Intra-and Inter-assay Coefficients Of Variation Research Articles

Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A.

Porcine viral diarrhea is caused by many pathogens and can result in watery diarrhea, dehydration and death. Various detection methods, such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), have been widely used for molecular diagnosis. We developed a triplex real-time quantitative reverse transcription PCR (qRT-PCR) for the simultaneous detection of three RNA viruses potentially associated with porcine viral diarrhea: porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus A (PoRVA). The triplex qRT-PCR had R2 values of 0.999 for the standard curves of PEDV, TGEV and PoRVA. Importantly, the limits of detection for PEDV, TGEV and PoRVA were 10 copies/μL. The specificity test showed that the triplex qRT-PCR detected these three pathogens specifically, without cross-reaction with other pathogens. In addition, the approach had good repeatability and reproducibility, with intra-and inter-assay coefficients of variation <1%. Finally, this approach was evaluated for its practicality in the field using 256 anal swab samples. The positive rates of PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 19.14% (49/256), respectively. The co-infection rate of two or more pathogens was 2.73% (7/256). The new triplex qRT-PCR was compared with the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018) and showed 100% agreement for PEDV and TGEV and 95.70% for PoRVA. Therefore, the triplex qRT-PCR provided an accurate and sensitive method for identifying three potential RNA viruses for porcine viral diarrhea that could be applied to diagnosis, surveillance and epidemiological investigation.

High throughput determination of free biogenic monoamines and their metabolites in urine using thin-film solid phase microextraction

UPLC-MS/MS methods are the gold standard for routine, high-throughput measurements of biogenic monoamines for the diagnosis of catecholamine-producing tumors. However, this cannot be achieved without employing efficient sample pretreatment methods. Therefore, two pretreatment methods, thin-film solid phase microextraction (TF-SPME) and packed fibers solid phase extraction (PFSPE), were developed and evaluated for the analysis of biogenic monoamines and their metabolites in urine. A hydrophilic-lipophilic balance (HLB) coating was chosen for the thin-film blade format SPME method and compared with a Polycrown ether (PCE) composite nanofiber used as an adsorbent for the PFSPE method. Under optimal conditions, the absolute extraction recovery and relative matrix effect of the newly developed TF-SPME method were determined to be 35.7–74.8% and 0.47–3.63%, respectively. The linearity was 0.25–500 ng mL−1 for norepinephrine, epinephrine, dopamine, normetanephrine 3-methoxytyramine, serotonin, histamine, and 0.1–500 ng mL−1 for metanephrine. The intra-and inter-assay coefficients of variation were 0.7–8.7%, and the respective accuracies were calculated to be 90.8–104.7% and 89.5–104.5% for TF-SPME. Compared with the PFSPE method, the TF-SPME method had a higher extraction efficiency, lower matrix effects and a wider linear range for eight target substances, which ensured higher accuracy of simultaneous detection of all compounds of interest. Therefore, the proposed TF-SPME method can be employed for the high throughput screening for neuroendocrine tumors in a routine clinical setting and other relative research by simultaneous quantitation of urine eight biological monoamines in a single run.

Application of element-tagged immunoassay combined with inductively coupled plasma mass spectrometry in HCG detection

Objective To establish an inductively coupled plasma mass spectrometry (ICP-MS) based immunoassay method for the quantitative detection of human chorionic gonadotropin (HCG), and evaluate the clinical applicability of this method. Methods Sm was selected as element tags, and the HCG quantitative detection system was established by double antibody sandwich method. The dosage of biotinylated antibody and reaction time were optimized. According to EP documents of Clinical and Laboratory Standards Institute (CLSI) and related standards, the analytical performance was evaluated after the establishment of the assay, including the limit of blank (LOB), linearity, precision, recovery, cross reactivity and interference test. And 88 clinical samples were measured using the new method compared to the electrochemical immunoassay (ECLIA) method. Results Total process completed within 30 min after optimization, and the optimal biotinylated antibody dosage was 0.5 μl. The LOB was 0.29 mIU/ml. The linearity was good within the range of 1.16-8 365.62 mIU/ml with the linear correlation coefficient greater than 0.995 (R2=0.998 0), the recovery was 97.53%-102.01%. Both intra-and inter-assay coefficients of variation of the high-value sample and the low-value sample were less than 10%. And there was no significant cross-reaction with Luteinizing hormone (LH), follicular stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The interference bias caused by different concentrations of interference substances was less than 10%. When compared with the ECLIA method for clinical sample detection, the proposed method showed a significant correlation(R2=0.960 0). Conclusion The proposed ICP-MS base immunoassay for HCG detection has good accuracy, high sensitivity and specificity, and the results of analytical performance verification meet the clinical requirements, which provides experimental basis for the clinical application of this method. Key words: Chorionic gonadotropin; Immunoassay; Spectrophotometry, atomic; Mass spectrometry

