progesterone-11-hemisuccinate--(11alpha)-isomer and isoluminol

The effect of varying the length of the alkyl bridge linking the chemiluminescent label isoluminol to progesterone on the light yield and binding affinity of progesterone chemiluminescent-marker conjugates was investigated. For this purpose five different derivatives of isoluminol, aminoethyl isoluminol (AEI), aminoethylethyl isoluminol (AEEI), aminobutyl isoluminol (ABI), aminobutyl-ethyl isoluminol (ABEI) and aminoethyl-ethyl isoluminol (AHEI) were covalently linked through a peptide bond to progesterone-11 alpha -hemisuccinate (P-11-HS). The resulting progesterone chemiluminescent-marker conjugates were then evaluated as potential labels for the development of an immunoassay based on monitoring chemiluminescence. These conjugates were able to compete with tritiated progesterone for the binding sites of an antibody raised against progesterone-11 alpha-HS bovine serum albumin, and they showed higher affinity than unaltered progesterone. These conjugates produced light upon oxidation by a hydrogen peroxide-microperoxidase system. The chemiluminescent reaction was optimized in terms of pH, concentration of oxidant, catalyst and conjugate design. Under optimal conditions, all the conjugates were detectable at femtomolar levels. The lowest detection limit was obtained using P-11-HS-ABEI (0.1 fmol). These results indicated that immunoassay techniques based on chemiluminescence can be developed with these labels.

Topics: Animals; Binding Sites, Antibody; Hydrogen-Ion Concentration; Hydroxyprogesterones; Immune Sera; Immunoassay; Luminescent Measurements; Luminol; Pyridazines; Rabbits

An immunoassay procedure for determination of progesterone in human plasma is described which utilizes chemiluminescence as the endpoint. The assay utilized progesterone-11-hemisuccinate-aminobutyl-ethyl-isoluminol (P-11-HS-ABEI) as the chemiluminescent marker conjugate and dextran coated charcoal for the separation of bound and free fractions of the ligand. An assay procedure for progesterone was established and validated in terms of sensitivity and precision, and assay results were compared with radioimmunoassay, using tritiated progesterone as the tracer. The two methods agreed well (n=35, r=0.96). The most important advantage of this assay is the elimination of problems inherent in the use of radioactive materials.

Topics: Azides; Humans; Hydroxyprogesterones; Immunoassay; Luminescent Measurements; Luminol; Pyridazines; Radioimmunoassay; Sodium Azide