Quantitative evaluation of indirect ELISA. Effect of calmodulin antagonists on antibody binding to calmodulin.
A simple linearization procedure has been developed to determine the apparent dissociation constant of the interaction between antigen and antibody from the data of indirect, non-competitive enzyme-linked immunosorbent assays (ELISA). Applying this dissociation constant the binding constant of ligands to antigen can be determined and the quantitative evaluation of the competitive ELISA experiments makes it possible to analyse the affinity of antibody to antigen on the surface and in solution. The binding of the monospecific anti-calmodulin antibody to calmodulin and to solid-phase bound calmodulin has been tested by non-competitive and competitive assays. We have developed an experimental system where binding of the antibody to the solid-phase bound calmodulin has been studied under equilibrium conditions. Competitive ELISA experiments showed that the affinity of antibody to calmodulin on the surface and in solution was almost the same. The binding constant of a hypothalamic neuropeptide to calmodulin was determined using the quantitative ELISA approach. The neuropeptide was found to be of very high inhibitory potency (Kd = 2 nM) and competed with the antibody for calmodulin binding. This simple and sensitive procedure is suitable for screening molecules with anti-calmodulin activity and comparing their efficacy.
Liliom
K
K
Biological Research Center, Hungarian Academy of Science H-1518, Budapest.
Orosz
F
F
Horváth
L
L
Ovádi
J
J
D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D008968:Molecular Conformation; D013997:Time Factors
D002147:Calmodulin