Rapid and sensitive label-free determination of aflatoxin M1 levels in milk through a White Light Reflectance Spectroscopy immunosensor

A miniaturized optical immunosensor based on White Light Reflectance Spectroscopy (WLRS) for the rapid and label-free detection of aflatoxin M1 (AFM1) in milk samples has been developed. WLRS sensing system consists of the measurement set-up and the biochip. The first encompasses the reflection probe, the light source and the spectrometer, while the latter is a Si chip with a SiO2 layer on top where an AFM1-bovine serum albumin conjugate has been immobilized. The assay was performed by running mixtures of rabbit polyclonal anti-AFM1 antibody with the calibrators or the samples, followed by reaction with biotinylated anti-rabbit IgG antibody and streptavidin. The assay cycle was completed in 25 min, the limit of detection was 6 pg/mL, and the linear working range extended from 0.012 to 2.0 ng/mL. The assay was repeatable (intra-and inter-assay coefficients of variation ranged from 2.1 to 6.3% and 3.5 to 8.2%, respectively) and accurate (percent recovery values ranged from 92.5 to 110%). AFM1 could be detected with the immunosensor developed in both processed and unprocessed milk of different animal species without any dilution. The excellent analytical characteristics and the small instrument size make the proposed sensor suitable for accurate low-cost AFM1 determination in milk samples at the point-of-need.

Automated measurement of 25-OH Vitamin D on the LUMIPULSE® G1200: analytical verification and method comparison

Background: Because of its potential value in several pathologies, clinical interest in 25-hydroxy Vitamin D (25OH-D) is increasing. However, the standardisation of assays remains a significant problem. Our aim was to evaluate the performance of the novel Lumipulse G 25-OH Vitamin D assay (Fujirebio), comparing results with the Liaison (Diasorin) method.Methods: Analytical verification of the Lumipulse G 25-OH Vitamin D assay was performed. Both methods were compared using sera from 226 patients, including 111 patients with chronic renal failure (39 on haemodialysis) and 115 patients without renal failure. In addition, clinical concordance between assays was assessed.Results: For Lumipulse G 25-OH Vitamin D assay, the limit of detection was 0.3 ng/mL, and the limit of quantification was 2.5 ng/mL with a 9.7% of coefficient of variation. Intra-and inter-assay coefficients of variation were <2.3 and <1.8% (25.4–50.0 ng/mL), respectively. Dilution linearity was in the range of 4.5–144.5 ng/mL. Method comparison resulted in a mean difference of −6.5% (95% CI from −8.8 to −4.1) for all samples between Liaison and Lumipulse G. Clinical concordance assessed by Kappa Index was 0.66.Conclusions: Lumipulse G 25-OH Vitamin D showed a good clinical concordance with the Liaison assay, although overall results measured in Lumipulse were higher by an average of 6.5%.

Higher levels of plasma matrix metalloproteinase-2 are associated with a significantly increased risk of arterial thrombosis in patients with the antiphospholipid syndrome

