Development of FITC-conjugated monoclonal anti-human liver HSPGs, F(ab’)2 for the investigation of acute myeloid leukemia by directimmunofluorescent technique

Abstract

Human liver HSPGs was first isolated from human liver and monoclonal antibodies were produced. 1E4-1C2, one of many clones obtained was proved to specifically reacted with cell membrane molecules of acute myeloid leukemia (AML) by indirect immunofluorescence. Since the method is time consumed and required FITC-conjugated anti-mouse Igs which is expensive and caused nonspecific reaction. To solve these problems, we aimed to develop 1E4-1C2 to F(ab’)₂ format and conjugate with FITC in order to investigate AML by direct immunofluorescence technique. The results showed that incubating at 37 ^oC for 8 hrs and 1500 units/ mg protein was optimal for pepsin digestion. F(ab’)₂ obtained has molecular weight of 116 kD. FITC was covalently coupled to primary amines (lysine) of the immunoglobulin. The result showed that F:P ratio of this product was 5.8. Staining of HepG2 cell lines by direct compared to indirect immunofluorescence showed significant positive results. It was concluded that FITC-conjugated anti-human liver HSPGs, F(ab’)₂ obtained was effectively reacted with interested cells. Bull Chiang Mai Assoc Med Sci 2009; 42: 85-93.