Production of a monoclonal dinitrophenyl-specific rat IgE and establishment of an IgE capture ELISA for estimating the concentration of rat IgE antibodies to dinitrophenyl-Ascaris suum.

Abstract

A hybridoma producing monoclonal rat IgE antibodies of antidinitrophenyl (anti-DNP) specificity was generated by fusion of Sp2/0-Ag 14 (SP2) mouse myeloma cells and spleen cells from a DNP-Ascaris-sensitized Brown-Norway rat. Subsequently, the supernatant of the hybridoma (FE-3) was applied to an affinity column of DNP-bovine serum albumin-Sepharose 4B. The adsorbed protein fraction was pooled, concentrated, and further purified using Sephadex G-200. The molecular weight of the isolated protein was approximately 200,000 by SDS-PAGE, and the protein reacted with peroxidase (POD) mouse antirat myeloma IgE on Western blotting. Rabbit antibodies against DNP-specific rat IgE were also prepared by immunizing Japanese white rabbits with monoclonal DNP-specific rat IgE. These antibodies against DNP-specific rat IgE were applied to an affinity column of normal rat serum-Sepharose 4B and monoclonal DNP-specific rat IgG2b-Sepharose 4B to remove any other reactive substances apart from IgE contained in the serum proteins of the rat sensitized with DNP-Ascaris. On ELISA, it was found that the specificity of POD rabbit antibodies against DNP-specific rat IgE for monoclonal DNP-specific rat IgE was the same as that for rat myeloma IgE (IR 162). In addition, determinations of the monoclonal DNP-specific rat IgE revealed that the sensitivity of ELISA using POD-rabbit antibodies against DNP-specific rat IgE [POD-RA(DNP)RE] was higher than that using POD goat antibodies against rat myeloma IgE. Furthermore, an IgE capture ELISA employing the above-mentioned RA(DNP)RE was established for estimating the rat IgE antibodies to DNP-Ascaris suum. A good correlation was found between the antigen-specific IgE antibodies in the serum of Wistar rats estimated by this IgE capture ELISA and those estimated by passive cutaneous anaphylaxis.