Alterations in the structure of extracellular matrix (ECM) play akey role in atherothrombosis. Matrix metalloproteinases (MMPs), afamily of zinc-dependent proteases produced and released by endo-thelial and smooth muscle cells, monocytes and platelets, degradeECM. There is evidence that MMPs are involved in cardiovascular dis-ease [1,2], but also in inflammatory disorders of the respiratory, gas-trointestinal, osteoarticular and central nervous systems [3].Plasma levels of some MMPs, and in particular of MMP-2, havebeen established as biomarkers of cardiovascular risk [1,2], but theyhave also been found to be increased in connective tissue disorders,likerheumatoidarthritis,systemiclupuserythematosusandSjogren'ssyndrome [3].No data are available instead on plasma MMPs in the anti-phospholipid syndrome (APS), an autoimmune condition characterizedby the occurrence of thrombotic events or pregnancy complications as-sociated with antiphospholipid antibodies (aPL). Among thromboticmanifestations of APS, both arterial and venous thrombosis may occur.However, so far no features have been identi fied distinguishing APS pa-tients undergoing arterial thrombotic events versus those developingvenous thromboembolism (VTE).AimofthepresentstudywastoexplorewhetherplasmaMMP-2isincreased in APS patients and to test if this marker can identifypatients undergoing arterial versus venous thrombosis.77 patients with APS (both primary and secondary), definedaccording to the international consensus criteria [4], (48 females,mean age 47±8 years), and 69 age- and sex-matched healthy con-trols (40 females, mean age 45±7 years) without concomitant riskfactors for cardiovascular disease, enrolled in two previously pub-lishedstudies[5,6], wereincluded inthepresentstudy.OftheAPSpa-tients,30 (22primaryand 8 secondary)(39%)had ahistoryof arterialthrombosis (mean time from event: 3.7±3 years), 41 (53%) (29 pri-mary and 12 secondary) a history of VTE (mean time from event:5±3 years) and six (8%) had obstetric manifestations. A group of pa-tients with history of arterial thrombosis (n=30, 16 females, 7 MI,5 stroke, 18 PAD) but no APS was alsostudied to distinguishthe role ofMMP-2 as a marker of vascular disease per se from a specific increaselinked to APS. Informed consent was obtained from each patient andthe study protocol, conformed to the ethical guidelines of the Declara-tion of Helsinki, was approved by the Local Ethic Committees (deliber-ation number 433/03). The authors of this manuscript have certifiedthattheycomplywiththePrinciplesofEthicalPublishingintheInterna-tional Journal of Cardiology.Plasma, obtained by centrifugation of 3.8% citrated venous bloodat 3000 g for 20 min, was immediately frozen and stored at −80 °Cfor later assay of MMP-2 by zymography, as described [7]. Intra-and inter-assay coefficients of variation of MMP-2 measurementswere 2.3% and 3.9%, respectively (n=8). All measurements were car-ried out by investigators blinded as to the patients' characteristics.The association of MMP-2 levels with history of arterial or venousthrombosis was evaluated by binary logistic regression analysis.Odds ratio (OR) and 95% confidence intervals (CIs) were calculatedconsidering the thrombotic event (arterial vs venous) as dependentvariable. Independent variables were: MMP-2 levels and age as con-tinuous variables, and sex (male vs female), oral anticoagulation(yes/no) and triple positivity for aPL (LAC, ACA, anti-β2GPI) (yes/no), as dichotomous variables. A two-sidedp-valueb0.05 was consid-ered as statistically significant. All analyses were performed with thestatistical package SPSS for Windows.PlasmaMMP-2wassignificantly higher in APSpatientsthanin con-trol subjects (Fig. 1A). When further subdivided in relation to clinicalmanifestations, MMP-2 levels were significantly higher in patientswith a previous arterial event than in patients with a previous VTE orin healthy controls (Fig. 1B). APS patients with obstetric manifesta-tions had MMP-2 levels similar to those of patients with VTE(740.9±94.2 ng/ml). MMP-2 levels in patients without APS but withprevious arterial thrombosis were higher than controls (682.6±84.3

Assessment of five ELISAs for measurement of leptin concentrations in dogs

To evaluate 5 commercially available ELISAs for determination of leptin concentrations in serum samples from dogs. Serum samples from overweight-obese and thin-ideal weight clientowned dogs. Serum samples with high and low leptin concentrations (n = 7 samples each) were used for validation of the assays. Intra- and interassay precision, linearity under dilution, spiking recovery, and limit of quantification were determined. In addition, leptin concentrations in thin-ideal weight (n = 8) and overweight-obese (37) dogs were quantified. Use of 2 of the 5 ELISAs (A and B) revealed reactivity with canine leptin. Intra-and interassay coefficients of variation were < 6.1% and 76%, respectively, for assay A and 14.0% and 13.7%, respectively, for assay B. In assays A and B, dilutions of canine serum pools were used to determine linear regression equations. Recoveries were 77% to 101% for assay A and 67% to 125% for assay B. Significant differences in leptin concentrations between thin-ideal weight and overweight-obese dogs were detected only when analyzed with assay A. Among 5 leptin ELISAs evaluated, a canine-specific leptin ELISA had adequate precision, linearity, and ability to discriminate between high and low leptin concentrations corresponding to overweight-obese and thin-ideal weight dogs, respectively.

