Production of an antibody to arterial fatty acid-binding protein (FABP) using a synthetic peptide as the antigen

Abstract 1. 1. A procedure is described for the preparation of an antibody to arterial FABP using a synthetic peptide as an antigen. In order to locate a highly conserved region located on the outer surface of FABP, computer analysis of primary and secondary structures of several proteins from the FABP family was undertaken and a 24 amino acid sequence beginning at the fifth position from the N -terminus of rat heart FABP was chosen. 2. 2. The synthetic peptide consisted of eight replications of the 24 amino acid sequence individually attached to the α and ϵ amino groups of each terminal lysine on an octalysine branched peptide. 3. 3. Antibody to the synthetic antigen was raised in New Zealand rabbits. Western analysis was conducted and detection was accomplished by using goat-anti-rabbit second antibody conjugated to alkaline phosphatase. 4. 4. The antibody produced from the previously described peptide, recognized purified rat heart FABP and demonstrated a high positive correlation ( r = 0.96) when known concentrations of purified hFABP were plotted against densitometric measurement of the bands. 5. 5. Additionally, the antibody recognized FABP from the 104,000 g supernates of rat atrial and arterial tissue fractionated by a Sephadex G-75 column. 6. 6. Therefore, the antibody produced from this particular protocol employing a synthetic peptide can be utilized qualitatively and quantitatively in the analysis of the heart and arterial FABP content.