Fatty acid-binding proteins as markers of brain injury

Fatty acid–binding proteins (FABPs) are abundant intracellular proteins that bind long-chain fatty acids with high affinity. 9 different FABPs, with tissuespecific distribution, have been identified so far. The primary role of all the FABP family members is regulation of fatty acid uptake and intracellular transport. Heart-type FABP (H-FABP) is considered one of the most sensible and efficient markers of heart and brain damage, but to date the cross-reactivity limits its specific use. Brain-type FABP (B-FABP) might be considered as a good marker of brain damage, being expressed only in the nervous tissue. The aim of this work was to generate a diagnostic reagent specific for human B-FABP, not cross-reacting with H-FABP. The work has been articulated in two parts: in the first one, recombinant protein B-FABP was expressed in E. coli cells and purified by two subsequent chromatographies. The protein was then utilized as immunogen in New Zealand rabbits and BalbC mice, obtaining two antisera and 8 different hybridoma cells. The antibodies in the rabbit serum and in culture supernatant of hybridoma cells displayed different degrees of affinity for B-FABP and H-FABP; only one hybridoma cell was able to produce antibodies specific and selective for brain-type FABP, but the antibody low-titer level and the activity decrease after subsequent subcloning steps affected its application on B-FABP detection in biological fluids. To overcome the limits encountered in the first part of work, a computer-assisted approach on B-FABP was employed in order to identify the potentially most antigenic regions of the protein. Two synthetic peptides were produced with the selected sequences and inoculated in rabbits. Two polyclonal antibodies were obtained, pAb 2979/2980 and pAb 2981/2982. They showed specific and selective reactivity for human B-FABP, without crossreactions with H-FABP. Taken together, our experimental approach was effective for the generation of specific α-B-FABP antibodies; these results suggest the antibodies obtained could be utilized in ELISA and immunoblot analyses in order to value B-FABP as marker of neurological disorders with ischemic, infective and degenerative etiology.