RESUMEN
This paper describes an original dot-enzyme-linked immunosorbent assay (ELISA) for predicting ovulation in women, based on the detection of the pre-ovulatory estrogen peak in urine. A monoclonal anti-estrogen antibody is used which recognizes not only free estrogens but also some of their urinary metabolites (17-glucuro- and sulfo-conjugates) allowing a direct assay on early morning urines. Antigen is immobilized as a spot on a nitrocellulose membrane which is immersed in urine in the presence of this antibody. A peroxidase-labeled second antibody allows the detection of the first antibody bound to the membrane. Antigen and anti-estrogen antibody concentrations are chosen to obtain a maximal enzymatic coloration of spots corresponding to basal urinary estrogen levels and no coloration corresponding to the pre-ovulatory surge. Six menstrual cycles were studied, comparing dot-ELISA results with patterns of: (1) urinary estrogens measured by RIA either directly or after hydrolysis and extraction, and (2) basal body temperatures. The validity of the pre-ovulatory signal obtained and the requirements for an adaptation of this methodology to a reliable home kit are discussed.
Asunto(s)
Detección de la Ovulación , Anticuerpos Monoclonales , Afinidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Estrógenos/inmunología , Estrógenos/orina , Femenino , Humanos , Hidrólisis , Radioinmunoensayo , Juego de Reactivos para DiagnósticoRESUMEN
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.
Asunto(s)
Inmunoglobulinas/inmunología , Testosterona/sangre , Testosterona/inmunología , Anticuerpos/inmunología , Unión Competitiva , Caseínas , Femenino , Humanos , Técnicas para Inmunoenzimas , Inmunoadsorbentes , Masculino , Microquímica/métodos , Radioinmunoensayo , Estándares de Referencia , Testosterona/análogos & derivadosRESUMEN
A competitive microtitre plate enzyme immunoassay for plasma aldosterone was developed using an immobilised aldosterone-bovine serum albumin conjugate and a monoclonal anti-aldosterone preparation, followed by the use of enzyme-labelled second antibodies to determine the degree of competition. The quantity of immobilised aldosterone-protein conjugate was adjusted to give optimum assay sensitivity with respect to the antibodies used. The lower limit of detection of aldosterone (55 fmol) was much less than that of an ELISA for aldosterone, using identical reagents but with an excess of immobilised aldosterone-protein conjugate, and up to 1400 fmol could be determined. Aldosterone levels in small amounts of male and female plasma could be assayed with good reproducibility.
Asunto(s)
Aldosterona/sangre , Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , RadioinmunoensayoRESUMEN
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.