Detection of mucopolysaccharidosis type II by measurement of iduronate-2-sulfatase in dried blood spots and plasma samples

BACKGROUND: Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder related to a deficiency in the enzyme iduronate-2-sulfatase (IDS). Clinical trials of enzyme replacement therapy are in progress, but effective treatment will require screening assays to enable early detection and diagnosis of MPS II. Our study evaluated the diagnostic accuracy of IDS protein and enzyme activity measurements in dried blood spots and plasma. METHODS: We collected dried-blood-spot and plasma samples from unaffected control individuals and from MPS II patients. We measured IDS protein concentration with a 2-step time-delayed dissociation-enhanced lanthanide fluorescence immunoassay. To measure enzyme activity, we immobilized anti-IDS antibody on microtiter plates to capture the enzyme and measured its activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. RESULTS: Dried-blood-spot samples from MPS II patients showed an almost total absence of IDS activity (0-0.075 micromol x h(-1) x L(-1)) compared with control blood spots (0.5-4.7 micromol x h(-1) x L(-1)) and control plasma (0.17-8.1 micromol x h(-1) x L(-1)). A dried-blood-spot sample from only 1 of 12 MPS II patients had detectable concentrations of IDS protein (24.8 microg/L), but no IDS protein was detected in plasma from MPS II patients. Ranges for IDS protein in control samples were 25.8-153 microg/L for blood spots and 22.8-349.4 microg/L for plasma. CONCLUSION: Measurement of the IDS protein concentration and enzyme activity (as measured by a simple fluorogenic assay with an immune capture technique) enables identification of the majority of MPS II patient samples from both dried blood spots and plasma samples.     

Development of an immunofluorometric assay and quantification of human kallikrein 7 in tissue extracts and biological fluids

BACKGROUND: Human kallikrein 7 (hK7), also known as human stratum corneum chymotryptic enzyme, is a chymotrypsin-like serine protease first identified in human skin extracts and predicted to be a secreted protease. The aim of this study was to develop a sensitive and specific immunoassay for hK7 and to examine the distribution of hK7 in tissue extracts and biological fluids. METHODS: Recombinant hK7 was produced in human embryonic kidney cells (HEK293T) and purified by a three-step column chromatographic procedure. The purified hK7 was injected into mice for antibody generation. A sandwich-type immunoassay was developed with the anti-hK7 monoclonal antibodies. RESULTS: The assay had imprecision (CV) <10% through the dynamic range of 0.2-20 microg/L and had no detectable cross-reactivity from other members in the human kallikrein gene family. Highest concentrations were found in skin, esophagus, and kidney. hK7 was also found in amniotic fluid, ascites from ovarian cancer patients, breast milk, cerebrospinal fluid, saliva, seminal plasma, serum, sweat, synovial fluid, and urine. CONCLUSIONS: This study describes the first ELISA-type immunoassay for hK7 protein quantification. hK7 is found many human tissues and in various biological fluids.     

Evaluation of a no-pretreatment cyclosporin A assay on the Dade Behring dimension RxL clinical chemistry analyzer

BACKGROUND: Monitoring whole-blood concentrations of cyclosporin A (CsA) is common practice in the management of solid organ and bone marrow transplant recipients. In a multicenter study we evaluated a new, direct (no pretreatment) CsA assay on the Dade Behring Dimension RxL system and compared results with those from the Abbott TDx CsA immunoassay and a HPLC method. METHODS: Whole-blood samples from heart (n = 111; 35 patients), liver (n = 201; 44 patients), kidney (n = 279; 65 patients), and miscellaneous organ (n = 77; 12 lung, 12 bone marrow, 5 kidney/pancreas, and 1 pancreas patient) recipients were obtained from patient populations of the participating institutions. Routine clinical monitoring of CsA was performed using either the TDx method or HPLC. RESULTS: The minimum detectable concentration of CsA averaged 9.4 microg/L, and the lower limit of quantification was 30 microg/L. The method was linear from 30 to 500 microg/L. Cross-reactivity with seven different CsA metabolites ranged from 0.0% to 5.7% for the Dimension RxL assay compared with 0.4-15.9% for the TDx assay. Total imprecision (CV) averaged 6.2%, and within-run imprecision averaged 4.9%. Passing-Bablok linear regression analyses of all samples from two sites yielded the following: RxL = 0.81 x TDx - 16.8; and RxL = 1.12 x HPLC - 1.7. CONCLUSIONS: The Dade Behring CsA assay for the random-access Dimension platform offers adequate performance characteristics for routine clinical use, does not require a manual pretreatment step, and demonstrates less cross-reactivity with CsA metabolites than another commonly used immunoassay.     

