13 research outputs found
Molecular characterization of buckwheat major immunoglobulin E-reactive proteins in allergic patients
ABSTRACTBuckwheat extract was analyzed by immunoblotting experiments using sera from nine allergic and three non-allergic individuals. Major IgE-reactive bands were 73, 70, 62, 58 and 54kDa under non-reducing conditions and were detected in allergic subjects, but not in non-allergic ones. Under reducing conditions, the 73, 70, 62 and 58kDa bands split to 56 and 24, 52 and 24, 45 and 24, and 43 and 24kDa, respectively. The 24kDa molecule was the most prominent band recognized with IgE as well as IgG or IgA. The FA02 cDNA clone, encoding the α and β subunits of the legumin-like storage protein, was isolated from a cDNA library made of immature buckwheat seeds. The deduced amino acid sequence of the cDNA clone is substantially identical to the N-terminal amino acid sequence of the 24kDa molecule, which may be identical to that of BW24KD reported by Urisu et al. Consistent with these results, the translation product of the cDNA encoding the putative β subunit was strongly recognized with serum IgE, IgG and IgA from buckwheat-allergic patients. These results suggested that the 24kDa molecule may be the β subunit of the legumin-like storage molecule of buckwheat
Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine
An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv) clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg) but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC50 values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe
Human Anti–Human IL-18 Antibody Recognizing the IL-18–Binding Site 3 with IL-18 Signaling Blocking Activity
Immunogenicity of M13 phage vaccine displaying N-terminal region of amyloid beta peptide: comparison of M13 phage vaccine expressed as g3p fusion and g8p fusion
Direct evidence of generation and accumulation of β-sheet-rich prion protein in scrapie-infected neuroblastoma cells with human IgG1 antibody specific for β-form prion protein.
We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935–1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology
Biopanning of Antibody-Phage Clones Using Immunoplates Coated with Gel Slices of Electrophoresis: Immunogel-Biopanning
Elevated Gene Expression of Argininosuccinate Synthetase in Peripheral Lymphocytes from Systemic Lupus Erythematosus (SLE) Patients
Discovery and Characterization of a Peptide Motif That Specifically Recognizes a Non-native Conformation of Human IgG Induced by Acidic pH Conditions*S⃞
In therapeutic antibody preparation, acidic pH conditions are generally
used for elution from Protein A affinity column of IgG or for its viral
inactivation. Exposing IgG to low pH conditions induces conformational
changes, leading to its functional damage or loss, although the mechanisms
have not been fully elucidated. In this study using random peptide T7 phage
display libraries, we isolated a unique and novel peptide motif that
specifically recognized the non-native conformer (acid conformer) of human IgG
that was generated by the low pH treatment, but not the native conformer. We
examined the generation conditions and biochemical properties of acid
conformer using the peptide motif as an affinity ligand. The acid conformer
was easily generated at acidic pH (<pH 3.0) and at moderate temperatures
(20-40 °C). The conformer was present in a monomeric form functionally
maintaining antigen or Fc receptor binding, but showed a tendency to aggregate
with a long incubation time at neutral pH (>25 °C). The peptides
isolated here could contribute to the elucidation of the mechanisms of
antibody dysfunction or aggregation during acid exposure as well as storage of
human IgG