cytotoxicity Tests, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D007075:Immunoglobulin M / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D007163:Immunosorbent Techniques; D007164:Immunosorbents / Q000276:immunology / Q000378:metabolism*; D008253:Macaca mulatta; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry; D011869:Radioligand Assay; D012685:Sepharose / Q000378:metabolism; D013552:Swine; D014183:Transplantation, Heterologous / Q000276:immunology*
D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007164:Immunosorbents; D008565:Membrane Proteins; D012685:Sepharose; D003488:Cyanogen Bromide