Terasaki-ELISA for murine IgE-antibodies. I. Quality of the detecting antibody: production and specificity testing of antisera specific for IgE.
In order to develop an ELISA for the quantitative determination of murine IgE, antisera specific for murine IgE were prepared in the goat and rabbit. As immunogen, monoclonal IgE antibody mixtures of several allotypically different hybridomas were used. Before use, these antibodies were purified employing procedures that allow maximum recovery of binding activity. The goat and rabbit mouse epsilon chain-specific antisera were adsorbed on normal mouse serum. The purified antisera were found to be free of allotypic activity. However, immunoadsorption on NMS could not always remove contaminating anti-idiotypic antibodies. Repeated adsorptions with monoclonal antibodies of different isotypes carrying a similar idiotype were necessary to remove all detectable anti-idiotypic activity. Only after these precautions were the antisera suitable for detecting IgE molecules on nitrocellulose blots as well as for quantitating circulating IgE antibodies (ELISA) and IgE-secreting cells (plaque assay and reverse ELISA plaque assay). The purity and reactivity of several commercially available anti-IgE preparations were tested in similar types of specificity assays. Since the specificity of antibodies used in ELISA determines the monospecificity of the assay, retesting for contaminating cross-reactivities in commercial preparations was shown to be necessary.
Savelkoul
H F
HF
Department of Cell Biology, Immunology and Genetics, Erasmus University, Rotterdam, The Netherlands.
Soeting
P W
PW
Radl
J
J
Van der Linde-Pressman
A A
AA
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology* / Q000302:isolation & purification; D000915:Antibody Affinity; D000918:Antibody Specificity; D001667:Binding, Competitive; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006462:Hemolytic Plaque Technique; D007073:Immunoglobulin E / Q000032:analysis*; D051379:Mice
D000888:Antibodies, Anti-Idiotypic; D007073:Immunoglobulin E