Jacques Levin

Fluorescence polarization immunoassay. Theory and experimental method

Abstract Fluorescence polarization measurements have been applied to diverse immunochemical systems for the purpose of directly following the reaction of fluorescent labeled haptens or antigens with antibody (Dandliker and de Saussure, Immunochemistry , 7 , 799 (1970)). Only rarely have systems been studied in which the hapten or antigen is present in a non-fluorescent as well as a fluorescent form, enabling a study of the inhibition of binding of the labeled form by the unlabeled form. The equations derived by us previously to describe the behavior of such systems (Kierszenbaum, Dandliker and Dandliker, Immunochemistry 6 , 125 (1969)) can be used to determined the association constant of the non-labeled form if its concentration is known or, conversely, to determine its concentration if the association constant is known. We have extended our previous equations so as to predict the form of standard curves for fluorescence polarization immunoassay, the region of optimal sensitivity and the sensitivity itself. Our results generalize previous theories of immunoassay in making specific allowance for an effect of labeling upon immunochemical reactivity, and in taking into account constraints imposed by instrumentation. Fluorescence polarization immunoassay is conceivably applicable to a wide variety of materials of biological interest both in the hapten as well as in the macromolecular size range. The sensitivity of the method depends strongly upon the binding affinity of the antibody. For the antibody preparations used, the sensitivity (polarization change per unit concentration change) for human chorionic gonadotropin was −0.0002 ml ng −1 while for the hapten penicilloyl-ϵ-aminocaproate it was −0.002 ml ng −1 .

[1] Equilibrium and kinetic inhibition assays based upon fluorescence polarization

Publisher Summary This chapter discusses the equilibrium and kinetic inhibition assays based upon fluorescence polarization. Assays, utilizing the high specificities of antibodies or other receptors make it possible to detect and quantify many substances present in only minute traces in complex biological materials. The polarization of fluorescence operates differently than the other methods of detection. In that, it gives a direct measure of the bound/free ratio instead of simply being a measure of amount of the label present. Fluorescence polarization experiments can be made, by observing, either the steady state, resulting from constant illumination of the sample or the transient state that follows a very short pulse of light. An analysis is made of the rates of the reactions that occur after preformed complexes are rapidly diluted so that the complexes dissociate to some extent and approach a new equilibrium state. The dilution jump assay affords a means of distinguishing between the specific and nonspecific interactions provided that the specific and nonspecific complexes dissociate with different rates. The assays, utilizing chaotropes to distinguish between the specific and nonspecific binding, are also discussed in the chapter.

CD-ROM based training: Interacting with the subject matter expert via the Web

We describe a project whose purpose is to bring together two technologies: CD-ROM technology, used widely to deliver training programs; and the Internet, used widely for communications. Bringing together these two technologies will make possible the ability to combine powerful training resources through CD-ROM with direct communications to the subject matter expert.