Enzyme-free electrochemical immunoassay with catalytic reduction of p-nitrophenol and recycling of p-aminophenol using gold nanoparticles-coated carbon nanotubes as nanocatalysts

A novel enzyme-free sandwich electrochemical immunoassay with an ultrahigh sensitivity was developed for detection of alpha-fetoprotein (AFP, as a model analyte) using carbon nanotube-enriched gold nanoparticles (CNT-AuNPs) as nanolabels/nanocatalysts on anti-AFP/glutaraldehyde/thionine-modified glassy carbon electrodes (GCEs). The assays were carried out in a pH 8.0 acetic acid-buffered solution containing 6 mM p-nitrophenol (NP) and 6 mM NaBH 4 after the formation of the sandwich-type immunocomplex. Initially, the NP molecules were reduced to p-aminophenol (AP) by the catalysis of the immobilized gold-nanoparticle labels on the CNT-AuNPs with the aid of NaBH 4, then the generated AP molecules were electrochemically oxidized to p-quinone imine (QI) by an electron mediator of thionine, and then the oxidized QI molecules were reduced back to APs by NaBH 4. The redox cycling of AP and QI continuously increased the signaling, leading to a high sensitivity. Compared with individual gold-nanoparticle labels, the immunosensor using CNT-AuNPs as labels displayed a wider linear range of 8.0 × 10 −7–2.0 × 10 2 ng/mL with a lower detection limit (LOD) of 0.8 fg/mL AFP at a signal-to-noise ratio of 3, which was lower 6 orders than that of commercially available ELISA. Intra-and inter-assay coefficients of variation were below 10%. In addition, the assay was evaluated with clinical serum samples, and no significant differences at the 5% confidence level were encountered in the analysis of real samples between the proposed immunoassay and commercially available Roche 2010 Electrochemiluminescent Automatic Analyzer for determination of AFP.

Salivary hormones and parturition in healthy, primigravid women

Hormonal predictors of labor initiation are of considerable interest but are not clearly elucidated. Current hypotheses suggest a surge in the ratio of estriol to progesterone from b1 to N1 marks the impending onset of labor [1–3]. Little research exists examining relationships between other steroid hormones and parturition in healthy, uncomplicated, late-term pregnancies [4]. The purpose of this study was to assess prospectively associations between salivary progesterone, DHEAS, testosterone, estrone, estradiol, and estriol concentrations and parturition. Twenty-four healthy, medication free, primigravid women were recruited from area childbirth education classes and gave their informed consent to participate. This study was approved by the UNLV institutional review board. Pre-prandial, un-stimulated saliva samples were collected from participants at 9:00 am at 37 weeks +/−2 days of pregnancy. Specimen were mailed via two-day courier to AllVia Diagnostic Lab in Phoenix, AZ for analysis, enzyme immunosorbent assays. Pearson's bivariate correlations were calculated to determine relationships between salivary hormones and length of gestation. Normality was tested using Shapiro–Wilks, pb .05. The mean age of the participants was 29.63, SD 4.16. The average length of gestation was 277 days +/−6.68 days. Participants were tested at 259 days +/−2 days. The mean number of days from test time to delivery was 18 days. All participants began labor naturally without assistance. After labor initiated 11 women required medical intervention either via medication (n=9) or cesarean section (n=2) to deliver. Thirteen women delivered without assistance. All delivered healthy infants. The intraand inter-assay coefficients of variation did not exceed 10% except for inter-day estrone (16.25%). Descriptive hormone data are presented in Table 1. Progesterone was correlated with longer pregnancies (r=.414, pb .05) while estradiol was associated with shorter pregnancies (r= −.519, pb .01). No significant correlations between estriol values or the mean ratio of estriol to progesterone (.57) and delivery was found. No other hormone was correlated with parturition. Researchers have identified a pre-delivery surge of estriol either alone [3] or relative to progesterone [1,2] associated with labor initiation, suggesting that fetal input contributes to the onset of labor and that an estriol surgemarks impending delivery. Our study does not support these findings. Here, progesterone and estradiol, but not estriol either alone or relative to progesterone were associated with parturition in healthy, late-

