Comparison of the two fully automated anti-HCMV IgG assays: Abbott Architect CMV IgG assay and Biotest anti-HCMV recombinant IgG ELISA.

OBJECTIVE AND BACKGROUND: To assess the performance characteristics of two fully automated human cytomegalovirus (HCMV) antibody tests. MATERIALS AND METHODS: Samples from negatively or not pre-screened blood donors were tested by the Biotest anti-HCMV recombinant IgG enzyme-linked immunosorbent assay (ELISA) in comparison to the Abbott Architect CMV IgG assay [chemiluminescent microparticle immunoassay (CMIA)]. For clarification, samples with discordant results between both assays were subjected to supplemental testing for anti-HCMV IgG, IgM and HCMV DNA in plasma. RESULTS: From 4938 samples tested, 362 delivered positive results in both assays (7.3%). 91 (1.8%) samples were discordant. Of 43 (two not further tested) samples positive only by ELISA, 41 were false positive, one true positive and one indeterminate. Of 45 (one not further tested) samples positive only by CMIA, 20 were false positive, 9 indeterminate and 16 true positive. Anti-HCMV IgM and HCMV DNA testing from the plasma were negative in indeterminate samples. Considering the results of supplemental testing, the CMIA achieved altogether better results concerning resolved sensitivity, resolved specificity as well as negative predictive value. Both assays had an inferior positive predictive value, with a better result for CMIA. CONCLUSION: Overall, the performance characteristics of the CMIA were better than those of the ELISA. Owing to the inferior positive predictive value, positive test results require confirmation if blood products from donors with remote HCMV infection should be administered.

MATRIX HCV--a newly developed semiautomated dot-immunoassay.

MATRIX HCV, a newly developed semiautomated assay for the detection of antibodies against individual antigens of the hepatitis C virus, was evaluated on blood donor specimens and compared with the results of another recombinant immuno-blot assay. 399 samples derived from a low-prevalence population revealed an assay specificity of 99.2% for MATRIX HCV. Investigation of 133 samples with repeat reactive screening results showed that the MATRIX HCV assay had a higher ability to discriminate between a clear reactive and nonreactive result compared to the recombinant immuno-blot assay.

Immuno-biochemical assay for determination of nuclear steroid receptors during tamoxifen therapy.

The exact knowledge of hormone receptor status is critical for therapeutic strategies in hormone-dependent tumors. The influence of tamoxifen on estrogen receptor concentration has to be taken into account when evaluating results in tamoxifen-treated patients. We studied the receptor modulation of tumors xenotransplanted into nude mice (one breast and one endometrial carcinoma) after injection of 50 micrograms tamoxifen/mouse. To differentiate between unoccupied and occupied receptors, determinations were done by an enzyme immunoassay for the estrogen receptor under low- and high-salt extraction. With low-salt extraction we found a temporary decrease of the estrogen receptor concentration within the first hours after tamoxifen treatment. This decrease lasted for several days before recovery to pretreatment levels occurred. The hormone-receptor complexes, tightly bound to acceptor sites of the DNA, increased more than 15 times within 24 h. These values remained at increased levels for 2-7 days, after which a decrease to initial level was observed.

Distribution of aromatase in the brain of the Japanese quail, ring dove, and zebra finch: an immunocytochemical study.

An immunocytochemical peroxidase-antiperoxidase procedure using a purified polyclonal antibody raised against human placental aromatase was used to localize aromatase-containing cells in the brain of three avian species: the Japanese quail, the ring dove, and the zebra finch. In quail and dove, immunoreactive cells were found only in the preoptic area and hypothalamus, with a high density of positive cells being present in the medial preoptic area, in the septal area above the anterior commissure, in the ventromedial nucleus of the hypothalamus, and in rostral part of the infundibulum. Immunoreactivity was weaker in zebra finches, and no signal could therefore be detected in the ventromedial and tuberal hypothalamus. The positive material was localized in the perikarya and in adjacent cytoplasmic processes, including the full length of axons always leaving a clear unstained cell nucleus. These features could be observed in more detail on sections cut from perfused brains and stained with an alkaline phosphatase procedure. The distribution of aromatase immunoreactivity was similar in the three species although minor differences were observed in the preoptic area. The localization of labelled neurons coincided with the distribution of aromatase activity as studied by in vitro radioenzyme assays on brain nuclei dissected by the Palkovits punch method. There was one striking exception to this rule: no immunoreactivity was detected in the zebra finch telencephalon, while assays had shown the presence of an active enzyme in several nuclei such as the robustus archistriatalis, the hyperstriatum ventrale pars caudale, and the hippocampus and area parahippocampalis. The origins of this discrepancy and the functional role of the aromatase observed in the axons are discussed.

Effects of castration and testosterone treatment on the activity of testosterone-metabolizing enzymes in the brain of male and female zebra finches.

