Immunoradiometric Analysis Research Articles

Endocrine and metabolic profiles in pregnant and non-pregnant Criollo mares

Gestation leads to metabolic changes, prioritizing nutrients’ delivery to the developing fetus (Fowden et al. Equine Veterinary Journal. 1984; 16(4), 239-246). Insulin, adiponectin, and leptin are key metabolic hormones that have been associated with pregnancy success in several species (Coverdale et al. J Anim Sci. 2015; 93(7), 3261-3267.; Meikle et al. Anim. Reprod., v.15, (Suppl.1), p.899-911. 2018). Information on these hormones and their metabolites during gestation of horses is lacking, specifically in native breeds. Therefore, the objective of this study was to establish the endocrine-metabolic profile in pregnant and non-pregnant Criollo mares, a native breed that is popular in South America. Mares (n=56) were categorized as pregnant (n=41) or non-pregnant (n=15). Animals were bled between days 200 and 250 of gestation and the Body Condition Score (BCS) was determined (scale 1-9, Henneke et al. Equine Veterinary Journal. 1983; 15(4), 371-372). The controls were assayed at the same time samples were taken, during the winter, between June and August. Insulin was determined by immunoradiometric analysis, leptin and adiponectin by Radioimmunoassay and other biochemistry variables by spectrophotometer. Hematology variables were determined by hematologic counter. Variables were analyzed by ANOVA using a mixed procedure. The reproductive status did not affect theleptin (9.4±1.6 vs 9.0±1.0 ng/mL, P=0.804 in pregnant and non-pregnant mares respectively), adiponectin concentrations (18.9±2.7 vs 15.5±1.6 ng/mL, P=0.304 in pregnant and non-pregnant mares respectively), nor the BCS (7.3±0.2 vs 7.6±0.1, P=0.356 in pregnant and non-pregnant mares respectively). Insulin (18.2±2.9 vs 17.7±1.7 UI/mL, P=0.644 in pregnant and non-pregnant mares respectively) and glucose (87.0±2.6 vs 83.0±1.6 mg/dL, P=0.180 in pregnant and non-pregnant mares respectively) concentrations were not affected either. Cholesterol concentrations were not affected by the reproductive status, while triglyceride concentrations were higher in pregnant when compared to non-pregnant mares (31.0±1.9 vs 21.6±3.1 mg/dL, P=0.012). Urea and creatinine concentrations were also higher in pregnant than in non-pregnant mares (52.8±1.0 vs 46.7±1.6 mg/dL, P=0.002 and 1.25±0.03 vs 1.01±0.05 mg/dL, P=0.0002 respectively). Hepatic enzymes and white blood cell count were not affected by the reproductive status. This study shows higher concentrations of triglycerides, urea and creatinine in pregnant mares. These changes could be associated with the metabolic prioritization of glucose delivery to the developing fetus, which leads to a differential nutrient partitioning, as reflected in different concentrations of specific metabolites of the protein and lipid pathways.

Changes in the T and B lymphocyte subset profiles upon treatment of patients with Graves’ disease with radioactive iodine

The aim of the present study was to evaluate the subpopulation profile of T and B lymphocytes, and their relationships during therapy of the patients with Graves’ disease (GD) treated by means of radioactive iodine. We have examined 36 women with verified diagnosis of GD. The contents of thyroid hormones were determined by immunoradiometric analysis. The levels of thyroid-stimulating hormone receptor autoantibodies (rTSH) were evaluated by enzyme-linked immunosorbent assay. On the basis of comprehensive pre-therapeutic examination, all patients were exposed to the fixed-activity therapy with radioactive iodine-131 at a dose of 400 to 700 MBq administered orally in isotonic aqueous solution of sodium iodide. 56 practically healthy women were examined as a control group. The phenotype of T and B cells in whole blood was studied by flow cytometry using direct immunofluorescence. It was shown that the patients, prior to treatment with radioactive iodine, had high levels of cellular functional activity, as determined by expression of CD25 antigen on T cells and CD23-antigen on B lymphocytes. Higher functional activity of the cells responsive for adaptive immunity in the patients with GD manifests in the presence of increased levels of autoantibodies to rTSH. By means of correlation analysis, we found that the patients with GD examined before the therapy had the thyroid status may determine the functional stimulation of T and B cells, thus increasing the levels of autoimmune processes. One month after radioiodine therapy (RIT), the GD patients, along with transient hyperthyroidism with increased concentration of autoantibodies to rTSH, showed a reduction of activated T lymphocyte contents (including T helpers and cytotoxic T cells) to control values. However, the level of cytotoxic T lymphocytes in the blood remained low, and the content of Treg cells was significantly increased in the patients. Decreased contents of B cells activated memory B cell to the control levels were found in patients with GD over 1 month after RIT when studying the phenotype of blood B lymphocytes. In this case, increased levels of naive B lymphocytes and B2 cells were detected, as well as decreased numbers of activated B1 lymphocytes. The observed changes in the subpopulation composition of T and B cells, and in their phenotype developed against the background of complete absence of relationships between the studied parameters, thus suggesting loss of thyroid control of immune processes and cooperative cell interaction during the development of the immune response. Generally, the phenotypic changes of T and B lymphocyte subsets in the blood of patients with GD through 1 month after treatment with radioactive iodine may reflect a trend for decreased functional activity of adaptive cellular immunity which may also account for inhibition of autoimmune processes.

