Serum levels of prostate-specific antigen in normal boys throughout puberty.

Serum levels of prostate-specific antigen (PSA) were measured in a group of 132 normal boys distributed in stages 1-5 of puberty. A highly sensitive (0.005 ng/mL) immunofluorometric assay was developed and used in the study. PSA levels were generally undetectable in state I and rose sharply from stage II to III and from stage III to IV. A significant positive correlation was found between PSA and testosterone levels, PSA and LH levels, PSA and age, PSA and testicular volume, as well as PSA and pubertal stage. Our findings indicate that PSA levels measured with highly sensitive assays can be of utility in the hormonal evaluation of puberty in boys.

Monoclonal antibody-based immunoenzymometric assay for serum human growth hormone.

A monoclonal antibody-based immunoenzymometric assay (IEMA) for the measurement of human serum growth hormone is described. Two high-affinity and complementary monoclonal antibodies were selected from a panel of 9 obtained upon fusion of SP2/O myeloma cells with spleen cells from a Balb/c mouse immunized against human growth hormone of pituitary origin. One monoclonal antibody was immobilized by attaching it to the walls of microtiter wells and the second was biotinylated. The reaction was quantitated by the addition of streptavidin-peroxidase. The sensitivity of the assay was 0.2 mIU/l and the intra- and interassay coefficients of variation for 4.6 to 46 mIU/l were less than 8.3 and 17.3%, respectively. Cross-reaction with human placental lactogen, human prolactin and rat growth hormone was less than 0.1% (w/w). Comparison of results obtained for 180 routine serum assays by radioimmunoassay and the assay described here had a correlation coefficient of 0.94 with a mean value of 16.3 +/- 1.3 (mean +/- SEM) and 13.3 +/- 1.2 mIU/l, with the IEMA providing values 18% lower than the RIA. The discrepancy emphasizes the necessity of redefining normal ranges before immunometric assays, like the one described, can be used routinely.

Development and clinical application of an immunofluorometric assay for intact parathyroid hormone.

Parathyroid hormone (PTH) is a linear peptide of 84 amino acids that is found in serum mainly in the form of carboxyl-terminal fragments. The biological activity of PTH depends on the presence of the amino-terminal portion and in circulation is limited to the intact molecule. We describe an immunofluorometric assay for the measurement of PTH-(1-84) based on a chicken egg yolk-derived amino-terminal antibody bound to microtiter plates by an anti-chicken Ig monoclonal antibody. As tracer antibody we employed a Europium-labelled carboxyl-terminal specific monoclonal antibody produced from a mouse immunized with hPTH-(53-84)-BSA conjugate. The assay included an initial overnight incubation of the sample and the solid phase-bound amino-terminal antibody, followed by washing and addition of the tracer antibody, and an additional two hours of incubation prior to fluorescence reading. The least-detectable dose was in the order of 2.5 pg/ml and preliminary studies in 40 normal adults showed values in the range of 4 to 70 pg/ml; for 12 patients with surgery-proven primary hyperparathyroidism values ranged from 109 to 743 pg/ml and for 34 patients with humoral hypercalcemia of malignancy from 2.5 to 66 pg/ml. We conclude that this assay, with its increased sensitivity and specificity, will be a valuable tool in the study of PTH secretion in normal and pathological situations.

Clinical utility of a 22-kDa growth hormone-specific assay.

Human growth hormone (hGH) circulates in different molecular forms, with the 22-kDa monomer being the predominant one and the 20-kDa variant corresponding to 5 to 15% of the serum hGH on a weight basis. Using monoclonal antibodies with different specificities we developed two immunoenzymometric assays, one with 22 + 20-kDa specificity and the other specific only for the 22-kDa form. Both assays used microtiter plates as solid phase and streptavidin-peroxidase for color development; intra-assay CV was less than 10% in the range of 1 to 100 mIU/l for the 22 + 20-kDa assay and in the range of 3 to 100 for the 22-kDa assay, with an inter-assay CV of less than 14% for both assays; sensitivity was 0.2 mIU/l for the 22 + 20-kDa assay and 0.5 mIU/l for the 22-kDa assay. The two assays were compared by measuring 200 serum samples with detectable hGH levels by both assays. Higher values were obtained with the 22 + 20-kDa assay (62.1 +/- 5.9 vs 59.2 +/- 6.1 mIU/l, mean +/- SD) with a correlation coefficient (r) of 0.99. In no clinical condition (28 patients with growth retardation and 14 acromegalics) did the two assays give discrepant values. We conclude that there was no practical advantage in using an assay with specificity restricted to the 22-kDa form for measuring hGH in clinical serum samples.

