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

Abstract

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.

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

“…When the allergens bind to IgE antibodies on the mast cells, the mast cells release chemical mediators such as histamine and other chemical mediators, which induce allergic symptoms. Contrary to this, the human monoclonal antibodies obtained in our previous study were all of the IgM class (Shinmoto et al 2004). This seems to be due to the fact that the population of cells transformed by the Epstein-Barr virus infection were B-cells expressing IgM antibodies on the cell surface (Crain et al 1989;Zhu et al 2008).…”

“…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.…”

“…Several peanut proteins have been identified as allergens by using sera from patients allergic to peanut, and Ara h1 and Ara h2 have been reported as major peanut allergens (de Jong et al 1998;Koppelman et al 2004;Burks et al 1991, Maleki et al 2000. We have established five clones of mouse-human hybridomas secreting IgM-class human monoclonal antibodies to peanut allergens from peripheral blood lymphocytes transformed with Epstein-Barr virus followed by cell fusion with mouse myeloma cells (Naganawa et al 2008;Shinmoto et al 2004). In this paper, we present the results of a linear B-cell epitope analysis with synthesized Ara h1 oligo peptides recognized by IgM-class human monoclonal antibody 92-2.…”

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

“…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 oligoclonal IgM antibody from human B-lymphoblastoid cells

“…At first, human peripheral B cells were transformed with EBV, and fused with mouse myeloma cell to form mouse-human hybridomas secreting two monoclonal antibodies to Ara h1. (Shimmoto et al 2004) However, making human monoclonal antibodies was very time consuming and we have failed to obtain additional monoclonal antibodies.…”