Intraoperative parathyroid hormone monitoring corroborates the success of parathyroidectomy in children.

Ob­jec­ti­ve: To assess the efficacy of intraoperative parathyroid hormone (PTH) monitoring in evaluating the outcome of parathyroidectomy in pediatric patients.Methods: Intraoperative PTH monitoring during parathyroidectomy was performed in five children (3M, 2F); three had parathyroid adenomas (single gland disease) and two had primary hyperplasia. One patient had undergone two previous surgical interventions to remove the parathyroid glands, but the PTH levels had remained high with persistence of symptoms. Immunoradiometric analysis was used for PTH measurements. Preoperative PTH values were obtained to monitor the baseline levels. Serum samples were collected 20 minutes after removal of the adenoma/parathyroid gland(s) and PTH levels were compared with preoperative values. Specimens were also confirmed by frozen sectional examination. Results: Mean age of the patients was 11 years (range: 3 months-16 years). Mean preoperative PTH values were 633.3±579 pg/mL (range: 143-1300 pg/mL). Intraoperative values decreased to 18.7±5.5 pg/mL (range: 8-27 pg/mL) following removal of the gland(s). Normal calcium levels were achieved with adequate management following surgery. One patient (with multiple surgeries and found to have an ectopic parathyroid gland) had hungry bone syndrome after the operation and was treated successfully. There were no major complications. All patients maintained normal calcium/phosphorus levels in the follow-up period, ranging from 2 to 5 years.Conclusion: An ectopic parathyroid gland or another undetected adenoma can be overlooked during surgery. Owing to the short life of the hormone, intraoperative PTH monitoring to determine PTH clearance proved to be a feasible marker for adequacy and safety of surgery and “cure”.

CA 15–3 Serum Levels in Patients with Ductal Breast Carcinoma: Relationship with Clinicopathological Parameters and Tumor Markers

Cancer antigen 15-3 (CA 15-3) is the most widely used serum marker in diagnosing and monitoring breast cancer. The aim of this work was to analyze preoperative CA 15-3 serum levels in patients with ductal breast carcinoma in relation to 1) clinicopathological parameters, 2) hormone receptors, and 3) tissue-based tumor markers. A group of 340 women with infiltrating ductal carcinoma of the breast who had undergone no prior treatment was studied. These women ranged in age between 27 and 83 years (mean age 61.5±9.9 years). Preoperative CA 15-3 serum levels were determined by an immunoradiometric method. Hormone receptors (estrogen, progesterone and androgen receptors), p53, bcl-2 and Ki67 were determined by different immunohistochemical methods. Epidermal growth factor receptor (EGFR) was analyzed in the cell membranes by radioligand assay whereas cathepsin D and pS2 were determined by immunoradiometric analysis. Tumor ploidy and S-phase fraction were studied by flow cytometry. CA 15-3 serum levels were higher in postmenopausal women (p=0.032), in patients with tumors exceeding 2 cm (p=0.003), lymph node involvement (p=0.026), distant metastases (M1) (p<0.0001), S-phase fraction <7% (p=0.015), EGFR <6 fmol/mg protein (p=0.025), and cathepsin D <50 pmol/mg protein (p=0.023). Preoperative CA 15-3 serum levels were associated with cell proliferation determined by the S-phase fraction, the concentration of cathepsin D in the cytosol, and the EGFR concentration in the cell membrane.