Radioimmunoassay for IgG and IgM rheumatoid factors.

The present paper describes two solid phase radioimmunoassays for detecting IgG and IgM rheumatoid factors (RF). Basically, serum rheumatoid factors were bound to rabbit IgG previously adsorbed to wells of flexible microtiter plates and quantitated after the addition of 125I-Fab anti-human gamma Fd for the IgG-RF assay or 125I-IgG anti-mu chain for the IgM-RF assay. The IgG and IgM rheumatoid factor assays were tested on 56 and 30 control sera, respectively. The upper normal limits for these assays were taken for cpm test serum/cpm control pool as 1.00 (0.76 + 0.24; X + 2SD) and 1.39 (0.73 + 0.66; X + 2SD) for IgG-RF and IgM-RF, respectively. When applied to sera of patients with rheumatoid arthritis, IgM-rheumatoid factor was detected in 15 of 15 (100%) patients, and IgG-rheumatoid factor in 15 of 17 (88%). The radioimmunoassay results for both rheumatoid factors were significantly higher for these patients than for the normal control sera (P less than 0.01).

Specific and nonspecific aspects of humoral immune response in leprosy.

1. We have studied some generic and specific aspects of the humoral immune response in 96 patients with leprosy (29 paucibacillary and 67 multibacillary individuals). We determined serum immunoglobulins (IgM, IgG and IgA), CH50, C1q, C3 and C4, circulating immune complexes (CIC), C-reactive protein (CRP), rheumatoid factor (RF) and antinuclear antibodies. No specific pattern of general humoral immune changes could be observed. 2. The specific immune response was studied by the detection of specific IgM anti-M. leprae antibodies. An immunoradiometric assay (IRMA) and an ELISA were compared for clinical effectiveness. IRMA showed greater sensitivity for the serodiagnosis of leprosy as compared to ELISA (88.1% vs 58.2% for multibacillary patients and 20.7% vs 10.3% for paucibacillary leprosy patients). Specificity was 96% for IRMA and 97% for ELISA. 3. Our results indicate that nonspecific changes in the humoral immune response are of little value in assessing leprosy patients and that immune assays for the detection of specific anti-M. leprae antibodies may be of value in the diagnosis, study and follow-up of these patients.

Production of anti-HBsAg monoclonal antibodies.

The production of monoclonal antibodies against the HBsAg is reported. Balb/c mice immunized against a commercial vaccine were used. Upon fusion of spleen cells from an animal having a high titer with the SP2/0 myeloma cell line, we obtained 6 stable cell lines, all of the IgG1 subclass. They showed a wide range of specificities against the classical HBsAg subtypes. These monoclonal antibodies can be used as the basis for the development of new methods for the screening and study of the hepatitis B virus.

Monoclonal antibodies specific for the free alpha subunit of glycoprotein hormones and their use in the development of a sensitive immunofluorometric assay.

Glycoprotein hormone free alpha subunit has been used as a marker for some pituitary tumors and to study the reactivity of glycoprotein hormone-producing cells under different circumstances. We describe a highly sensitive and specific immunofluorometric assay for the measurement of serum free alpha subunit levels. The assay is based on a monoclonal antibody, specific for free alpha subunit, bound to microtiter plates. As tracer antibody we employed an europium-labelled free/complexed alpha subunit specific monoclonal antibody. Using overnight incubation and 50-microliters samples, the least detectable dose was of the order of 4 ng/l. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively. Normal adult males showed values ranging from 120 to 790 ng/l, not different from normal adult premenopausal females (88 to 604 ng/l). In post-menopausal females, serum concentrations were significantly higher, ranging from 341 to 4071 ng/l. In 56 patients with untreated pituitary tumors (18 "non-secreting", 25 GH-producing and 13 prolactin-producing tumors), 10 showed high values, 3 of them from the first group, 3 from the second and 4 from the third. We conclude that this highly sensitive assay can be a valuable tool for the diagnosis and follow-up of selected patients with pituitary tumors and in other circumstances in which the glycoprotein hormone-producing cells of the pituitary require evaluation.

Serum leptin concentration during puberty in healthy nonobese adolescents.

