Nonspecific Radioimmunoassay Research Articles

Quantification of BK1-5, the Stable Bradykinin Plasma Metabolite in Humans, by a Highly Accurate Liquid-Chromatographic Tandem Mass Spectrometric Assay

Bradykinin is a vasoactive nonapeptide involved in cardiorenal physiology and inflammatory states. It has been linked to the pathophysiology of hypertension and diabetes. Correlating levels of bradykinin with disease states has been hampered by its rapid degradation, artifactual production during blood sampling, and nonspecific radioimmunoassay techniques. We previously identified BK1-5 as the stable in vivo plasma metabolite of systemic bradykinin in humans. We now report a sensitive and specific assay method for BK1-5 in human blood utilizingliquid chromatography–tandem mass spectrometry(MS) with electrospray ionization. [13C2,15N]Glycine was incorporated into chemically synthesized BK1-5 for use as an internal standard. Blood samples (5 ml) were collected into 15-ml chilled ethanol to prevent artifactual kinin production and degradation. BK1-5 in ethanolic plasma supernatant was purified on a polymeric solid phase extraction cartridge. MS analysis was in the selective reaction monitoring mode. Precision of the assay is ±7.5% and accuracy is 99%. Recovery of BK1-5 through sample preparation was 43% and the lower limit of detection is 4 fmol/ml blood. Concentrations of BK1-5 in 12 normal volunteers were 44.2 ± 7.1 fmol/ml blood (mean ± SE). During blood sampling, no artifactual production of BK1-5 was detected for up to 60 s prior to denaturing the sample. This assay provides the first accurate and precise method using MS to quantify BK1-5 in human blood as a marker for the production of systemic bradykinin in humans.

Progress Report of an External Quality Assessment Scheme for Cyclosporine Assay

Beginning in 1987, an external quality assessment (EQA) scheme for cyclosporine (CsA) assay was organized by our Institute and supported by National Research Council (CNR); in 1991, the CNR EQA joined the French CsA interlaboratory program organized by Service de Radiopharmacie et Radioanalyse, University of Lyon. During the last 2 years (1993 and 1994 cycles), > 170 laboratories (from seven European countries) participated in this survey, assaying 44 control samples prepared from pooled blood of heart- and renal-transplant patients; normal pools added with known amounts of CsA were also distributed. During the whole EQA period, a trend toward the use of specific and nonisotopic techniques has been observed. In 1994, 91% of the collected results have been produced by specific methods [high-pressure liquid chromatography (HPLC) or specific immunoassays developed to assay the native molecule of CsA without its metabolites];in the same cycle, the fully automated techniques [TDx, fluorescence polarization immunoassay (FPIA), and enzyme-multiplied immunoassay (EMIT)] accounted for 73% of total results. CsA concentration measured by monoclonal specific immunoassays [(TDx FPIA, radioimmunoassay (RIA) Cyclo-Trac, and EMIT] was well correlated with those from HPLC (r = 0.99-1.00); results from EMIT were very close to those from HPLC, whereas results from RIA Cyclo-Trac and TDx were slightly overestimated (10-20%). Nonspecific methods (TDx polyclonal and nonspecific RIA Cyclo-Trac) measured CsA concentrations 3-4 times higher than those found by HPLC. The cross-reactivity of CsA metabolites in specific immunoassays was estimated from data of patients' samples and spiked samples; an interference of 6% in RIA Cyclo-Trac, 7% in EMIT, and 26% in TDx FPIA was found. The precision coefficient of variation (CV% between laboratory and between assay) of the methods observed in the 1994 EQA cycle was 9.5 for TDx polyclonal FPIA, 10.4 for TDx monoclonal FPIA, 10.5 for EMIT, 10.6 for RIA specific Cyclo-Trac, 14.2 for RIA nonspecific Cyclo-Trac, and 15.2 for HPLC.

Comparison of Nonspecific Radioimmunoassay, High-Performance Liquid Chromatography, and Fluorescence Polarization Immunoassay for Cyclosporine Monitoring in Renal Transplantation

Three methods, i.e., nonspecific radioimmunoassay (RIA; Incstar), fluorescence polarization immunoassay (FPIA; TDx Abbott), and high-performance liquid chromatography (HPLC), have been used for monitoring cyclosporine blood levels in renal transplantation patients. The levels obtained from 135 samples showed a modest correlation between RIA and HPLC, FPIA and HPLC, RIA and FRIA. The mean ratios of RIA to HPLC, FPIA to HPLC, and RIA to FPIA were 2.96, 4.14, and 0.73. The significant variations in cyclosporine levels result from the cross-reaction of antibody with some cyclosporine metabolites, by which these two methods often overestimate the true blood cyclosporine level. HPLC is a more effective and reliable method for pharmacokinetic studies and blood level monitoring of cyclosporine in clinical practice.

