Epitope-Based Chicken-Derived Novel Anti-PAD2 Monoclonal Antibodies Inhibit Citrullination.

The aberrant upregulation of protein arginine deiminase 2- (PAD2-) catalyzed citrullination is reported in various autoimmune diseases (rheumatoid arthritis and multiple sclerosis) and several cancers. Currently, there are no anti-PAD2 monoclonal antibodies (mAbs) that can inhibit the citrullination reaction. Here, an epitope YLNRGDRWIQDEIEFGY was examined as an antigenic site of PAD2.

Generation of intracellular single-chain antibodies directed against polypeptide GalNAc-transferase using a yeast two-hybrid system.

Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary.

[Characterization of anti-mouse prion peptide single chain Fv antibody by phage display].

Antibodies can distinguish not only differences in amino acid sequences (primary structure), but also differences in three-dimensional structure and thus may be useful for detecting the conversion of prion proteins, especially in vivo. For diagnosis, we prepared chicken single chain variable fragment (scFv) antibodies that specifically recognized a prion protein using a phage display approach. As antigen, mouse prion protein (MoPrP) 138-153 containing YYR residues was conjugated with KLH.

Development of recombinant chicken IgY from single chain fragment of variable region for diagnosis of BSE.

We generated two recombinant chicken IgYs, designated Ab3-15 and Ab4-19, against mammalian prion protein (PrP) from the single chain fragment of variable region (scFv) antibodies. These two antibodies recognized PrP(Sc) from bovine spongiform encephalopathy (BSE) in cattle and were more sensitive than the corresponding scFv antibodies. These antibodies also recognized PrP(Sc) from other scrapie-infected mammals.

Stable production of recombinant chicken antibody in CHO-K1 cell line.

When compared with mammalian IgG, chicken IgY is advantageous in terms of cross-reactivity. In this study, two plasmids were constructed for expression of recombinant chicken IgY derived from a chicken hybridoma. The first was for expression of the light (L) chain, and the other was for the heavy (H) chain with a histidine (His) tag at the carboxy-terminal.

Inhibition of prion propagation in scrapie-infected mouse neuroblastoma cell lines using mouse monoclonal antibodies against prion protein.

We screened six mouse monoclonal antibodies (mAbs) against prion protein (PrP), which were previously established in our laboratory, for inhibitory activity against PrP(Sc)-accumulation in scrapie-infected cell lines and identified two mAbs, 3S9 and 2H9, as possessing this inhibitory activity. mAb 3S9 recognized an epitope including 154th tyrosine in the helix 1 region of PrP, while mAb 2H9 recognized a discontinuous region that included helix 1. In three scrapie-infected cell lines infected with different mouse-adapted scrapie strains, mAb 3S9 strongly inhibited accumulation of PrP(Sc), while mAb 2H9 moderately inhibited accumulation of PrP(Sc), indicating that inhibition of prion propagation by mAbs may be dependent on PrP(Sc) characteristics.

Biological activity of recombinant chicken interleukin-6 in chicken hybridoma cells.

Interleukin-6 (IL-6), a multipotential cytokine that plays roles in regulating immune responses, acute phase reactions and hematopoiesis, induces proliferation and antibody production in hybridoma cells. The biological activities of the recombinant chicken IL-6 (rchIL-6) were determined using murine and chicken hybridoma cells. Cell proliferation and tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT3) were induced by rchIL-6 in the IL-6-dependent murine hybridoma cell line MH60, whereas the recombinant protein exhibited no significant cell proliferation activity in chicken hybridoma cells but induced antibody production and tyrosine phosphorylation of STAT3.

Characterization and expression analysis of the chicken interleukin-11 receptor alpha chain.

Interleukin-11 (IL-11) is a multifunctional cytokine involved in various pathways in blood cells, their precursors and many other cell types in vitro and in vivo. The effects of IL-11 are largely mediated by the IL-11 receptor alpha-chain (IL-11Ralpha). In this study, a putative cDNA sequence encoding the 414 amino acid propeptide of chicken IL-11R (chIL-11R) was identified.

Generation of a mouse monoclonal antibody against chicken interleukin-6.

A monoclonal antibody (MAb) specific for chicken interleukin-6 (chIL-6) was generated by using Balb/c mice immunized with recombinant chIL-6 (rchIL-6). On Western blot analysis, the MAb, designated E3, reacted with rchIL-6 but not with recombinant murine IL-6 (rmIL-6). The MAb E3 also reacted with supernatant of the chicken macrophage-like cell line HD11 stimulated with lipopolysaccaride.

Excision of foreign gene product with cathepsin D in chicken hepatoma cell line.

To easily and rapidly recover exogenous gene products from chicken egg yolk, we constructed pVTG-catD (VTG, vitellogenin; catD, cathepsin D), a vector cassette carrying two catD-recognition signal peptides (catD-RSPs) in addition to the cloning site. An enhanced green fluorescence protein (EGFP)-encoding DNA fragment was ligated into the pVTG-catD. When the resultant construct pVTG-EGFP-catD containing histidine- and myc-tags was transfected into the chicken hepatoma cell line LMH, EGFP-expression at 24h post-cultivation was confirmed by fluorescence microscopy.

Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state.

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available.

Application of tyramide signal amplification for detection of N-glycolylneuraminic acid in human hepatocellular carcinoma.

Background: N-Acetylneuraminic acid and N-glycolylneuraminic acid (NeuGc) are the most common sialic acids in mammals, and NeuGc has attracted attention as a tumor-associated antigen.

Methods: In frozen liver sections from patients with hepatocellular carcinoma, glycolipid-type NeuGc was detected on the surface of liver cancer cells in 9 of 17 samples (52.9%) by immunostaining, using two chicken monoclonal antibodies against NeuGc and the tyramide signal amplification method.