The periurethral glands do not significantly influence the serum prostate specific antigen concentration.

PURPOSE: The periurethral glands are known to produce prostate specific antigen (PSA). With ultra-sensitive assays now routinely available, it is necessary to determine if the periurethral glands significantly influence serum PSA concentration after radical prostatectomy. MATERIALS AND METHODS: Serum PSA levels of 46 men, 51 to 89 years old (median age 67) who underwent radical cystoprostatectomy and total urethrectomy, were compared with those of 92 men 46 to 91 years old (median age 67) who underwent radical cystoprostatectomy only. All men had transitional cell carcinoma of the bladder without gross or microscopic evidence of prostate cancer and all underwent ileal conduit diversion. Serum was obtained at least 1 year postoperatively. Each specimen was analyzed using the Tosoh, Immulite, and Yu and Diamandis ultra-sensitive PSA assays with analytical detection limits of 0.02 ng./ml., 0.004 ng./ ml. and 0.002 ng./ml., respectively. RESULTS: Median PSA for the radical cystoprostatectomy with urethrectomy group was 0.00 ng./ml. (range 0.00 to 0.14) for each of the 3 assays. For the radical cystoprostatectomy only group the median Tosoh and Immulite PSA assay levels were 0.01 ng./ml. (range 0.00 to 0.22), and median Yu and Diamandis PSA assay level was 0.00 ng./ml. (range 0.00 to 0.31). CONCLUSIONS: The greatest difference in median PSA levels that could be found between men with and without periurethral glands when using 3 different ultra-sensitive assays was 0.01 ng./ml., indicating that the periurethral glands do not have a clinically significant effect on serum PSA concentration after radical prostatectomy. Thus, a serum PSA level above the residual cancer detection limit following radical prostatectomy, even if obtained with a ultra-sensitive assay, reflects either malignant or benign residual prostatic tissue, rather than the presence of periurethral glands.

Detection of immunoglobulins G and M to rubella virus by time-resolved immunofluorometry.

We describe new methods for the detection of immunoglobulin G (IgG) and IgM rubella-specific antibodies in serum. The IgG assay was based on a solid-phase rubella antigen immobilization approach, and the IgM assay was based on the IgM capture assay principle. Both assays used biotinylated antibodies (anti-human IgG and antirubella monoclonal antibody, respectively). The tracer system was based on streptavidin labeled with a fluorescent europium chelate. The final measurements were done by using time-resolved fluorescence. Both assays were thoroughly evaluated with clinical samples and compared successfully with established techniques. We anticipate that these assays are suitable for routine clinical use.

Sensitive time-resolved immunofluorometric assay of thyrotropin in serum.

We have developed a time-resolved solid-phase immunofluorometric assay for thyrotropin (TSH). The assay is performed in white opaque microtitration wells which are coated with a monoclonal capture antibody. Serum TSH binds simultaneously to the solid phase and to a biotinylated monoclonal detection antibody. The degree of biotinylated antibody binding is quantitated with streptavidin conjugated to thyroglobulin which is heavily labelled with the Eu3+ chelator 4,7-bis [chlorosulfophenyl] -1,10-phenanthroline -2,9-dicarboxylic acid (BCPDA). The final fluorescent complex is measured on the solid phase with time-resolved fluorometry. The assay requires two incubation steps and can be completed in 5 hours. The detection limit is 0.03 milli-int. units/L. The present assay was compared with two immunoradiometric assays and gave satisfactory results.

Multiple fluorescence labeling with europium chelators. Application to time-resolved fluoroimmunoassays.

Multiple fluorescence labeling with conventional probes like fluorescein, to improve the detection limit of labeled reactants, is not usually successful because of fluorescence quenching. In contrast, we found that the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) can be incorporated into proteins at very high molar ratios. Working with thyroglobulin as a model protein, we found that when 160 BCPDA molecules are incorporated into one thyroglobulin molecule, the fluorescence emitted by the labeled protein in the presence of excess Eu3+, is equivalent to that emitted by approximately 900 molecules of unconjugated BCPDA:Eu3+ complexes. We took advantage of the lack of any quenching effects and of the enhancement observed with the multiply labeled protein, to develop a universal reagent system consisting of (a) streptavidin covalently coupled to BCPDA labeled thyroglobulin and (b) excess Eu3+. With this approach, streptavidin is heavily labeled through thyroglobulin and retains its full biotin binding activity. We used the reagent to develop a highly sensitive time-resolved heterogeneous immunofluorometric assay of alpha-fetoprotein (AFP) in serum, using monoclonal antibodies. One antibody is immobilized in white microtitration wells (solid-phase) and the other is biotinylated. We demonstrate that this assay, using the newly developed reagent, is 25-fold more sensitive than the one using directly BCPDA labeled antibody and 5-fold more sensitive than an assay that uses BCPDA-labeled streptavidin. The detection limit of the assay with the new reagent was down to 60 amol of AFP per well. We conclude that multiple fluorescence labeling with europium chelators is an effective method of extending the sensitivity of currently used fluorescence immunoassay procedures.

