[Preparation and preliminary identification of Fc-silent anti-human CD36 chimeric antibody]

Objective To prepare a Fc-silent chimeric antibody against human CD36 and analyse its bioactivity and phagocytic effect on CD36(+) platelets. Methods The genes encoding V_H and V_L fragments of the monoclonal antibody (mAb) 32-106 were obtained through RNA extraction from hybridoma cell lines, PCR amplification and sequence analysis. By using gene synthesis and recombination techniques, the vectors expressing light and heavy chains of the chimeric antibody against human CD36 were constructed. Stable cell lines secreting the chimeric antibody were established by selective culture using Zeocin and Blasticidin. Antibodies were purified by an affinity chromatography column. The purity and molecular weight of the chimeric antibody were detected by SDS-PAGE. The activity of the antibody binding to CD36 antigen was determined by ELISA and flow cytometry. The ability of the chimeric antibody to participate in CD36(+) platelet phagocytosis was analyzed by a platelet phagocytosis assay. Results The vectors expressing light and heavy chains of chimeric antibody were successfully constructed. After co-transfection into HEK293 cells, stable cell lines secreting chimeric antibody were obtained by screening and cloning. The high purity and correct molecular weight of the chimeric antibody were confirmed by protein silver staining. The results of Flow cytometry and ELISA showed that the chimeric antibody had the activity of binding to human CD36 antigen. Platelet phagocytosis assay showed that the Fc-silent chimeric antibody against human CD36 basically lost the ability of mediating monocyte phagocytosis of CD36(+) platelets. Conclusion In this study, the Fc-silent chimeric antibody against human CD36 was successfully prepared, and its loss of ability to mediate CD36(+) platelet clearance was confirmed in vitro, which provides a preliminary basis for the study of modified antibodies for the therapy of CD36-antibody-mediated fetal and neonatal alloimmune thrombocytopenia(FNAIT).