Immunological comparison of the proteins of chicken and rat liver ribosomes.
A comparison of the proteins of chicken and rat liver ribosomes using immunochemical techniques was undertaken. The procedures included quantitative precipitation, passive hemagglutination, and immunodiffusion on Ouchterlony plates. The results indicate that antisera specific for chicken or rat liver ribosomes recognize only about 20% of common determinants. While there are important reservations, the results suggest extensive differences in the proteins of rat and chicken liver ribosomes. Despite those differences, rat and chicken liver ribosomal proteins maintain some homologous sequences present in bacterial ribosomal proteins. An enriched antibody preparation against chicken 80 S ribosomes inhibited the poly(U)-directed synthesis of polyphenylalanine and the elongation factor G (EF-G)-catalyzed binding of [3H]GDP to Escherichia coli ribosomes. Thus, chicken liver ribosomes, like ribosomes from rat liver and yeast, must have proteins homologous with those of E. coli ribosomes. (+info
Performance and specificity of monoclonal immunoassays for cyclosporine monitoring: how specific is specific?
BACKGROUND: Immunoassays designed for the selective measurement of cyclosporin A (CsA) inadvertently show cross-reactivity for CsA metabolites. The extent and clinical significance of the resulting overestimation is controversial. A comprehensive assessment of old and new methods in clinical specimens is needed. METHODS: In a comprehensive evaluation, CsA was analyzed in 145 samples with the new CEDIA assay and compared with the Emit assay with the old and new pretreatments, the TDx monoclonal and polyclonal assays, the AxSYM, and HPLC. All samples were from patients with liver and/or kidney transplants. RESULTS: The CEDIA offered the easiest handling, followed by the AxSYM, which showed the longest calibration stability. The TDx monoclonal assay provided the lowest detection limit and the lowest CVs. The mean differences compared with HPLC were as follows: Emit, 9-12%; CEDIA, 18%; AxSYM, 29%; and TDx monoclonal, 57%. The CycloTrac RIA paralleled the Emit results. In contrast to the mean differences, substantial (>200%) and variable overestimations of the CsA concentration were observed in individual patient samples. Metabolic ratios, estimates of the overall concentrations of several cross-reacting metabolites (nonspecific TDx polyclonal/specific reference method), correlated with the apparent biases of the various monoclonal assays. Metabolic ratios varied up to 10-fold, which translated into biases for individual samples between -7% and +174%. The higher the cross-reactivity of an assay was, the higher was the range of biases observed. The interindividual differences markedly exceeded other factors of influence (organ transplanted, hepatic function). CONCLUSION: Because assay bias cannot be predicted in individual samples, substantially erratic CsA dosing can result. The specificity of CsA assays for parent CsA remains a major concern. (+info
Automated homogeneous immunoassay for gentamicin on the dimension clinical chemistry system.
BACKGROUND: Monitoring of the concentration of gentamicin in serum and plasma during therapy is widely recommended and practiced in hospitals. Our aim was to develop a homogeneous immunoassay based on particle-enhanced turbidimetric inhibition immunoassay technology to quantify gentamicin on the Dimension clinical chemistry system. METHODS: Assay performance was assessed on each of the Dimension models in a 15-instrument interlaboratory comparison study. A split-sample comparison (n = 1171) was also performed between the gentamicin methods on the Dimension system and the Abbott TDx analyzer, using multiple reagent and calibrator lots on multiple instruments. RESULTS: The Dimension method was linear to 25.1 micromol/L (12.0 microg/mL) with a detection limit of 0.63 micromol/L (0.3 microg/mL). Calibration was stable for 30 days. The within-run imprecision (CV) was <1.3%, and total imprecision ranged from 1.8% to 3.2% between 4.2 micromol/L (2.0 microg/mL) and 16.7 micromol/L (8.0 microg/mL) gentamicin. Linear regression analysis of the results on the Dimension method (DM) vs the Abbott TDx yielded the following equation: DM = 0.98TDx - 0.42; r = 0.987. Minimal interference was observed from structurally related compounds such as sagamicin, netilmicin, and sisomicin. CONCLUSION: The monoclonal antibody used in this method has similar reactivities toward the individual gentamicin subspecies C1, C1a, and C2, thus providing analytical recovery not significantly dependent on relative subspecies concentrations. (+info
Serum IGF-binding protein-6 and prostate specific antigen in breast cancer.
OBJECTIVE: Recent studies have demonstrated the presence of the IGF-binding proteins (IGFBPs) and prostate specific antigen (PSA), an IGFBP protease. in human breast tissue. We sought to investigate the differences in serum IGFs, IGFBP-1, -3 and -6, and PSA between patients with surgically proven breast cancer and patients with benign breast disease. DESIGN AND METHODS: Concentrations of IGFs, IGFBP-1, -3 and -6, and PSA were determined in the sera from 57 patients with breast cancer (CA), and 46 women with benign breast disease (BBD) using immunoassays for IGFs and IGFBPs and an ultrasensitive ELISA for PSA. RESULTS: The mean (+/- S.E.M.) serum IGFBP-6 level in the CA group, 127 (16) ng/ml, was statistically significantly lower than in the BBD group, 157 (10) ng/ml (P = 0.016). Patients with CA had an elevated geometric mean serum PSA level of 0.018 (range: 0.0015-0.107) ng/ml, compared with 0.007 (range: 0.0015-0.019) ng/ml in women with BBD (P = 0.025). Mean serum IGFBP-1 concentrations were significantly lower in the CA group, 16 (2) ng/ml, versus 37 (4) ng/ml in the BBD group (P = 0.001). Mean serum IGFBP-3 concentrations were also lower in the CA group versus the BBD group, at 1981 (65) ng/ml, versus 2603 (140) ng/ml (P = 0.002) respectively. In the CA group, statistically significant correlations between PSA and IGFBP-6 (r = 0.413; P = 0.001), and between PSA and IGFBP-1 (r = -0.329; P = 0.021) were seen. Differences in IGF-I and -II between the two groups were not statistically significant. CONCLUSION: Lower serum concentrations of IGFBP-6, -3 and -1, but higher PSA concentrations were seen in the breast cancer group, and collectively these would suggest that there is an increase in bioavailable IGF-I in breast cancer. (+info
Differential immunodiagnosis between cystic hydatid disease and other cross-reactive pathologies.