Time-resolved fluoroimmunoassay of cortisol in serum with a europium chelate as label.

A non-isotopic heterogeneous competitive immunoassay of serum cortisol is described. Cortisol present in the sample competes with immobilised cortisol (cortisol-thyroglobulin conjugate) for binding to a monoclonal anti-cortisol biotinylated antibody. The amount of antibody bound is measured on the dry solid-phase by time-resolved fluorometry after adding streptavidin labeled with the Eu3+ chelate 4,7-bis(chlorosulfophenyl)-1,10 phenanthroline-2,9-dicarboxylic acid (BCPDA), in the presence of excess Eu3+. The assay is simple to perform, its characteristics are similar to those of radioimmunoassay techniques, and is suitable for routine clinical use.

Effects of quinine on Ca++-induced K+ efflux from human red blood cells.

The Ca++-mediated increase in K+-permeability of intact red blood cells (Gardos effect) was initiated by exposing cells to know concentrations of Ca++ (using EGTA buffers) in the presence of the ionophore A23187. The potency of quinine, an inhibitor of the response, was found to depend on the external K+ concentration. In K+-free solutions the concentration of quinine to achieve 50% inhibition (K50) was 5 microM, but at 5 mM K+ the required concentration was increased 20-fold to 100 microM. An increase in internal Na+ had the opposite effect, allowing a high potency of quinine despite the presence of external K+. Alterations in the internal K+ level, on the other hand, were without effect on the K50, suggesting that the membrane potential is not a factor. This conclusion is supported by the lack of effect on quinine inhibition of substitution of Cl- by NO3-, a considerably more permeant anion. The data are consistent with the hypothesis that quinine inhibits by competitively displacing K+ from an external binding site, the reported K+-activation site for the Ca++-mediated K+-permeability.

Decreased iodination of the red cell surface following phospholipase C treatment.

Human red blood cells were treated with phospholipase C from Clostridium welchii. Lipase concentrations which produced less than 1% hemolysis and 10-15% hydrolysis of the membrane phospholipids reduced markedly (greater than 80%) the accessibility of membrane proteins to the external surface as measured by lactoperoxidase-catalyzed iodination.

Arrangement of human erythrocyte membrane proteins.

The orientation of human erythrocyte membrane protein was examined by enzymic iodination using lactoperoxidase with the glucose-oxidase system for generating peroxide, followed by proteolytic digestion. The outer surface of intact cells was labeled with 125I and the cytoplasmic surface of either resealed ghosts containing lactoperoxidase or of inside-out vesicles was labeled with 131I. Following iodination, the outer surface (resealed ghosts) or the cytoplasmic surface (outer surface of inside-out vesicles) was digested with trypsin, chymotrypsin, or pronase. Sodium dodecyl sulfate gel electrophoresis of the isolated membranes revealed three major and several minor peaks of radioactivity. Their surface orientation, defined within the limits of the specificity of the probes used, was as follows: the three major peaks consist of: (a) a 90,000 to 100,000 molecular weight component labeled on both surfaces; its proteolytic digestion profile indicated that it spans the membrane in an asymmetric manner and that it is composed of more than one peptide; (b) the major red cell membrane glycoprotein (apparent molecular weight 60,000) which is labeled and digested at only the outer surface; and (c) peptide(s) of high molecular weight (approximately 200,000), labeled and digested at only the cytoplasmic surface. The minor components include a glycoprotein of approximately 25,000 (apparent molecular weight) accessible to both surfaces and peptides of 60,000 to 70,000, 45,000, and 20,000 molecular weight labeled only on the inner surface.