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Preparation of rabbit anti immuno globulin e for use in radio immunoassays of total immuno globulin e and specific immuno globulin e antibodies


Journal of Immunological Methods 20: 185-200
Preparation of rabbit anti immuno globulin e for use in radio immunoassays of total immuno globulin e and specific immuno globulin e antibodies
Detailed methodology for preparing and testing isolated rabbit anti-human Ig[immunoglobulin]E suitable for radioimmunoassays of total IgE and specific IgE antibodies was presented. A method for obtaining a product free of anti-idiotype antibodies was also described. IgE was isolated from E-myeloma serum (PS) by 40 and 50% saturated ammonium sulfate fractional precipitation, followed by DEAE Sephadex ion-exchange chromatography. The purified product was digested with papain, and the Fc fragments were separated from the Fab fragments by G-150 gel filtration and ion-exchange chromatography. Rabbits were immunized with IgE (Fc), and the antisera with the highest precipitin titers were pooled. A globulin fraction was prepared from the pooled antiserum by 50% saturated ammonium sulfate precipitation, and the fraction was absorbed using an immunosorbent prepared from whole human serum having a very low IgE level; this globulin fraction was also absorbed with immunosorbents prepared from D-myeloma protein and from IgE (Fab). Following absorption, the antibodies were demonstrated by micro-Ouchterlony technique to have no cross-reaction with IgG, IgA, IgM, IgD or any serum protein other than IgE. More than 200 mg of isolated anti-IgE (Fc) was prepared from antiserum globulin by consecutive affinity chromatography on columns of insolubilized IgE. Elution peaks appeared following the application of 0.1 M glycine-HCl, 1.0 M NaCl buffer at pH 2.5, as well as following a subsequent flush with 0.1 M phosphate, 1.0 M NaCl buffer at pH 7.4. The eluates were tested, pooled, radiolabeled with iodine-125 and were demonstrated to be effective in RAST [radioallergosorbent test] analyses. Antibodies to idiotypic determinants of the IgE molecule were eliminated by preparing the affinity chromatography column from a 2nd E-myeloma protein (HL) rather than from the E-myeloma protein (PS) used for immunization of the rabbits. Anti-IgE preparations which were free of anti-idiotype antibodies displayed less nonspecific binding to IgG and IgD coated discs than preparations containing such antibodies.


Accession: 006173758



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