Isolation and immunological characterization of fatty acid binding protein isoforms from Fasciola hepatica.
A combination of molecular sieving chromatography and 2-step preparative isoelectric focusing showed that native Fh12, a fatty acid-binding protein isolated from Fasciola hepatica adult worms, is a protein complex of at least 8 isoforms with identical molecular mass but different isoelectric points. Using enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA assays, immunological differences were observed between native (nFh12) and a recombinant molecule denoted rFh15 that was obtained after screening a cDNA library from F. hepatica adult worms with an anti-Fh12 monospecific polyclonal antibody. It was confirmed that in infected rabbits, antibodies to nFh12 appear by the second week postinfection, whereas antibodies to rFh15 appear much later, by 6 wk postinfection. Four acidic forms (Fh12(1-4)) showed more immunological identity with rFh15 than with nFh12, based on the observation that they inhibited ELISA activity by nearly 50% when they were added to the anti-rFh15 polyclonal antibody at 20 microg/ml of protein concentration. Moreover, the Fh12(1-4) isoforms were poorly reactive with sera from rabbits 2-4 wk postinfection. However, the 2 acidic forms, denoted Fh12(5) and Fh12(6), and the neutral/basic forms, denoted Fh12(7) and Fh12(8), showed more immunological identity with the native nFh12 molecule than with the recombinant rFh15 because they were highly reactive with sera of rabbits with early 2-wk F. hepatica infection and inhibited ELISA activity nearly 50% when they were quantitatively added to the anti-nFh12 polyclonal antibody. These results suggest that rFh15 could be one of the acidic forms of nFh12, and that it, in fact, may be one of the less immunogenic or immunoprotective members, or both, of the nFh12 protein complex.
Espino
A M
AM
Department of Pathology and Laboratory Medicine, University of Puerto Rico, School of Medicine, San Juan 00936, USA.
Rodríguez Medina
J R
JR
Hillyer
G V
GV
D000818:Animals; D000909:Antibodies, Helminth / Q000096:biosynthesis / Q000097:blood; D000947:Antigens, Helminth / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002417:Cattle; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005210:Fasciola hepatica / Q000737:chemistry / Q000276:immunology*; D050556:Fatty Acid-Binding Proteins; D015801:Helminth Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008970:Molecular Weight; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D015801:Helminth Proteins; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C074842:Fh15 protein, Fasciola hepatica