Stability and immunological reactivity of recombinant membrane CD4 electroinserted into the plasma membrane of erythrocytes

[摘要]

Concentration-dependent electroinsertion of recombinant human membrane CD4 in human erythrocytes shows a saturation at an average of about 3,500 inserted CD4 epitopes per cell, detectable by flow cytometry. The erythrocyte recovery drops to 10% at this high level of electroinsertion. Experimentally an optimum for cell recovery and insertion rate was found at about 2,500 CD4 epitopes per red blood cell. In vitro stability assays by flow cytometry indicated a temperature- and medium-dependent decrease in the number of CD4 epitopes inserted per cell. This decrease is biphasic, with an exponential part during the first 24 h after electroinsertion followed by a much slower linear decay.

[学科分类] 生物化学/生物物理

[关键词] Electroinsertion;Recombinant CD4;Erythrocyte;Number of epitopes;Stability;Flow cytometry;RBC;red blood cell;rmCD4;recombinant membrane CD4;sCD4;soluble CD4;RBC-rmCD4;RBC bearing electroinserted rmCD4;QIFA;quantitative immunofluorescent assay;PBS 7.2;phosphate buffered saline at pH 7.2;-FITC;fluorescein isothiocyanate conjugated;QSC-beads;Quantum Simply Cellular micro-beads;PM;Adsol red cell preservation solution;PMhigh;low;high and low cell density in PM;FBS;fetal bovine serum;RPMI/FBS;RPMI 1640 medium with 5% heat-inactivated FBS;RAMF;FITC-conjugated rabbit antimouse antibody