Terasaki-ELISA for murine IgE antibodies. II. Quantitation of absolute concentration of antigen-specific and total IgE.
A Terasaki tray-based ELISA system was developed for the quantitative measurement of antigen-specific and total IgE antibodies in 5 microliter samples of mouse serum dilutions. The assay was based upon non-competitive binding of mouse IgE antibodies between the immobilized appropriate antigen or capture antibodies and the detecting rabbit antibodies. A conjugate of protein A-labelled beta-galactosidase and the fluorigenic substrate methylumbelliferyl-beta-D-galactoside were used as a detecting system. The resulted fluorescence could be measured rapidly and automatically using an inverted micro-fluorimeter. These measurements were automatically transformed into absolute concentrations by a microprocessor-based program using a four-parameter logistic function and an absolute IgE standard. The assay was shown to have a detection limit of 0.04 ng/ml and a range of linearity of 0.04-20 ng/ml, which is sufficient to measure IgE concentrations in mouse serum.
Savelkoul
H F
HF
Department of Cell Biology, Immunology and Genetics, Erasmus University, Rotterdam, The Netherlands.
Soeting
P W
PW
De Josselin De Jong
J E
JE
Pathak
S S
SS
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000032:analysis; D000918:Antibody Specificity; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007073:Immunoglobulin E / Q000032:analysis*; D007700:Kinetics; D051379:Mice; D001616:beta-Galactosidase / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007073:Immunoglobulin E; D001616:beta-Galactosidase