K.M. Rajkowski

The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.

An enzyme-linked immunoassay of testosterone.

An enzyme-linked immunoassay of testosterone is described. The conjugation of testosterone with high specific activity horseradish per-oxidase and a highly sensitive assay for this enzyme having previously been studied, here we describe the immobilization of the anti-testosterone antibody and the development of assay conditions permitting the determination of 50 pg to 1.5 ng testosterone in one working day. In this work attention has been paid to keeping the assay method as simple as possible. The method is discussed in terms of other enzyme-linked immunoassays for steroids.

A competitive microtitre plate enzyme immunoassay for plasma testosterone using polyclonal anti-testosterone immunoglobulins☆

An enzyme immunoassay for plasma testosterone was developed based on competition between an immobilised testosterone-casein conjugate and the analyte for polyclonal anti-testosterone immunoglobulins, followed by the use of enzyme-labelled second antibodies to determine the degree of competition. The quantity of immobilised testosterone-casein conjugate was optimised so that the lower affinity anti-testosterone antibody populations present did not affect the assay. The assay standard curve covered a range of 11-300 fmol/well. Testosterone levels in small amounts of male and female plasma could be assayed with good reproducibility and correlated well with results obtained by radioimmunoassay. By comparison with an analogous assay using monoclonal antibodies it appears that, given that the assay sensitivity is the most important criterion for choice, the use a polyclonal antiserum with this type of reactive antibody selection is preferable to the use of monoclonal antibodies.

Enzyme-linked immunosorbent assay (ELISA) for steroid hormones with polyclonal and monoclonal antibodies: an assay for urinary aldosterone

The development of non-competitive microtitre plate enzyme-linked immunosorbent assays for steroids was investigated using immobilised steroid as immunosorbent and enzyme-labelled second antibodies. An assay for testosterone with polyclonal anti-testosterone immunoglobulins was optimised with respect to a number of parameters but remained unsatisfactory for clinical assays. The results were applied to developing an aldosterone assay using monoclonal anti-aldosterone immunoglobulins. The latter method was used for the determination of urinary aldosterone and the results are compared with those obtained by a classical radioimmunoassay. Problems concerned with the use of sulphur-containing proteins and with the presence of low affinity antibodies in polyclonal preparations are discussed.

Investigation of human chorionic somatomammotropin--antihuman chorionic somatomammotropin antibody binding by two physicochemical methods: phase partition and fluorescence polarization.

Abstract The determination of binding parameters for interactions between proteins and nondialyzable ligands is studied by two physicochemical methods, both rigorously respecting the binding equilibrium: two-polymer aqueous phase partition and fluorescence polarization. These two methods are applied to an antigen/antibody system (human chorionic somatomammotropin/antihuman chorionic somatomammotropin immunoglobulins). A fluorescein-labeled antigen is used for both methods, permitting their comparison. The optimization of the phase partition system is described as is the mathematical treatment of the fluorescence measurements. The results obtained for the intrinsic apparent association constant ( k a ) and the number of sites ( n ) are k a = 0.75 × 10 7 m −1 , n = 0.16 by phase partition, and k a = 0.83 × 10 7 m −1 , n = 0.15 by fluorescence polarization. At low ligand concentrations (10 −8 m) fluorescence polarization measurements permitted detection of an antibody population of higher affinity ( k a = 1.2 × 10 8 m −1 , n = 0.05). Given that, at similar ligand concentrations, the two methods yield identical results while respecting the binding equilibrium, their relative practicability is discussed.

The interpretation of ligand displacement experiments: calculations for multisite acceptors

A method is described for calculating the degree of competition for binding between two ligands which are bound at any number of site classes on a binding protein from a generalization of the equilibrium competitive binding equations, the proteins binding parameters for each of the ligands, and the total protein and ligand concentrations. Theoretical displacement curves thus obtained for each of the possible competitive binding models with a multisite protein can then be compared with experimentally determined ligand displacements in order to find which model is most realistic or if measured displacements are due rather to negative cooperativity effects. The binding parameters used for the calculations have a statistical error attached to them, since they have been obtained experimentally, so here we also propose a method for calculating the standard deviations of the theoretical displacement curves deriving from these errors. This permits the use of statistical hypothesis testing in the comparison of theoretical and experimental results. An example is shown in which this method permits the verification that two drugs (phenylbutazone and azapropazone) are both bound by the same high- and low-affinity sites of a protein (alpha-fetoprotein).