Data obtained during the past five years have indicated that there are important age- and gender-based differences in the regulation and action of leptin in humans. To study the physiological changes of leptin during puberty in both sexes, and its relationship with body composition and sexual maturation, we measured leptin concentrations in 175 healthy adolescents (80 girls, 95 boys, 10-18 years of age), representing all pubertal stages. We excluded individuals with a body mass index (BMI) below the 5th or above the 95th percentile relative to age. Serum concentrations of leptin were determined by a monoclonal antibody-based immunofluorimetric assay, developed in our laboratory. Body composition was determined by dual-energy X-ray absorptiometry. Pubertal stage was assigned by physical examination, according to Tanner criteria for breast development in females and genital development in males. Leptin concentration in girls (N = 80) presented a positive linear correlation with age (r = 0.35, P = 0.0012), BMI (r = 0.65, P < 0.0001) and %fat mass (r = 0.76, P < 0.0001). In boys (N = 95) there was a positive correlation with BMI (r = 0.49, P < 0.0001) and %fat mass (r = 0.85, P < 0.0001), but a significant negative linear correlation with Tanner stage (r = -0.45, P < 0.0001) and age (r = -0.40, P < 0.0001). The regression equation revealed that %fat mass and BMI are the best parameters to be used to estimate leptin levels in both sexes. Thus, the normal reference ranges for circulating leptin during adolescence should be constructed according to BMI or %fat mass to assure a correct evaluation.

Comparison of the determination of insulin by a monoclonal antibody-based immunofluorometric assay and by radioimmunoassay.

This paper describes an immunofluorometric assay (IFMA) for insulin and compares it with the classical radioimmunoassay (RIA). Monoclonal antibodies against insulin were produced and used to develop the IFMA. One, immobilized on microtiter plates, was used for capture, the other, labelled with Europium, was used as tracer antibody. The IFMA presents sensitivity to an amount of insulin of 3 pmol/l and acceptable values for intra- and interassay error. The IFMA presented superimposable curves for human insulin, Arg65/Gly66-split proinsulin and des-Lys64,Arg65, and no cross-reactivity with human proinsulin, Arg32/Glu33-split and des-Arg31,Arg32. The RIA showed 100% cross-reactivity with human proinsulin, 90% with Arg32/Glu33-split, 193% with Arg65/Gly66-split, 340% with des-Arg31,Arg32 and 170% with des-Lys64,Arg65. The assays were used to measure insulin in 300 serum samples from 50 subjects submitted to an oral glucose tolerance test (OGTT). Twenty were normal, 10 had impaired glucose tolerance and 20 non-insulin-dependent diabetes mellitus. The mean value (+/- SEM) obtained by IFMA was 166.7 +/- 12.1 pmol/l and the mean value obtained by RIA was 339.6 +/- 18.6, with a correlation of r = 0.80 (P < 0.01). Comparison of basal insulin levels of the different groups of individuals using IFMA or RIA led to the same conclusions. The area under the curve showed statistically significant differences only for the comparison between normal lean subjects and individuals with impaired glucose tolerance, when measured by RIA. Our data stress the importance of methodology definition when comparing insulin results.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific and highly sensitive time-resolved fluoroimmunoassay for human proinsulin.

We describe a time-resolved fluoroimmunoassay specific for human proinsulin using a combination of two high-affinity monoclonal antibodies, one against insulin and the other specific for intact proinsulin and for split 65-66 and des 64-65 proinsulin forms. The assay employs only 200 microl of serum, with a detection limit of 0.1 pmol/l. The intra-assay variation coefficient was less than 3% between 3 and 1000 pmol/l. There was 0% cross-reaction with insulin, C-peptide, split 32-33 and des 31-32 proinsulin. Serum concentration of proinsulin was analyzed in 50 subjects during an oral glucose tolerance test (10 non-obese control, 10 obese controls, 10 subjects with impaired glucose tolerance, 10 patients with type II diabetes mellitus (DM) and fasting blood glucose (FBG) < 140 mg/dl, and 10 patients with type II DM and FBG > 150 mg/dl). Mean fasting serum proinsulin levels measured by this assay in non-obese controls (0.84 - 0.90 pmol/l; 0.1-2.4 pmol/l) were lower than the results reported by other investigators. There was an increase of proinsulin related to obesity and increased glucose levels, suggesting that proinsulin levels increase with insulin resistance.

[Serum beta 2 microglobulin (beta 2M) following renal transplantation]. / beta 2 microglobulina (beta 2M) sérica na monitorização do transplante renal.

