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

• Published in: Journal of immunoassay & immunochemistry Vol. 29; no. 1; pp. 19 - 41
• Main Authors: Kajiura, Satoshi, Yashiki, Tetsuya, Funaoka, Hiroyuki, Ohkaru, Yasuhiko, Nishikura, Ken, Kanda, Tatsuo, Ajioka, Yoichi, Igarashi, Michihiro, Hatakeyama, Katsuyoshi, Fujii, Hiroshi
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis Group 01-01-2008
• Subjects: Animals; Antibodies - immunology; Antibodies - isolation & purification; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antibody Affinity; Blotting, Western; Characterization; Establishment; Fatty Acid-Binding Proteins - analysis; Fatty Acid-Binding Proteins - chemical synthesis; Fatty Acid-Binding Proteins - chemistry; Fatty Acid-Binding Proteins - immunology; Humans; Immunohistochemistry; Intestinal fatty acid-binding protein (I-FABP); Jejunum - chemistry; Jejunum - immunology; Mice; Monoclonal antibody; Peptide Fragments - chemical synthesis; Peptide Fragments - chemistry; Peptide Fragments - immunology; Peptides - chemistry; Peptides - immunology; Polyclonal antibody; Recombinant Proteins - immunology; Synthetic regional peptide
• ISSN: 1532-1819; 1532-4230
• DOI: 10.1080/15321810701735005

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.