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

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

“…In the previous results of pin-peptide ELISA with overlapping icosa peptides, a human monoclonal antibody to Cry j1 bound three regions (AA51-80 (AA51-70 and AA61-80), AA171-299 (AA171-190 and AA 181-200), and AA251-280 (AA251-270 and AA261-280)) [4]. Those results might include non-specific binding of antibodies used.…”

“…A human-mouse hybridoma 2-103H6-26 [4] secreting human IgM monoclonal antibody to the cedar pollen allergen Cry j1 was cloned twice by a limiting dilution method, and a hybridoma clone 4701-1 was established. The hybridoma cells were cultured with ERDF medium (Kyokuto Seiyaku, Co. Ltd., Japan) supplemented with 10% fetal calf serum (FCS), (Nichirei Biosciences Inc., Japan).…”

“…There are several allergens in cedar pollen; the major one is Cry j1, which is located in the Ubisch body of the pollen particles [3]. In a previous study [4], we established three human-mouse hybridomas secreting IgM class anti-Cry j1 human monoclonal antibodies from patients' peripheral blood lymphocytes using infection with Epstein-Barr virus [5][6][7] followed by cell fusion with mouse myeloma cells [8,9]. In the present study, we chose one hybridoma clone secreting monoclonal antibody that recognizes an epitope on the primary structure of Cry j1 in order to analyze in detail the amino acid sequence of the epitope.…”

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

“…Preparation of a human-mouse hybridoma secreting anti-Ara h1 human monoclonal antibody Peripheral blood lymphocytes (PBLs) prepared from peripheral blood from a healthy donor were transformed by Epstein-Barr virus infection as described in a previous report (Naganawa et al 2005;Shinmoto et al 1992;Shinmoto et al 1991). The resulting human B-lymphoblastoid cells (BLCs) secreting IgM-class antibody to peanut allergen Ara h1 were detected by Enzyme-Linked Immunosorbent Assay (ELISA).…”

“…Then, the fusion mixture was diluted with 10 ml of ERDF medium and centrifuged again. The fused cells were suspended in 30 ml of ERDF medium supplemented with 15% FCS and the cells were cultured in two 96-well microculture plates for 24 h. The fused hybridoma cells were selected with O-HAT medium (ERDF medium supplemented with 10% FCS, 0.1 mmol/L hypoxanthine, 0.0003 mmol/L aminopterin, 0.016 mmol/L thymidine, and 0.002 mmol/ouabain) by replacing half of the culture medium three times a week (Shinmoto et al 2004;Naganawa et al 2005;Shinmoto et al 1991). Antibody-producing hybridomas were analyzed by ELISA.…”