Production and immunological characterization of the novel single-chain variable fragment (scFv) antibodies against the epitopes on Opisthorchis viverrini cathepsin F (OvCatF).

BACKGROUND: Opisthorchis viverrini infection is a significant health problem in several countries, especially Southeast Asia. The infection causes acute gastro-hepatic symptoms and also long-term infection leading to carcinogenesis of an aggressive bile duct cancer (cholangiocarcinoma; CCA). Hence, the early diagnosis of O. viverrini infection could be the way out of this situation. Still, stool examination by microscopic-based methods, the current diagnostic procedure is restricted by low parasite egg numbers in the specimen and unprofessional laboratorians. The immunological procedure provides a better chance for diagnosis of the infection. Hence, this study aims to produce single-chain variable fragment (scFv) antibodies for use as a diagnostic tool for O. viverrini infection. METHODS: This study uses phage display technologies to develop the scFv antibodies against O. viverrini cathepsin F (OvCatF). The OvCatF-deduced amino acid sequence was analyzed and predicted for B-cell epitopes used for short peptide synthesis. The synthetic peptides were used to screen the phage library simultaneously with OvCatF recombinant protein (rOvCatF). The potentiated phages were collected, rescued, and reassembled in XL1-blue Escherichia coli (E. coli) as a propagative host. The positive clones of phagemids were isolated, and the single-chain variable (scFv) fragments were sequenced, computationally predicted, and molecular docked. The complete scFv fragments were digested from the phagemid, subcloned into the pOPE101 expression vector, and expressed in XL1-blue E. coli. Indirect ELISA and Western analysis were used to verify the detection efficiency. RESULTS: The scFv phages specific to OvCatF were successfully isolated, subcloned, and produced as a recombinant protein. The recombinant scFv antibodies were purified and refolded to make functional scFv. The evaluation of specific recognition of the particular epitopes and detection limit results by both computational and laboratory performances demonstrated that all three recombinant scFv antibodies against OvCatF could bind specifically to rOvCatF, and the lowest detection concentration in this study was only one hundred nanograms. CONCLUSION: Our produced scFv antibodies will be the potential candidates for developing a practical diagnostic procedure for O. viverrini infection in humans in the future.

Alteration of Extracellular Matrix Components in the Anterior Pituitary Gland of Neonatal Rats Induced by a Maternal Bisphenol A Diet during Pregnancy.

The extracellular matrix (ECM) plays crucial roles in the anterior pituitary gland via the mechanism of cell-ECM interaction. Since bisphenol A (BPA), a well-known endocrine disruptor, can cross through the placenta from mother to fetus and bind with estrogen receptors, cell populations in the neonatal anterior pituitary gland could be the target cells affected by this chemical. The present study treated maternal rats with 5000 µg/kg body weight of BPA daily throughout the pregnancy period and then investigated the changes in ECM-producing cells, i.e., pericytes and folliculostellate (FS) cells, including their ECM production in the neonatal anterior pituitary at Day 1. We found that pericytes and their collagen synthesis reduced, consistent with the increase in the number of FS cells that expressed several ECM regulators-matrix metalloproteinase (MMP) 9 and the tissue inhibitors of metalloproteinase (TIMP) family. The relative MMP9/TIMP1 ratio was extremely high, indicating that the control of ECM homeostasis was unbalanced. Moreover, transmission electron microscopy showed the unorganized cell cluster in the BPA-treated group. This study revealed that although the mother received BPA at the "no observed adverse effect" level, alterations in ECM-producing cells as well as collagen and the related ECM balancing genes occurred in the neonatal anterior pituitary gland.