[Preparation and application of a polyclonal antibody against the peptides of β₂glycoprotein 1]

To prepare a polyclonal antibody against human and murine β2 glycoprotein 1 (β2GP1) antigen with chemically synthesized β2GP1 peptides, and identify its specificity and pathogenicity.

The peptides from the NH₂-terminal 35th-51th amino acids of β₂GP1 were synthesized by standard Fmoc assay, and then used to immunize New Zealand white rabbits after coupling with keyhole limpet hemocyanin (KLH). The polyclonal antibody in the rabbit sera was purified by protein G column. The titer and specificity of the polyclonal antibody were determined by ELISA and Western blot analysis. The total RNA was extracted and the protein lysates were collected from C3H/HeN mouse peritoneal macrophages treated with the above anti-β₂GP1 peptides antibody/β₂GP1 complexes in vitro. And the tissue factor (TF) mRNA and protein expression in the peritoneal macrophages were detected by real-time quantitative PCR and Western blotting. The activation of p38 and NF-κB p65 induced by anti-β₂GP1 peptides antibody/β₂GP1 complexes was determined by Western blotting using phosphor-specific antibodies. Experimental antiphospholipid antibody syndrome (EAPS) mouse model was established in C3H/HeN mice by intraperitoneal injection of anti-β₂GP1 peptides antibody in vivo. The titers of anti-β₂GP1 antibodies in the mouse peripheral blood and the activated partial thromboplastin time (APTT) were detected.

The purity of chemically synthesized β₂GP1 peptides was 94%, which met the immunogen standard. The titer of antiserum of the rabbit immunized with β₂GP1 peptide coupling with KLH was over 1:32 000. Western blotting showed that the anti-β₂GP1 peptides antibody could specifically recognize both human and mouse β₂GP1. Furthermore, ELISA showed that the antibody could specifically bind to β₂GP1 cryptic epitope. In vitro experiments demonstrated that the anti-β₂GP1 peptides antibody/β₂GP1 co mplexes could enhance p38 and NF-κB p65 phosphorylation in mouse peritoneal macrophages and induce TF mRNA and protein expression. Moreover, EAPS mouse model induced by anti-β₂GP1 peptide antibody was successfully established, in which the titers of anti-β₂GP1 antibody in mouse peripheral blood were greater than 1:3200 and APTT was significantly shorter that that of control group.

The anti-β₂GP1 peptide antibody we prepared could specifically recognize both human and mouse β₂GP1 and specifically bind to β₂GP1 cryptic epitope. It was also proved to have the pathogenic effect.