Terasaki-ELISA for murine IgE. III. Determination of concentration and functional affinity by sequential equilibrium binding analysis.
A simple Terasaki tray-based ELISA technique with a fluorescent detecting system has been used to determine the affinity of murine IgE antibodies. The system was shown to be sensitive enough to measure affinities in the range of 10(-6)-10(-10) M as well as detect IgE antibodies down to a limit of 0.1 ng/ml. The results, expressed as arbitrary fluorescence units (AFU), were compared with those obtained using equilibrium dialysis for several DNP-specific IgE monoclonal antibodies of known affinities yielding KD values. The relationship between KAFU and KD established a conversion factor which could then be used to compute KD from KAFU, provided the detection system remained identical. Based on the equations proposed, an alternative method for the quantitation of murine IgE is described which is independent of the affinity of IgE for the coated antigen.
Pathak
S S
SS
Department of Immunology, Erasmus University, Rotterdam, The Netherlands.
Vos
Q
Q
Savelkoul
H F
HF
D000818:Animals; D000915:Antibody Affinity; D003956:Dialysis; D004797:Enzyme-Linked Immunosorbent Assay; D007073:Immunoglobulin E / Q000032:analysis*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA
D007073:Immunoglobulin E