Cissus quadrangularis augments IGF system components in human osteoblast like SaOS-2 cells

Osteoporosis is a public health problem which is associated with significant morbidity and mortality. Growth factors are produced locally in the bone and control cellular events such as induction of bone growth. Signaling through the Insulin-like growth factor (IGF)-I receptor (IGF-IR) by locally synthesized IGF - I or IGF-II in osteoblast is considered crucial for normal development and for bone remodeling. Traditional use of Cissus quadrangularis ( C. quadrangularis) in the treatment of bone disorders have been documented, however its regulatory effects on IGF system components remain largely unknown. The present study is employed to delineate the effects of ethanolic extract of C. quadrangularis on the regulation of IGF system components in human osteoblast like SaOS-2 cells. RT-PCR analysis revealed an increase in the mRNA expression of IGF-I, IGF-II, IGF-IR in cells treated with C. quadrangularis when compared with control cells. The mRNA expression of IGF binding protein-3 (IGFBP-3) did not differ significantly between control and C. quadrangularis treated cells. Immunoradiometric analysis revealed increased levels of IGF-I, IGF-II and IGFBP-3 in the conditioned medium of C. quadrangularis treated cultures when compared with control. Western blotting analysis revealed increase in protein levels of IGF-IR in cells treated with C. quadrangularis. These results indicate positive regulation of C. quadrangularis on the IGF system components of human osteoblast like SaOS-2 cells.

Growth Hormone, Insulin Like Growth Factor-1, and Insulin-like Growth Factor-Binding Protein-3 Levels in the Neonatal Period: A Preliminary Study

Growth hormone (GH), insulinlike growth factor-1 (IGF-1), and IGF-binding protein-3 (IGFBP-3) are used in the neonatal period for evaluation of diseases such as GH deficiency, intrauterine growth restriction, and hypoglycemia. However, GH level has been reported as a single value during the neonatal period. In this study, we attempted to determine the changes of GH, IGF-1, and IGFBP-3 in the neonatal period for each week. One hundred and two appropriate-for-gestational age (AGA) term neonates who did not have any diseases that would interfere with GH-IGF axis were included in this cross-sectional study. Blood samples were collected and serum GH, IGF-1 and IGFBP-3 levels were measured by immunoradiometric analysis (IRMA) and weekly values were obtained for the first postnatal four weeks. We showed that GH level [median (1st-3rd quartile)] decreases from the first to the fourth postnatal week [25.1 (18.5-37.4); 17.2 (13.2-22.8); 17.6 (12.2-20.2); 14.4 (6.60-19.8) ng/mL, respectively)], IGF-1 and IGFBP-3 levels [median (lst-3rd quartile)] increase [41.7 (18.0-70.0); 55.9 (39.0-103); 53.0 (40.0-97.7); 71.7 (44.3-137) and 1852 (1597-2451); 2430 (1645-2838); 2841 (2280-3675); 3018 (2151-4189) ng/mL, respectively)]. GH, IGF-1 and IGFBP-3 values can be evaluated for each week separately instead of for the entire neonatal period.

A joint detection of CEA and CA-50 levels in saliva and serum of patients with tumors in oral region and salivary gland

To detect the levels of carcinoembryonic antigen (CEA) and carcinoma associated antigen CA-50 in the patients with oral or salivary malignant tumors. The concentrations of salivary CEA and CA-50 were assayed in 80 patients of oral and salivary malignant tumors, 40 patients of benign tumors and 80 health controls. In 80 patients with malignant tumors, serum CEA and CA-50 were also assayed by enzyme-linked immunoabsorbent assay and immunoradiometric analysis, respectively. Salivary CEA and CA-50 levels were significantly higher in malignant tumors than in benign tumors and in health controls, respectively (P < 0.001). Only 7 cases and 3 cases of 80 patients with malignant tumors were found having increased serum CEA and CA-50 levels, respectively. The measurement of CEA and CA-50 levels in saliva were more sensitive than in serum. This may be more useful as prognostic indicators in early diagnosis of oral and salivary malignant tumors.

Immunoradiometric determination of thyroglobulin in serum samples by time calibration transfer

A limitation of isotopic assays is decay of the radioactive signal and the need for a calibration curve in each run. Thus, calibration transfer has been proposed for immunoradiometric analysis (IRMA). The present study was undertaken to establish the main methodological issues applied to the determination of thyroglobulin (Tg). Serum samples were analyzed at a different experimental time than Tg standards. Tg concentrations were then determined using a transferred calibration curve estimated from a calibration model based on a three-parameter antigen-antibody interaction model and a decay kinetic constant. A kinetic constant for decay of the radiolabeled antibody signal was calculated (-0.018+/- 0.002 day(-1)). This strategy avoided full recalibration, despite degradation of the radiolabeled immunoreagents or experimental variations. The calibration transfer was validated using addition, dilution and recovery protocols. Repeated analyses of serum samples were performed during the life of the commercial immunoreagents, and concentrations were determined using daily and transferred calibration curves. Results were statistically comparable, but significant reductions in reagent consumption, analysis time, radioactive waste and staff time were obtained. Transferred curves can be used to analyze samples by IRMA without simultaneous measurement of standards, and provide reliable concentrations. This principle has been demonstrated for Tg determination, but the key issues to extend to other IRMA methods have been established.