Although there was an important improvement in graft and patient survival the last 10 years, graft rejection continues to be a major barrier to the success of renal transplantation. Identification of a laboratory test that could help to diagnose graft rejection would facilitate the management of renal transplanted patients. PURPOSE--To evaluate the utility of monitoring serum beta 2M in recently transplanted patients. METHODS--We daily determined serum beta 2M levels in 20 receptors of renal grafts (10 from living related and 10 from cadaveric donors) and compared them to their clinical and laboratory evolution. RESULTS--Eight patients who presented immediate good renal function following grafting and did not have rejection had a mean serum beta 2M of 3.7 mg/L on the 4th day post transplant. The sensitivity of the test for the diagnosis of acute rejection was 87.5%, but the specificity was only 46%. Patients who presented acute tubular necrosis (ATN) without rejection had a progressive decrease in their serum levels of beta 2M, while their serum creatinine changed as they were dialyzed. In contrast, patients with ATN and concomitance of acute rejection or CSA nephrotoxicity presented elevated beta 2M and creatinine serum levels. CONCLUSION--Daily monitoring of serum beta 2M does not improve the ability to diagnose acute rejection in patients with good renal function. However, serum beta 2M levels seemed to be useful in diagnosing acute rejection or CSA nephrotoxicity in patients with ATN.

Renal evaluation in women with preeclampsia.

BACKGROUND/AIMS: Preeclampsia (PE) is a cause of glomerulopathy worldwide. Urinary retinol-binding protein (RBP) is a marker of proximal tubular dysfunction, albuminuria is an endothelial injury marker, urine protein:creatinine ratio (PCR) may have a predictive value for renal disease later in life, and, recently, podocyturia has been proposed as a sensitive tool in pregnancy, but it needs to be tested. The aim of this study was to evaluate renal involvement in PE and healthy pregnancy. METHODS: Case-control study with 39 pregnant women assessed after 20 weeks of gestation (25 in the control group, CG, and 14 in the PE group) by performing urinary tests. RESULTS: Mean (±SD) age and gestational age of the CG were 26.9 ± 6.4 years and 37.1 ± 5.0 weeks, and of the PE group 26.4 ± 6.9 years and 30.6 ± 5.6 weeks, respectively (p = 0.001). Mean (±SD) urinary RBP (p = 0.017), albuminuria (p = 0.002), and urinary albumin concentration (UAC) ratio (p = 0.006) of the CG were 0.4 ± 0.7 mg/l, 7.3 ± 6.9 mg/l, and 8.2 ± 6.7 mg/g and of the PE group 2.0 ± 4.4 mg/l, 2,267.4 ± 2,130.8 mg/l (p = 0.002), and 3,778.9 ± 4,296.6 mg/g (p = 0.006), respectively. Mean (±SD) urine PCR in the PE group was 6.7 ± 6.1 g/g (p < 0.001). No statistical differences were found between podocyturia in the CG and PE group (p = 0.258). CONCLUSIONS: Urinary RBP, PCR, albuminuria, and UAC ratio were elevated in the PE group in comparison to the CG. Podocyturia did not predict PE.

PP019 The role of renal markers in women with and without preeclampsia: Evaluation of urinary excretion of podocytes and proteins.

INTRODUCTION: Preeclampsia (PE) is an important cause of glomerulopathy. Assessment of renal markers during pregnancy may have a predictive value for glomerular disease later in life. The early detection of PE may prevent the complications of this syndrome. OBJECTIVES: Assess the glomerular involvement in PE and in normal pregnancy by evaluating renal markers such as podocyturia and proteinuria. METHODS: Case-control study with 39 pregnant women after 20 weeks of gestation (control group - CG with n=25 and PE with n=14), we assessed podocyturia (cytospin method) and proteinuria (albuminuria, urine protein:creatinine - PCR, urinary retinol protein - RBP and albumin/creatinine ratio - ACR). (Grant FAPESP 08/56338-1) RESULTS: Mean±standard deviation of age and mean gestational age of CG were 26.9±6.4years and 37.1±5.0weeks and of PE, 26.4±6.9 and 30.6±5.6, respectively (p=0.001). No statistical differences were found between podocyturia in CG and PE although it was more frequent in this last group (p=0.258). Podocyte cells and parietal epithelial cells were detected in the slides. Mean±standard deviation of urinary RBP (p=0.017), albuminuria (p=0.002) and UAC ratio (p=0.006) of CG were 0.4±0.7mg/L, 7.3±6.9mg/L and 8.2±6.7mg/g and of PE, 2.0±4.4mg/L, 2267.4±2130.8mg/L (p=0.002) and 3778.9±4296.6mg/g (p=0.006), respectively. Mean value±standard deviation of urine PCR in PE was 6.7±6.1g/g (p=< 0.001). CONCLUSION: Urinary RBP, PCR, albuminuria and UAC ratio were elevated in PE in comparison to CG indicating its glomerular involvement but there was no correlation between those renal parameters and podocyturia. RPC and UAC ratios were good predictors of PE, but not podocyturia. Either podocyte cells as parietal epithelial cells were detected in the urine, these findings may indicate a non-invasive marker for renal disease activity but more studies are required to determine its role in PE.