A Direct Enzyme Immunoassay for the Measurement of Furosemide in Horse Plasma

A new enzyme immunoassay (EIA) for the measurement of furosemide in horse plasma is described. The lower limit of detection of this EIA method was 7.8 ng/ml. The intra-and inter-assay coefficients of variation ranged from 2.5% to 4.9% and 7.5% to 9.8%, respectively. Cross-reactivity with other compounds was not observed. There was a high correlation (r2=0.987) between the high-performance liquid chromatography and EIA results obtained for furosemide concentrations in horse plasma. These results indicate that the newly developed EIA method is useful for the quantitative analysis of furosemide in horse plasma.

Simultaneous determination of androstenedione and testosterone in human serum by liquid chromatography-tandem mass spectrometry

We aimed to develop a sensitive assay to quantitate serum concentrations of both androstenedione and testosterone within the female range simultaneously, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for use in the routine clinical laboratory and to compare this method with immunoassay. Samples (200 microL) were prepared by liquid-liquid extraction using (1 mL) methyl-tert-butyl-ether. Deuterated androstenedione and testosterone were used as internal standards. The standard curve was linear to 50 nmol/L, the lower limit of quantitation was 0.25 nmol/L, and intra- and inter-assay coefficients of variation were < 10% for both androgens over the range 0.3-35 nmol/L. There was a poor relationship between the LC-MS/MS and the radioimmunoassay methods for androstenedione with the LC-MS/MS generally giving lower results. For testosterone, the LC-MS/MS and immunoassay methods compared well at all concentrations. However, when female samples only were examined, the agreement deteriorated. We have developed a sensitive and precise LC-MS/MS method, which gives more accurate results for all androstenedione measurements and low testosterone concentrations than immunoassay.

Quantitative Sandwich ELISA for the Determination of Lupine (Lupinusspp.) in Foods

The use of lupine in foods has increased considerably during the past decade, reflected by a corresponding increase in reported lupine-induced allergic incidents. Lupine allergy may arise either by primary sensitization or by clinical cross-reactivity in peanut-allergic persons. Detection of lupine proteins in food has previously been based on the use of patient serum. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of lupine in processed foods was developed, using a polyclonal rabbit antilupine capture antibody and a biotinylated conjugate of the same antibody for detection. The antibody was highly specific for lupine, apart from minor cross-reactivities to other legumes. The assay had a detection limit of 1 mug/g and was successfully used to quantify lupine protein in various food matrixes. Recoveries ranged from 60 to 116%, while the intra-and interassay coefficients of variation were <6% and <21%, respectively.

A sensitive and practical competitive radioassay for plasma biotin.

Biotin is a water-soluble vitamin which plays an important biochemical role in a variety of carboxylase-mediated metabolic reactions. Determination of biotin status for the diagnosis of biotin deficiency is crucial. We describe a solid-phase protein-binding assay involving [(125)I]iodostreptavidin as a tracer for the determination of biotin in plasma. The assay was conducted in one step by incubation of a fixed amount of [(125)I]iodostreptavidin as binding reagent with varying amounts of biotin, diluted in biotin-free plasma (standard curve) or unknown samples in tubes previously coated with biotin linked to goat antirabbit IgG. Increasing amounts of biotin in the standard or unknown samples in tubes previously coated with biotin occupy more sites on iodostreptavidin, resulting in fewer counts bound to tubes. The effects of the incubation time and temperature on the competitive binding of biotin with iodostreptavidin were tested. The detection limit of the plasma assay for biotin was 100 pmol/L. Only 100 microL of plasma were necessary for the assay, which was performed within 6 h. The dilutions of plasma and synthetic biotin gave a parallel response. Plasma biotin levels ranged from 0.49 to 1.33 nmol/L (mean 0.76 nmol/L) in healthy subjects. The intra and inter-assay coefficients of variation were 3.5% and 10%, respectively, at a concentration of 0.27 nmol/L. This assay was suitable for the direct measurement of biotin in human plasma and was robust and sensitive enough for screening for biotin deficiency.

