Production of IgE antibody to Japanese cedar pollen allergen Cry j1 by short term culture of human peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBL) from a patient allergic to Japanese cedar pollens, were stimulated with IL-4, IL-13, CD40-Ligand and/or hydrocortisone in the presence of Epstein-Barr virus in 96-well round bottomed culture plates, and the secretion of IgE-class antibody against a Japanese cedar pollen allergen Cry j1 in the supernatants were examined. PBL cultured with IL-4, and IL-4 + CD40-Ligand showed the highest IgE secretion and the cultures were maintained for 30 days. However, we failed to expand the culture with high IgE secretion. It was suggested that patient's PBL stimulated with IL-4 were useful for short term IgE production to Cry j1.

Tomato endo beta-mannanase: A candidate of potential tomato allergen protein detected with human monoclonal antibody established from a patient suffered from Japanese cedar pollinosis.

Peripheral blood lymphocytes from a patient allergic to Japanese cedar pollens were transformed by Epstein-Barr virus infection. Some transformed B-lymphoblastoid cells (BLCs) secreted IgM class antibodies to cedar pollen extracts and tomato fruit extracts. One stable human-mouse hybridoma clone Y-22-3-3 secreting IgM class monoclonal antibody to tomato fruit extracts was established by cell fusion of BLCs with mouse myeloma cells. Western blot analysis of tomato extracts showed Y-22-3-3 monoclonal antibody recognized a tomato protein with a molecular weight of 40 kDa. The CBB-stained 40 kDa protein from antibody-affinity chromatography was analyzed by MALDI-TOF/TOF, and identified as tomato endo-beta-mannanase, which was previously reported as one of the potential candidates for tomato allergens.

Epitope analysis of Japanese cedar pollen allergen Cry j2 with a human IgM class monoclonal antibody 404-117.

Japanese cedar pollen allergen Cry j2 is a causal allergen of seasonal pollinosis in Japan. To analyze B cell epitopes of Cry j2, we established two human-mouse hybridomas secreting IgM class human monoclonal antibodies to Cry j2. A pin-peptide enzyme-linked immunosorbent assay with synthesized icosa peptides showed that 404-117 monoclonal antibody bound to peptides #11-13 with cry j2 amino acid sequence of 101F-L140. Detailed analysis with octa peptides and alanine substituted peptides indicated that an amino acid sequence of 118FKVD121 was an essential for antibody binding. When K119 (Asn) was substituted with alanine, 404-117 monoclonal antibody did not bind to the alanine substituted peptide. We concluded that the 118FKVD121 sequence might have a very important role in early recognition by Cry j2-specific B cells, which could act as antigen presenting cells.

Generation of mouse-human hybridomas secreting human monoclonal antibodies to Japanese cedar pollen allergen Cry j1.

Peripheral blood lymphocytes from 13 donors were transformed with Epstein-Barr virus (EBV) to establish immortalized human B-cells secreting antibodies to Japanese cedar (Cryptomeria japonica) pollen allergens. EBV-transformed B-lymphocytes were then fused with mouse myeloma SP2/O3, and three clones of mouse-human hybridomas secreting human IgM class monoclonal antibodies to a cedar pollen allergen Cry j1 were established.

Epitope analysis of Japanese cedar pollen allergen Cry j1 with the human monoclonal antibody 4701-1.

We obtained a stable human-mouse hybridoma clone 4701-1 secreting IgM class human monoclonal antibody to Japanese cedar pollen allergen Cry j1. A pin-peptide enzyme-linked immunosorbent assay (ELISA) with synthesized pentadeca peptides showed a peptide with an amino acid sequence of LYTVT NSDDD PVNPA was found to be positive. Detailed analysis with deca to tetra peptides indicated that an amino acid sequence of TVTN was an essential sequence for antibody binding. When N (Asn) was substituted with A (Ala) of the TVTN epitope, the resulting peptide did not have antibody binding ability. We concluded that the TVTN sequence might have a very important role in early recognition of Cry j1 allergen by Cry j1-specific B cells, which act as antigen presenting cells.

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody 92-2.

A human-mouse hybridoma clone 92-2 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was established. To detect antibody-binding sequences on Ara h1, we synthesized a series of peptides of the Ara h1 protein on a multi-pin apparatus for the pin-peptide ELISA. The 92-2 human monoclonal antibody was found to recognize a sequence of GREGEQEWGTPGSHVREETS. Further analysis with shorter pin-peptides with eight amino acid-long showed that the sequence of QEWGTPGS was an essential linear sequence of this epitope. When the QEW part of the sequence was replaced by alanine, the 92-2 monoclonal antibody did not bind to the substituted peptide, showing that those amino acids play an important role in the binding of the 92-2 monoclonal antibody.

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody clone #86.

A human-mouse hybridoma clone #86 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was newly established. To detect an antibody-binding sequence (epitope) on Ara h1, the monoclonal antibody #86 was reacted with multi-pin apparatus with a series of peptides synthesized from the amino acid sequence of Ara h1. The antibody #86 was found to bind to a peptide with amino acid sequence of 481EEEEDEDEEEEGSNREVRRY500. Further analysis with shorter pin-peptides with ten amino acid-long showed that the peptides reacted with the antibody #86 contained a sequence of 485DEDEEEE491. This might be an essential linear sequence of this epitope. When the 485DED487 part of the peptide was replaced by alanine, decreased binding of antibody #86 was observed.

Epitope analysis of peanut allergen Ara h1 with oligoclonal IgM antibody from human B-lymphoblastoid cells.

To analyze epitopes of peanut allergen Ara h1, Epstein-Barr virus-transformed human peripheral oligoclonal B-cells were cultured to obtain antibodies to Ara h1. The combined reaction pattern with six oligoclonal antibodies showed there were six antibody binding areas named a to f in Ara h1. We found the novel antibody binding area named "area c" (171-230aa).

Generation of mouse-human hybridomas secreting antibodies against peanut allergen Ara h1.

Two clones of mouse-human hybridomas, secreting human monoclonal antibodies to a peanut allergen Ara h1, were generated from human peripheral blood lymphocytes transformed with Epstein--Barr virus, followed by cell fusion with mouse myeloma cells. Epitope analysis with overlapping peptides synthesized on a multi-pin apparatus revealed antibody-binding sequences of Ara h1 protein.

Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes.

Three novel missense mutations in the human lysosomal sialidase gene causing amino acid substitutions (P80L, W240R. and P316S) in the coding region were identified in two Japanese sialidosis patients. One patient with a severe, congenital form of type 2 sialidosis was a compound heterozygote for 239C-to-T (P80L) and 718T-to-C (W240R). The other patient with a mild juvenile-onset phenotype (type 1) was a homozygote for the base substitution of 946C-to-T (P316S). None of these mutant cDNA products showed enzymatic activity toward an artificial substrate when coexpressed in galactosialidosis fibroblastic cells together with protective protein/cathepsin A (PPCA). All mutants showed a reticular immunofluorescence distribution when coexpressed with the PPCA gene in COS-1 cells, suggesting that the gene products were retained in the endoplasmic reticulum/Golgi area or rapidly degraded in the lysosomes. Homology modeling of the structural changes introduced by the mutations predicted that the P80L and P316S transversions cause large conformational changes including the active site residues responsible for binding the sialic acid carboxylate group. The W240R substitution was deduced to influence the molecular surface structure of a limited region of the constructed models, which was also influenced by previously identified V217M and G243R transversions.