Immunoradiometric analysis of circulating human glycosylated and nonglycosylated prolactin forms: spontaneous and stimulated secretions

Immunoradiometric analysis of circulating human glycosylated and nonglycosylated prolactin forms: spontaneous and stimulated secretions

Immunoradiometric analysis of circulating human glycosylated and nonglycosylated prolactin forms: spontaneous and stimulated secretions.

The monoclonal antibodies (MAbs) obtained in mice immunized with human PRL coupled to an anti-PRL MAb were screened for their ability to distinguish the glycosylated (G-) and nonglycosylated (NG-) forms of PRL. The 431-29 MAb exhibited high affinity binding for NG-PRL but little or no cross-reactivity to G-PRL. Using this antibody in conjunction with other MAbs which equally recognized both forms, we developed 2 immunoradiometric assays which were used to determine the amount of G- and NG-PRL in plasma. In 85 normal subjects, NG-PRL baseline levels averaged 6.6 +/- 3 micrograms/L, and represented 76 +/- 8% of the total PRL immunoreactivity. In 74 pregnant women, this proportion was significantly higher during the last 2 trimesters (84 +/- 4% and 85 +/- 6%), as compared to the first trimester (76 +/- 7%). In 6 healthy volunteers studied over 24 h, 79% of the NG-PRL peaks detected using the cluster algorithm occurred concomitantly to a G-PRL peak. The mean NG-PRL/PRL ratio was significantly higher during NG-PRL pulses (81 +/- 9%) than during valleys (71 +/- 12%). Similarly, this ratio was significantly increased during TRH or metoclopramide stimulated PRL secretion (to 88 +/- 7% and 86 +/- 6%, respectively). We conclude that 1) NG-PRL is the predominant immunoassayable form of PRL in plasma; 2) both G- and NG-PRL are cosecreted but NG-PRL is the main PRL form released during spontaneous or pharmacologically induced PRL secretion.

Quantitative and qualitative assessment of pro-opiocortin related peptides by immunoradiometric analysis

Quantitative and qualitative assessment of pro-opiocortin related peptides by immunoradiometric analysis

Ferritin concentrations in plasma from capillary (finger prick) blood and venous blood compared.

Investigating the feasibility and validity of determining plasma ferritin concentration in blood obtained by finger prick, we studied 29 adults (ages 21-49 years) and 35 children (ages 14-66 months). Blood was sampled simultaneously in the same subject from both the antecubital vein (venous blood) and by finger pricking (capillary blood). The plasma was obtained by centrifugation. Ferritin concentration was determined by immunoradiometric analysis. Ferritin concentration in plasma from capillary blood was significantly higher than in venous plasma (p less than 0.01). This difference was more marked in children. The correlation between ferritin from the two blood sources was highly significant (r2 = 0.945 and 0.994 for samples from adults and children, respectively), and the slopes of the respective regression lines in both children and adults were significantly different from 1 (p less than 0.0001). We conclude that, despite the close association between the two procedures, the determination of ferritin concentration in capillary blood plasma overestimates the concentration of ferritin in venous blood plasma.

Immunoradiometric serum ferritin concentration compared with stainable bone-marrow iron as indices to iron stores.

Immunoradiometric analysis has made it possible to measure ferritin reliably in serum. The determination is a sensitive means of assessing body iron because concentrations of ferritin closely parallel the body's reticuloendothelial iron stores. The information provided is similar to that obtained from bone-marrow aspirates or biopsies stained for iron with Prussian Blue. Comparative studies indicate good correlation between iron content in the marrow and serum ferritin, except possibly in patients with leukemia and metastatic tumors to the bone marrow. The clinical value of the assay in relation to other indicators of iron status (i.e., hemoglobin, erythrocyte indices, serum iron and iron-binding capacity, and erythrocyte protoporphyrin) is in its specificity and sensitivity for assaying physiological iron stores.