Preparation and application of fgl2 prothrombinase peptide antibody
ObjectiveTo prepare a peptide polyclonal antibody against both murine and human fgl2 prothrombinase/fibroleukin based on the chemically synthesized peptide 3 (Pep3) and peptide 4 (Pep4) from exon 1 and 2 of the predicted amino acid sequence of mfgl2 protein separately. MethodsThe Pep3 and Pep4 were coupled to keyhole limpet hemocyanin (KLH) to form a complex of Pep3-KLH and Pep4-KLH. The anti sera were developed by immunizing rabbit with Pep3-KLH or Pep4-KLH after emulsified in Freund′s complete adjuvant (FCA). The purified anti sera were also utilized to detect the hfgl2 expression in the liver tissue of hepatitis B patients. ResultsThe anti sera react with Pep3 and Pep4,and the titer around 1∶320 000 (Pep3) and 1∶160 000 (Pep4) respectively. Western blot displayed a specifically M_r (molecular mass relatively) 65 000 reactive band in murine hepatitis virus strain 3 infected macrophages from BALB/cJ mice when incubated with anti sera. PCA assay showed that the anti sera inhibited functional activity of mfgl2 prothrombinase expressed on the MHV-3 infected BALB/cJ macrophages in a dose-dependent fashion. The purified anti sera also recognized hfgl2 molecule highly expressed in liver tissue from patients with active acute on chronic hepatitis B. ConclusionPeptide polyclonal antibody against fgl2 prothrombinase was successfully raised with expected specificity,titers and neutralization activity. This study provided an important tool to explore the roles of this newly discovered molecule from human disease and animal model.