Measurement of antigen-specific mouse IgE by a fluorometric reverse (IgE-capture) ELISA.
A reverse, or IgE-capture, enzyme-linked immunosorbent assay (ELISA) for measuring ovalbumin-specific IgE antibody in the serum of immunized mice has been developed. Microplate wells were first coated with a commercial anti-mouse IgE rat monoclonal antibody, and then incubated with two-fold serial dilutions of test sera with 10% normal mouse serum as diluent for the capturing of only IgE class molecules. Biotinylated ovalbumin and then beta-D-galactosidase-conjugated streptavidin were added and, finally, 4-methylumbelliferyl-beta-D-galactoside was used as the enzyme substrate. The fluorescence intensity of the reaction product (4-methylumbelliferone) was determined on a microplate fluorescence reader. The sensitivity of this assay was equal to that of passive cutaneous anaphylaxis (PCA). In contrast to indirect ELISAs this IgE-capture assay is free from competition by non-IgE antibodies. Furthermore, it requires much less antigen than the PCA assay.
Sakaguchi
M
M
Department of Microbiology, Institute of Public Health, Tokyo, Japan.
Inouye
S
S
Miyazawa
H
H
Tamura
S
S
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006358:Hot Temperature; D007073:Immunoglobulin E / Q000032:analysis*; D051379:Mice; D010047:Ovalbumin / Q000276:immunology; D010323:Passive Cutaneous Anaphylaxis
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007073:Immunoglobulin E; D010047:Ovalbumin