Association of leisure-time physical activity on inflammation markers (C-reactive protein, white cell blood count, serum amyloid A, and fibrinogen) in healthy subjects (from the ATTICA study)

T the 20th century, many researchers focused their interest on prevention and therapy of cardiovascular diseases. Among the factors that may influence the occurrence of disease is the beneficial effect of physical activity, which has been discussed in several studies.1–3 In addition, recent studies have provided evidence that inflammation plays a role in the pathogenesis of cardiovascular disease.4,5 Several investigators have addressed the association between fitness and the inflammation process,6–9 but the strength of this relation has not been fully investigated. The aim of this study is to evaluate the effect of various levels of leisure-time physical activity on inflammation markers, such as high-sensitivity C-reactive protein (CRP), fibrinogen, amyloid A, and white blood cell (WBC) counts, in a population-based sample of healthy adults. • • • The ATTICA study is a health and nutrition crosssectional survey that was carried out in the province of Attica from 2001 to 2002. A sample of 891 men and 965 women, aged 18 years old, was drawn from the general population, which excluded persons living in institutions, or subjects with mobility problems or who had chronic disease that could restrict their physical activity status (e.g., arthritis). Also, all subjects entered into this study were without any clinical evidence of coronary heart disease, stroke, or any atherosclerotic disease according to a detailed medical history, a physical examination, and electrocardiography as performed by a cardiologist. The stratification was random and based on the age/gender/city distribution of the Attica area (census of 2001). The study’s design anticipated enrolling only 1 participant per household (78% of the selected subjects participated). The number of the participants was determined by power analysis. All participants were interviewed by trained personnel who used a standard questionnaire. Physical activity was defined as any type of nonoccupational physical exercise 1 time per week, during the past year. A self-reported questionnaire was applied that was based on a special questionnaire for the assessment of leisure-time physical activity.10 Physical activity was graded in qualitative terms as follows: light (expended calories 4 kcal/min, i.e., walking slowly, stationary cycling, light stretching, and so forth), moderate (expended calories 4 to 7 kcal/min, i.e., walking briskly, cycling outdoors, swimming with moderate effort, and so forth), and high (expended calories 7 kcal/min, i.e., walking briskly uphill, long distance running, cycling fast or racing, swimming fast crawl, and so forth). The remaining subjects were defined as physically inactive. The duration of physical activity, in years of exercise, was also taken into account. Venous blood samples were collected between 8 and 10 A.M., with patients seated after 12 hours of fasting and avoidance of alcohol. High-sensitivity CRP levels, as well as fibrinogen levels, were measured by BNII Dade Behring Inc. (Leiderbach, Germany) automatic nephelometry. For the determination of plasma fibrinogen, blood was anticoagulated with 3.8% trisodium citrate (9:1 volume/volume) and cooled on ice until centrifugation. The intraand interassay coefficients of variation of fibrinogen did not exceed 9%, total cholesterol 8%). All other biochemical examinations (uric acid, urea, creatinine, total cholesterol, low-density lipoprotein cholesterol, highdensity lipoprotein cholesterol, triglycerides) were measured using a chromatographic enzymatic method with a Technicon RA-1000 automatic analyzer (TexLab Inc., Houston, Texas). Blood pressure at rest was measured with subject sitting comfortably for 5 to 10 minutes and the cuff arm supported at the heart level. Hypertension was defined as systolic blood pressure 140 mm Hg, diastolic blood pressure 90 mm Hg, or the use of any antihypertensive medication. Hypercholesterolemia was defined as total cholesterol levels 220 mg/dl or the use of antilipidemic medication. Diabetes mellitus was defined as a fasting blood sugar 125 mg/dl or the use of antidiabetic medication. The questionnaire given to subjects included the following demographic characteristics: age, gender, financial status (classified as low, moderate, high, and very high), and educational level (as an index of social status) that was measured in years of schooling. DiFrom the Cardiology Clinic, School of Medicine, University of Athens; Hellenic Heart Foundation, Athens, Greece; and Cardiology Division, Georgetown University, Washington, DC. The ATTICA study is funded by research grants from the Hellenic Cardiological Society and the Hellenic Heart Foundation, Athens, Greece. Dr. Panagiotakos’ address is: 48-50 Chiou Str., Glyfada, 165 61, Attica, Greece. E-mail: D.b.Panagiotakos@usa.net. Manuscript received July 29, 2002; revised manuscript received and accepted September 24, 2002.