Predictive value of cyclosporin A level for efficacy or renal dysfunction in psoriasis

Cyclosporin A (CyA) trough (pre-dose) levels were measured in whole blood with a radioimmunoassay (RIA) using specific monoclonal antibody in 193 patients receiving CyA for the treatment of psoriasis. These patients received either CyA 2.5 mg/kg/day (n = 134) or 5 mg/kg/day (n = 59). In addition, a subgroup of 94 patients also had CyA trough levels measured using a non-specific polyclonal RIA. Within each CyA dose group, no difference was detected between mean CyA trough levels in relation to success or failure nor to the presence or absence of renal dysfunction with the use of either the specific or non-specific RIA. Based on the experience in transplantation, CyA thresholds of 100 and 200 ng/ml (specific) were selected for the assessment of efficacy and renal dysfunction. The success rate was higher by 10-15% when the CyA level was above, rather than below, 100 ng/ml in both the CyA 2.5 mg and 5 mg/kg/day groups. A slightly increased incidence of renal dysfunction was only found in the 5 mg/kg/day group when the CyA level was above 200 ng/ml. Because of its low predictive value, measurement of the level of CyA was not particularly useful for monitoring patients with psoriasis treated with low-dose CyA.

Cyclosporine Blood Concentrations Determined by Different Assay Methods in Heart Transplant Patients

Cyclosporine (CsA) through blood concentrations were determined by polyclonal nonspecific radioimmunoassay (RIA) (Sandoz), monoclonal specific and nonspecific RIA (Sandimmun, Sandoz), monoclonal specific and polyclonal nonspecific RIA, (Cyclo-Trac, INCSTAR) and fluorescence polarization immunoassay (TDx, Abbott), at multiple points in time, in two patients receiving CsA for immunosuppression after heart transplantation. Results obtained by the different nonspecific methods have a correlation (r) from 0.76 to 0.94. Concentrations determined by Sandimmun nonspecific RIA, Cyclo-Trac nonspecific RIA and TDx were consistently higher than those by Sandoz nonspecific RIA, owing to different cross-reactivity with CsA metabolites, giving ratios of 1:8, 1:4, and 1:6, respectively. In the first patient, the ratios between nonspecific and specific results were higher during days 1-20 than later on, which coincides with the high serum bilirubin level observed (r = 0.72, n = 31). This observation was not made in the second patient, whose liver function was within normal limits. Because CsA and its metabolites are eliminated primarily in the bile, the high values obtained with the nonspecific assay indicate that CsA metabolites accumulate in blood when liver function is impaired.

High Performance Liquid Chromatographic Separation of Estradiol-17 α and -17/β in Biological Fluids; Application to Plasma, Milk and Urine of Cows

Abstract A rapid HPLC technique was developed to separate estradiol epimers. In order to improve the sensitivity of the detection, a radioitmmunoassay was used. Estrone, estradiol-17α and estradiol-17β were separated within 20 min using 10 ml of chloroform: acetone (90:10), as the mobile phase. The efficiency of the technique was assessed with 3 steroids and the assay of collected fractions with antlsera specific to each estrogen. Using a non-specific radioimmunoassay, profiles of endogenous estrogens in different biological fluids (blood plasma, milk, urine) were obtained. The efficiency of HPLC as a separation method and the high sensitivity of radioimmunoassay as a detector allows us to obtain profiles of estrogens from biological samples where steroid concentration is below lOOpg/ml.

Chorionic Gonadotropin Concentration in Early Human Pregnancy: Comparison of Specific and Nonspecific Assays

Daily blood samples were collected during 10 menstrual cycles in which conception had occurred, and changes in circulating concentrations of human chorionic gonadotropin (hCG) were measured simultaneously in specific and nonspecific radioimmunoassays. The hCG concentration profiles were not identical, and the fundamental differences could not be related to differences in technique. Both specific and nonspecific parameters of hCG concentration rose exponentially for the first 2 to 3 weeks following first detection and were thus amenable to linear regression analysis. Doubling times were significantly different at 2.3 and 1.8 days, respectively. Extrapolation of the regression data gave a (theoretical) plasma concentration of 3 IU/liter at 9.5 days after the luteinizing hormone peak using the specific assay but at only 5.5 days using the nonspecific assay. This difference is sufficiently large to suggest the presence of other forms of hCG or its subunits about the time of implantation and during early pregnancy.

The use of a radioimmunoassay specific for human chorionic gonadotropin in patients with molar pregnancy and gestational trophoblastic disease

Serum human chorionic gonadotropin (hCG) activity is compared in 14 patients under treatment for gestational trophoblastic disease (GTD) using both a radioimmunoassay (RIA) specific for hCG and a nonspecific rapid solid-phase RIA which measures both luteinizing hormone (hLH) and hCG. The results indicate that the nonspecific RIA is adequate for the diagnosis and management of patients with GTD when the hCG titer is above endogenous hLH levels, but a specific RIA is required to ensure complete remission and to detect early recurrence during follow-up.