Establishment and characterization of monoclonal and polyclonal antibodies against human intestinal fatty acid-binding protein (I-FABP) using synthetic regional peptides and recombinant I-FABP.
We have succeeded in raising highly specific anti-human intestinal fatty acid-binding protein (I-FABP) monoclonal antibodies by immunizing animals with three synthetic regional peptides, i.e., the amino terminal (RP-1: N-acetylated 1-19-cysteine), middle portion (RP-2: cysteinyl-91-107) and carboxylic terminal (RP-3: cysteinyl-121-131) regions of human I-FABP, and the whole I-FABP molecule as antigens. We also raised a polyclonal antibody by immunizing with a recombinant (r) I-FABP. To ascertain the specificity of these antibodies for human I-FABP, the immunological reactivity of each was examined by a binding assay using rI-FABP, partially purified native I-FABP and related proteins such as liver-type (L)-FABP, heart-type (H)-FABP, as well as the regional peptides as reactants, and by Western blot analysis. In addition, the expression and distribution of I-FABP in the human gastrointestinal tract were investigated by an immunohistochemical technique using a carboxylic terminal region-specific monoclonal antibody, 8F9, and a polyclonal antibody, DN-R2. Our results indicated that both the monoclonal and polyclonal antibodies established in this study were highly specific for I-FABP, but not for L-FABP and H-FABP. Especially, the monoclonal antibodies raised against the regional peptides, showed regional specificity for the I-FABP molecule. Immunoreactivity of I-FABP was demonstrated in the mucosal epithelium of the jejunum and ileum by immunohistochemical staining, and the immunoreactivity was based on the presence of the whole I-FABP molecule but not the presence of any precursors or degradation products containing a carboxylic terminal fragment. It is concluded that some of these monoclonal and polyclonal antibodies, such as 8F9, 4205, and DN-R2, will be suitable for use in research on the immunochemistry and clinical chemistry of I-FABP because those antibodies can recognize both types of native and denatured I-FABP. In order to detect I-FABP in blood samples, it is essential to use this type of antibody, reactive to native type of I-FABP. It is anticipated that, in the near future, such a method for measuring I-FABP will be developed as a useful tool for diagnosing intestinal ischemia by using some of these antibodies.
Kajiura
Satoshi
S
Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan. satoshi-kajiura@ds-pharma.co.jp
Yashiki
Tetsuya
T
Funaoka
Hiroyuki
H
Ohkaru
Yasuhiko
Y
Nishikura
Ken
K
Kanda
Tatsuo
T
Ajioka
Yoichi
Y
Igarashi
Michihiro
M
Hatakeyama
Katsuyoshi
K
Fujii
Hiroshi
H
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000915:Antibody Affinity; D015153:Blotting, Western; D050556:Fatty Acid-Binding Proteins / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D007583:Jejunum / Q000737:chemistry / Q000276:immunology; D051379:Mice; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D050556:Fatty Acid-Binding Proteins; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; C527661:intestinal fatty acid-binding protein (1-19), human; C527663:intestinal fatty acid-binding protein (121-131), human; C527662:intestinal fatty acid-binding protein (91-107), human