Rapid and Sensitive Screening of Sulfamethazine in Porcine Urine with an Enzyme-Linked Immunosorbent Assay and a Field-Portable Immunofiltration Assay

An enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunofiltration assay (ELIFA) were developed for the screening of sulfamethazine (SMZ) in porcine urine. Incurred urine samples were measured by ELISA with a working range of 0 to 10 ng of SMZ per ml. The assay showed good accuracy and precision, with recoveries above 99.8% and intra-and interassay coefficients of variation (CVs) ranging from 2.6 to 5.6% and from 5.9 to 12.7%, respectively. Good agreement was observed when the results of the immunoassay were compared with those of liquid chromatography/tandem mass spectrometry analysis. For the ELIFA, a nylon membrane is placed on top of an absorbent material and covalently coated with rabbit anti-rat immunoglobulins. Free binding sites are blocked, and monoclonal anti-SMZ antibodies, SMZ standard or urine, and SMZ–horse radish peroxidase conjugate are subsequently dropped onto the membrane. During the assay, the reactants are drawn through the membrane because of its close contact with the absorbent pad. Finally, a substrate solution is added for blue color development. The blue spot produced can be visually evaluated or instrumentally measured (numeric ▪ value), and the intensity of its color is inversely proportional to the analyte concentration. When a blue dot appears on the membrane, even if its color is less intense than that of the negative control, the sample is considered “negative,” i.e., it is thought to contain a concentration of SMZ that is below the visual detection limit. If no color appears on the test membrane, the sample is considered “positive,” i.e., it is thought to contain a concentration of SMZ that is equal to or above the visual detection limit. Validation of the assay showed good inter- and intra-assay precision (CV < 10%). Because samples can be analyzed after a simple dilution in <30 min with this assay format, it has strong potential for application in the field.

Measurement of free and complexed soluble vascular endothelial growth factor receptor, Flt-1, in fluid samples: development and application of two new immunoassays

Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogenesis and enhances vascular permeability. VEGF interacts with the endothelium via two membrane-spanning receptors, fms-like tyrosine kinase (Flt)-1 and kinase domain receptor. A soluble form of Flt-1 (sFlt-1) was isolated from endothelial cell media; however, its biological significance is still unknown, with limited data on plasma sFlt-1 levels in disease states. We have developed two new ELISAs for detecting free and VEGF-complexed sFlt-1, which were tested in accordance with standard validation and assessment methodologies employed in commercial settings. The intra-and inter-assay coefficients of variation are <5% and 10% respectively, and results are highly reproducible. Applying these ELISAs in a clinical setting, we measured levels of VEGF, free and complexed sFlt-1 in citrated plasma from 40 patients with cardiovascular disease and 40 healthy controls. Median (interquartile range) plasma levels of VEGF in patients were significantly greater than controls [403 pg/ml (158--925 pg/ml) versus 113 pg/ml (33--231 pg/ml), P< or =0.05]. Free sFlt-1 was significantly lower in patients compared with controls [8 ng/ml (2--22 ng/ml) versus 21 ng/ml (10--73 ng/ml), P< or =0.05]. There was no significant difference in the levels of complexed sFlt-1 between the two groups. Plasma levels of VEGF-complexed sFlt-1 are minimal, despite the presence of excess free sFlt-1. Thus unbound plasma VEGF detected by ELISA may represent the majority of circulating VEGF, and justifies the measurement of plasma VEGF as an indicator of circulating VEGF levels. Furthermore, these results suggest that circulating sFlt-1 may serve as a selective inhibitor of VEGF activity, and that this regulatory mechanism may be altered by pathological conditions.