We assessed an Echinococcus granulosus hydatid fluid antigen-ELISA (EgHF-ELISA) as a serologic prescreening test for E. granulosus infections, supplemented by more specific confirmatory tests, including arc-5 immunoprecipitation and antigen B subunit 8-kD immunoblotting. The diagnostic sensitivity of the EgHF-ELISA was 91%. With regard to the test specificity of the EgHF-ELISA (overall = 82%), we observed relatively frequent cross-reactions in tumor patients (6%) and in patients with other parasitic diseases. Cestode-related cross-reactivity can be resolved by the complementary use of E. multilocularis-specific antigens or Taenia solium cysticercosis-specific immunoblotting. Immunoblotting based upon the detection of antibody reactivity to the 8-kD antigen of EgHF, or if appropriately detectable, to the 29-kD and 34-kD bands exhibited a 91% diagnostic sensitivity and an overall specificity of 97% or 94%, respectively. Thus, immunoblotting provided a 99% discrimination between seropositive pre-operative cystic hydatid disease cases and cross-reactive non-cestode parasitic infections or malignancies. (+info
An immunoluminometric assay for N-terminal pro-brain natriuretic peptide: development of a test for left ventricular dysfunction.
Measurement of plasma levels of brain natriuretic peptide (BNP) has been used to assess left ventricular dysfunction and prognosis. Levels of the N-terminus of the precursor of BNP (NT-proBNP) have been reported to be elevated to a greater extent than BNP in left ventricular dysfunction. We have devised a non-radioactive sensitive and specific assay for NT-proBNP based on a competitive ligand binding principle. The chemiluminescent label 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium 9-carboxylate fluorosulphonate was used to label peptides representing domains in the middle and C-terminal sections of NT-proBNP. Assay of the C-terminal section of NT-proBNP (amino acids 65-76) in patients with proven left ventricular dysfunction [left ventricular wall motion index median 0.9 (range 0.3-1.4)] revealed elevated values [median 639 (386-911) fmol/ml] compared with normal controls [left ventricular wall motion index of 2 in all, NT-proBNP median 159 (120-245) fmol/ml, P<0.001]. Measurement of the middle section of NT-proBNP (amino acids 37-49) was not a discriminating test. It is thus possible to derivatize small peptides with a methyl acridinium label and preserve immunodetection with specific antibodies. Such methodology may allow non-radioactive immunoluminometric assays to be devised. (+info
Endometrial evaluation is not predictive for in vitro fertilization treatment.
PURPOSE: The main purpose of this study was to evaluate ovarian function by clomiphene citrate (CC) challenge test in a group of tubal infertile women and to study endometrial morphological maturation in the early luteal phase of CC-stimulated cycles as compared to in vitro fertilization (IVF) treatment outcome. METHODS AND RESULTS: Four women presented with strongly retarded, proliferative endometrium in the luteal phase. Of these, three presented with impaired ovarian function, high basal follicle-stimulating hormone, and high follicle-stimulating hormone levels after clomiphene stimulation on cycle day 10. In the remaining 30 women, showing an in-phase endometrium after CC stimulation, a comparison of six morphological characteristics did not reveal any significant differences between the 14 women who did become pregnant and the 16 who did not. No significant differences in endometrial thickness were observed between the groups. Significant differences were found when comparing estradiol and progesterone area under the curve during the luteal phase (P < 0.001 and P < 0.01, respectively) between those who did and those who did not become pregnant. CONCLUSIONS: Luteal endometrium morphology was not a sharp instrument to detect differences between women who did and women who did not become pregnant following IVF treatment, while ovarian function, as measured by hormonal markers, seemed to be a more reliable prognostic factor for IVF treatment outcome. (+info
Malignant transformation of human prostatic epithelium is associated with the loss of androgen receptor immunoreactivity in the surrounding stroma.
The cellular pathways involved in the pathogenesis of hormone resistance remain unclear. Studies evaluating the role of changes in human androgen receptor (hAR) expression in the progression of prostatic tumors have been inconclusive. Androgenic influence over prostatic growth is mediated via the regulation of interactions between stromal and epithelial cells. We hypothesized that neoplastic transformation of the prostate would be associated with alterations in hAR expression in the adjacent stroma. Using immunohistochemical techniques, we determined hAR positivity in the epithelium and adjacent stroma of sections from 17 benign and 39 malignant prostatic glands. We found that whereas the expression of the receptor decreased in both cellular compartments as the tissues dedifferentiated, the depletion was more pronounced in the stromal nuclei (P<0.0001). However, in sections from both untreated and hormone-resistant prostate cancer tissues, although heterogeneity of hAR expression in malignant epithelia was increased, there appeared to be a unique field effect around the cancerous prostate glands that resulted in a decreased expression of the receptor in the adjacent benign glands and its total loss in the surrounding stroma. The loss of hAR in the stroma adjacent to malignant prostatic epithelium may play an important role in prostate cancer progression. Furthermore, the similarity of the lack of stromal hAR expression in newly diagnosed and hormone-resistant prostate cancer (P = 0.85) may be an indication that the mechanisms responsible for the acquisition of hormone independence are established early in the malignant transformation process. (+info