PP090. The contribution of podocyturia to the evaluation of ongoing glomerular disorder in pregnant women with kidney graft (preliminary results).

INTRODUCTION: Progressive proteinuria and glomerulosclerosis characterize chronic allograft nephropathy. However, the causes are not fully elucidated. Injury of parietal epithelial cells in glomeruli, the podocytes, is the initiating cause of many renal diseases, leading to proteinuria with possible progression to glomerulosclerosis. Podocytes are highly specialized cells, with an important role in maintaining the glomerular filtration barrier and producing growth factor for mesangial cells and endothelial cells. With their foot processes they cover the glomerular basement membrane, and form slit diaphragms with neighboring podocytes. The potential role of podocytes in the failing transplanted kidney is unknown. OBJECTIVES: To evaluate podocyturia as a functional marker in pregnant women with kidney grafts. METHODS: Twenty pregnant women with kidney grafts had their urine samples cytocentrifugated and evaluated by indirect immunofluorescence. The slide was incubated for 45' at room temperature with fluorescein (FITC) anti-rabbit IgG secondary antibody (Sigma-Aldrich, EUA). Then Vectashield (mounting medium for fluorescence) with DAPI (4'6-diamino-2-fenilindol dihidrocloreto) were applied H-1200 (Vector laboratories, inc, USA). The podocytes and the total number of cells were counted in 15 fields photographed under 400x magnification with a digital camera coupled to an epifluorescence microscope DM1000 (Leica, Germany) connected to a computer. The results were expressed as podocyte/total cells (%) per area of higher cell concentration (hot spots) in the field of 400x detected by staining of nuclei and cytoplasm. (Grant FAPESP 08/56338-1). RESULTS: The mean age of the women was 26years. The urinalysis was performed at the third trimester of gestation; 11 did not exhibit urinary podocytes and 9 had podocyturia. There was also a relationship between blood pressure levels, proteinuria and the excretion of podocytes. CONCLUSION: Urinary podocyte number, blood pressure and proteinuria were associated. We observed that urinary podocyte excretion occurs in pregnant women with kidney transplant almost synchronously with higher systolic and diastolic blood pressure and higher mean levels of proteinuria. The detection of podocyturia in these women could be useful for early diagnosis and follow-up of glomerular injury, eventually preeclampsia. It may be also associated to its severity or activity, although additional studies are necessary to confirm these aspects.

Renal abnormalities in leprosy.

We have evaluated laboratory and clinical manifestations of renal disease in 96 patients with leprosy, looking for a sensitive and early marker for detection and possibly follow-up of nephropathy in these patients. Microscopic hematuria was observed in 21.9% of the cases (with dysmorphic erythrocytes in 71.4% of them). Abnormal microalbuminuria and urinary beta 2-microglobulin were found in 15.8 and 19.8% of the cases, respectively. We have observed a high frequency of hematuria, abnormal microalbuminuria and elevation of urinary beta 2-microglobulin in these patients still with normal serum creatinine.

Characterization of circulating immune complexes detected by monoclonal rheumatoid factor and conglutinin radioimmunoassays in SLE nephritis.

By utilizing a monoclonal rheumatoid factor (mRF) and bovine conglutinin (K) in radioimmunoassays, ICs detected in sera of patients with lupus nephritis were partially characterized. The mRF-RIA detected high levels of ICs in 86.9% of patients with active SLE and in only 22.7% of patients with inactive disease. Positive association was observed with clinical scores and significant negative correlation was found with serum levels of C1q, C3 and C4. The mRF-reactive ICs were shown to be cryoprecipitable and analysis by gel filtration through Sephacryl S-300 disclosed a material eluting between IgM and IgG, being dissociated in acidic pH. On the other hand, no association could be demonstrated between levels of ICs detected by K-RIA and clinical activity. Positivity in this assay was only 8.7% and 31.8% for active and inactive groups. Differently from the ICs detected by mRF-RIA, the K-reactive material was not precipitated by 3.5% PEG, nor by centrifugation in the cold, and EDTA did not reduce the binding of IgG to K in positive sera. The reactive IgG in Sephacryl S-300 chromatography eluted in the same position as monomeric IgG both in neutral and dissociating conditions. No C3 could be detected in ICs reactive in both assays.