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.

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.

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.