Recently, we described the distribution of testosterone-metabolizing enzymes (i.e., aromatase, 5 alpha- and 5 beta-reductases) in the zebra finch (Taeniopygia guttata) brain using a sensitive radioenzyme assay combined to the Palkovits punch method. A number of sex-differences in the activity of these enzymes were observed especially in nuclei of the song-control system. The hormonal controls of these differences have now been analyzed by gonadectomizing birds of both sexes and by giving them a replacement therapy with silastic implants of testosterone (T). Five nuclei of the song system (Area X [X], nucleus magnocellularis of the anterior neostriatum [MAN], nucleus robustus archistriatalis [RA], nucleus intercollicularis [ICo], hyperstriatum ventrale, pars caudalis [HVc]) and three preoptic-hypothalamic areas (preoptic anterior [POA], periventricular magnocellular nucleus [PVM], and posterior medial hypothalamic nucleus [PMH]) were studied as well as other limbic and control non-steroid-sensitive areas. The activity of the 5 alpha-reductase was higher in males than in females for the five song-control nuclei and was not affected by the hormonal treatments. The overall activity of this enzyme was not sexually dimorphic in POA and PVM. It was higher in males than in females in intact birds only, and was reduced by gonadectomy and enhanced by T. The activity of the 5 beta-reductase was higher in females than in males in all nuclei of the song system and in POA, but was not influenced by the changes in T level. Both sex and treatment effects were observed in the control of aromatase. The production of estrogens was dimorphic (females greater than males) in RA and PMH. It was increased by T in POA, PVM, and PMH, and also in RA. These data show that some of the sex differences in T-metabolizing enzymes result from the exposure to different levels of T in adulthood (e.g., 5 alpha-reductase in POA and PVM or aromatase in PVM), whereas others persist even if birds are exposed to the same hormonal conditions. These are presumably the result of organizational effects of steroids. The steroid modulation of the aromatase might be related directly to the activation of sexual, aggressive, and nest-building behaviors, whereas the stable dimorphism in 5 alpha- and 5 beta-reductase observed in the nuclei of the song system might be one of the neurochemical bases of the sex differences in the vocal behavior of the zebra finch.

Sex- and age-related differences in the activity of testosterone-metabolizing enzymes in microdissected nuclei of the zebra finch brain.

Many effects of testosterone (T) in the zebra finch (Taeniopygia guttata) can be mimicked by T-metabolites, mainly estradiol and 5 alpha-dihydrotestosterone. We have therefore studied the neuroanatomical distribution of testosterone-metabolizing enzymes by means of the Palkovits punch technique combined with radioenzyme assay in the brain of adult and young male and female zebra finches. The activity of these enzymes was studied by a one-point assay in 5 nuclei of the song system (X, MAN, HVc, RA, ICo), 2 nuclei of the visual system (ectostriatum, nucleus rotundus) and in limbic and hypothalamic areas. Very noticeable was the presence of a very high aromatase activity in the hippocampal and parahippocampal region and in the nucleus taeniae and the absence of this enzyme in ICo. We found a higher aromatase activity in female than male HVc and RA and a higher 5 alpha-reductase activity in MAN, HVc, RA and ICo of males compared to females. The 5 alpha-reductase was more active in the preoptic area of females. A few sex-related differences in the activity of the 5 beta-reductase were also observed (higher activity in females than in males for area X and RA, but difference in the opposite direction for the ectostriatum). The statistical significance of these differences depended, to some extent, on the statistical technique used to demonstrate them, with the sex differences in RA being by far the most robust ones. Many age-related metabolic differences were also detected but these do not have a clear interpretation since the Km of these enzymes also changes with age. Extremely low levels of 5 beta-reductase activity were found in the nuclei of the visual system in adult birds while this enzymatic activity was very high in young birds. The biological significance of this change with age remains obscure. Correlations are thus observed between the neuroanatomical distribution of T-metabolizing enzymes and of androgen and estrogen receptors with the important exception of ICo which has no aromatase but contains high concentrations of estrogen receptors. Testosterone-metabolizing enzymes are however also present in areas which are not known as steroid targets.

Changes in the activity of testosterone-metabolizing enzymes in the brain of male and female zebra finches during the post-hatching period.

The kinetic properties of the testosterone-metabolizing enzymes were studied in the hypothalamus of adult and young zebra finches of both sexes. Estradiol, 5 alpha-dihydrotestosterone, 5 beta-dihydrotestosterone and 5 beta-androstane-3 alpha,17 beta-diol were identified as metabolites of testosterone in males and females at different ages between 5 days post-hatch and adulthood. During maturation, the maximum velocity (Vmax) of the aromatase and 5 beta-reductase decreased in males and females while the affinity of these enzymes for the substrate increased (decrease in km). These changes were more pronounced for 5 beta-reductase than for aromatase. The affinity change of the 5 beta-reductase occurred progressively during the post-hatching development between 5 and 30 days and is thus probably a true developmental process. In the case of the aromatase, the change in affinity occurred much later (after 30-40 days) and is thus probably related to the sexual maturation. These kinetic changes during development are directly related to the roles played by testosterone and its metabolites, in particular estradiol, in the differentiation and activation of reproductive behavior in the zebra finch. In particular, the dramatic decrease in 5 beta-reductase activity during sexual maturation should correspond to a potentiation of testosterone action in the brain.