Novel Immunogenic Epitopes in the NaPi-IIb Protein: Production of Monospecific Antibodies Using Synthetic Peptides Outlined on Isoform Specific Regions of the Type IIb Sodium-Dependent Phosphate Transporter (NaPi-IIb)

Abstract

NaPi-IIb is a multiple passage protein membrane which is primarily responsible for phosphate uptake in the kidney and in the small intestine. Beyond its physiological functions, their involvement with carcinogenesis was initially described in mid-2003, due to its distinct level of expression in normal and tumor cells of the ovary. Although less common than cervical cancer, epithelial ovarian cancer is considered the most lethal gynecologic malignancy, which is mainly due to diagnosis in the advanced stages as a result of the absence of symptoms during the onset of the disease and the lack of tools for early detection. Here, we produce antibodies that are anti-synthetic peptides that are derived from the regions of second extracellular loop of NaPi-IIb, which is a non-overlapping portion of MX35 epitope. These two 15 distinct amino acid residue peptides, designated as Let#1 and Let#2, are engineered in a very thorough way to detect specific sites only in this isoform, thus excluding cross-reactivity with other carriers of the same family. The lack of immunogenicity of small peptides is surpassed by the conjugation with carrier proteins. Using immunochemical methods, we demonstrate that polyclonal antibodies that are mono-specific for the Let#1 and Let#2 peptides recognize proteins that express similar amino acid sequences during blood circulation. Additionally, using flow cytometry, we identify NaPi-IIb in NIH:OVCAR-3 cells. The clear identification of two shorter peptides on the extracellular loop of NaPi-IIb, which are far from the monoclonal antibody MX35-recognizing epitopes, adds new promising tools for ovarian cancer follow-up and staging.