Evaluation of the “Hamilton Thorn computer-based automated system” for dog semen analysis

An objective evaluation of semen is warranted to assess the canine male fertility and to select appropriate techniques and extenders for semen preservation. With conventional microscopic evaluation, the subjectivity of the analysis makes any comparison of results difficult. In the present study, we validated the Hamilton Thorn computer-aided semen analyzer (HTR-IVOS10 analyzer) for objective assessment of canine semen. A description of fertile canine motility parameters using this analyzer is reported. Semen analysis at 38°C is found to be more optimal and accurate than 30°C. The Makler chamber was preferred to the Cell-vu, which induced a decrease of all semen motility parameters. The repeatedly of the measures was good with intra-and inter-assay coefficients of variation below 10% and 20%, respectively, for most of measured parameters. An overestimation of semen concentration, increasing with dilution of semen, was observed when HTR-IV0S10 results were compared with the classical manual Makler cell evaluation. A significant decrease in semen motility parameters was recorded when high semen dilutions were used. Generated from the analysis of 42 mature fertile male beagle dogs, a description of semen motility parameters using the CASA system is presented to serve as reference when comparing Beagle ejaculates both in clinical and experimental studies.

Quantitative automated particle-enhanced immunonephelometric assay for the routinary measurement of human cystatin C.

Human cystatin C is a low molecular mass protein of 13359 Dalton recently proposed as a new very sensitive marker of changes in glomerular filtration rate. Serum cystatin C concentration correlates negatively with glomerular filtration rate as well as or better than creatinine. We evaluated a recently introduced automated nephelometric immunoassay for cystatin C in serum or EDTA-plasma samples on the Behring Nephelometer System. The assay consists of incubating the 100-fold diluted sample for 6 minutes with latex particles covalently coated with anti-human cystatin C antibodies, and then quantifying the change of light-scatter produced. Method reproducibility is satisfactory, the intra- and inter-assay coefficients of variation ranging from 1.58% to 3.77% and from 5.6% to 11.47% respectively. Rheumatoid factor (< or = 1116 IU/ml), bilirubin (< or = 418 micrommol/l), triglycerides (10.47 mmol/), and haemoglobin (12 g/l) do not significantly interfere in the assay. No significant difference was found in cystatin C concentration between serum and EDTA-plasma samples. Cystatin C is stable in serum samples stored under different conditions up to one month. This method correlates well (mean difference=-0.536+/-0.307 mg/l) with another commercially available particle-enhanced turbidimetric immunoassay. Cystatin C offers better clinical sensitivity than creatinine for discriminating patients with normal renal function and those with mild-to-moderate reduction in renal function. This method is suitable for routine cystatin C measurement, including emergencies.

Recombinant somatolactin as a stable and bioactive protein in a cell culture bioassay: development and validation of a sensitive and reproducible radioimmunoassay.

A recombinant somatolactin (SL) obtained by cloning and expression of sole SL cDNA was analyzed and used to develop a sensitive and specific RIA. In contrast to native proteins, which tend to dimerize and aggregate immediately after pituitary isolation, the majority of recombinant sole SL (rsSL) remained as a monomeric protein after long-term storage, as shown by size exclusion chromatography and Western blot. Using rsSL as a tracer and standard in the RIA, the minimum detectable dose and the midrange (ED50) of the assay were 0.15 and 1.8-2.1 ng/ml respectively. Intra-and interassay coefficients of variation were 4.3% and 6.5% at ED50 levels. Recombinant gilthead sea bream GH and recombinant trout GH did not show cross-reactivity, whereas a good parallelism between rsSL standard and serial dilutions of plasma and sole pituitary extracts was observed. In order to demonstrate some biological activity of rsSL, the ability of this recombinant product to prime gilthead sea bream phagocytes for in vitro enhancement of mitochondrial activity was examined by a chromogenic assay. A bell-shape dose-response curve was obtained with a maximum at 50 nM (1.2 micrograms/ml), similar to that reported previously for GH. Therefore, taking together all these data, it appears conclusive that rsSL is a long-term stable protein which retains, at least in part, biological activity, providing a useful tool to clarify the physiological role of fish SL.

Changes in Plasma Sex Steroids in Females of Two Sympatric Leptodactylus from Subtropical South America

Changes in Plasma Sex Steroids in Females of Two Sympatric Leptodactylus from Subtropical South America