Nicole Cittanova

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.

A new approach for quantitative evaluation of cross-reactivity of steroids with an antiserum by radioimmunoassay: Application to a highly specific antiestriol

Abstract The study of several antiestriol antisera in the presence of a series of analogues of estriol has led to a critical discussion of the classical cross-reaction test. A more accurate test (CR 1 ng) which permits a more precise determination of specificity of antisera is described.

Immunoassay of the human chorionic gonadotrophin using fluorescence polarization.

Classical immunoassay methods are based on the displacement of a labelled antigen by the antigen to be measured from sites on an antibody and necessitate the separation of the free and bound antigen fractions. The fluorescence polarization method detects variations in the hydrodynamic volumes of fluorescent molecules; the larger the molecule, the greater the increase in the degree of polarized light emitted. This permits interactions between a fluorescent antigen and an antibody to be detected directly, without recourse to a separation step. In the presence of a specific antiserum the antigen, labelled with a fluorochrome, is bound, resulting in an increase in the degree of fluorescence polarization. Upon addition of unlabelled antigen, bound labelled antigen will be displaced from the antibody and the fluorescence polarization will decrease accordingly. This article describes the application of such a method to the measurement of human chorionic gonadotrophin.

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.

Determination of progesterone and of free and conjugated estrogens in pregnant and pseudo-pregnant rats ☆

Abstract Rat progesterone and estrogen levels have been determined in peripheral, ovarian, foetal plasma and in amniotic fluid, during estrous cycle, pseudo-pregnancy and pregnancy. From the progesterone levels, it was concluded that the predominant source of this hormone is the ovary. Though an ovarian contribution could not be excluded, placental origin of estrogens during pregnancy appeared evident. The estrogen levels showed the preponderance of the free form in a ratio E 2 /E 1

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.

Adaptation of fluorescence polarization immunoassay to the assay of macromolecules.

This paper describes an original methodology for determining macromolecular antigen levels by polarization of fluorescence. it involves the use of fluorescent derivatives of Fab fragments of a monoclonal antibody (Mr 50,000), whose fluorescence polarization rises significantly when it combines with a macromolecular antigen. An experimental system (Fab anti-aldosterone and aldosterone--bovine serum albumin (BSA)) is studied to test this methodology, which was then used to develop an immunoassay for human immunoglobulin M (IgM), using anti-mu chain Fabs. In the two assays, the binding stoichiometry of Fab/antigen was 10/1 and 8/1 for aldosterone--BSA and IgM, respectively. The lower limit of detection of the IgM assay was 0.8 microgram/ml and thus it was applicable to clinical detection of IgM concentrations.

Murine monoclonal antibody against aldosterone: production, characterization and use for enzymoimmunoassay

Hybridomas secreting monoclonal antibodies to aldosterone were obtained by fusion of myeloma cells and spleen cells from Balb/c mice immunized with aldosterone-3-carboxylmethyloxime-bovine serum albumin. A monoclonal antibody was purified from ascites fluid and characterized. An affinity constant of 1.61 x 10(9) M-1 has been measured and no cross-reactivity with tetrahydroaldosterone (THA), cortisol, cortisone, corticosterone, deoxycorticosterone (DOC), dehydroepiandrosterone (DHA), progesterone and estrone, could be detected. A peroxidase conjugated-antibody (1.5 mole of enzyme per mole of antibody) was obtained and used for microwell enzyme immunoassay and Immun-Blot assay. The high affinity and specificity of this antibody should make the direct determination of aldosterone in biological fluids possible at concentrations as low as 5 x 10(-10) M.

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.