Identification of pro-MMP-7 as a serum marker for renal cell carcinoma by use of proteomic analysis

BACKGROUND: No validated renal cell carcinoma (RCC) marker is known for detection of asymptomatic disease in selected populations or for prognostic purposes or treatment monitoring. We identified immunogenic proteins as tumor markers for RCC by combining conventional proteome analysis with serological screening, and we investigated the diagnostic clinical value of such markers in serum. METHODS: We studied the immunogenic protein expression profile of CAL 54, a human RCC cell line, by 2-dimensional electrophoresis combined with immunoblotting using sera from healthy donors compared with RCC patients. We developed a homogeneous, fluorescent, dual-monoclonal immunoassay for metalloproteinase 7 (MMP-7) and used it to measure MMP-7 in sera from 30 healthy donors, 30 RCC patients, and 40 control patients. RESULTS: Pro-MMP-7 (29 kDa; pI 7.7) in the CAL 54 cell line secretome was an immunogenic protein reactive with RCC patient sera but not with control sera. The concentrations of pro-MMP-7 were increased (P <0.0001) in sera of RCC patients (median 7.56 microg/L; range 3.12-30.5 microg/L) compared with healthy controls (median 2.13 microg/L; range 0.17-3.5 microg/L). Serum pro-MMP-7 had a sensitivity of 93% (95% CI 78%-99%) at a specificity of 75% (59%-87%) for RCC in the samples tested. CONCLUSION: Proteomics technology combined with serology led to the identification of serum pro-MMP-7 as a marker of RCC and represents a powerful tool in searching for candidate proteins as biomarkers.     

Development of a dual monoclonal antibody immunoassay for total human kallikrein 2.

BACKGROUND: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-alpha(1)-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. METHODS: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions. RESULTS: The minimum detectable concentration in the thK2 assay was 0.008 microg/L, and PSA cross-reactivity was <0.001%. The assay detected prohK2 and three different hK2-serum protease complexes. The median serum concentration of thK2 in control samples (0.013 microg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 microg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at -70 degrees C. CONCLUSIONS: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.     

高迁移率族蛋白B1对人T淋巴细胞免疫功能影响的体外研究

目的观察高迁移率族蛋白B1（HMGB1）对健康人T淋巴细胞增殖的影响，并对其机制进行初步探讨。方法分离健康人外周血单个核细胞（PBMCs），调整细胞浓度后接种于细胞培养板并加入重组人高迁移率族蛋白B1（rhHMGB1）进行刺激。以四甲基偶氮唑盐微量Ⅱ酶反应比色法（MTT）检测细胞数量和细胞活性，观察HMGB1对T淋巴细胞增殖活性的影响。采用四色流式细胞术（FCM）分析CD3^＋淋巴细胞CD4表达。细胞中自细胞介素-2（IL-2）、IL-2α受体（IL-2Rα）基因表达水平采用逆转录一聚合酶链反应（RT—PCR）分析。结果①500～1000μg／L rhHMGB1作用48h后T淋巴细胞增殖反应显著抑制，低于这一剂量对其增殖活性影响不显著。②不同rhHMGB刺激时间和作用剂量对CD4^＋T淋巴细胞未造成明显改变，但rhHMGB1能时间-剂量依赖性增加Th2亚群比例，并因此降低Th1／Th2比值，刺激后T淋巴细胞免疫功能出现Th1优势向Th2优势偏移。③经植物血凝素激活后12hT淋巴细胞IL-2和IL-2Ra基因表达达到峰值；rhHMGB1与T淋巴细胞共同培养12h后，10～100μg／L剂量可明显上调IL-2和IL-2Ra基因表达；而较高剂量rhHMGB1（100～1000μg／L）刺激持续48h上述效应衰竭，并表现出相反的变化趋势。结论HMGB1对T淋巴细胞包括增殖、分化和细胞因子分泌等免疫功能具有直接调节效应。剂量蓄积和持续刺激可诱导T淋巴细胞功能亚群从促炎优势向抗炎优势转化。     

Development of a rapid and sensitive immunofluorometric assay for glutathione S-transferase A

BACKGROUND: The short half-life of the alpha-class glutathione S-transferases (GSTAs) in plasma combined with their even distribution throughout the liver lobule suggests that they may be useful complements to the more traditionally used liver markers. However, the currently available assays for measuring GSTAs in biological fluids have a poor dynamic range and are cumbersome, requiring multiple steps and prolonged incubation times. METHODS: Hybridomas that secrete monoclonal antibodies to human GSTAs were produced and used to develop a rapid one-step immunometric assay for the determination of GSTA in serum. The assay uses a time-resolved immunofluorometric assay (TR-IFMA) format and requires 35 min of incubation. The reference interval was determined using 208 serum samples from healthy blood donors. We also compared our TR-IFMA with a commercially available enzyme immunoassay (EIA) for GSTAS: RESULTS: The assay had a detection limit of 0.07 microg/L with a measuring range up to 625 microg/L. Within-run imprecision (CV) was 1.8-2.6% over the concentrations of GSTA tested (2.5-311 microg/L), with a between-run CV of <5%. In healthy blood donors, the median values and reference intervals were 2.0 microg/L and 0.6-7.2 microg/L for females and 2.6 microg/L and 0.7-9.8 microg/L for males, respectively. GSTA concentrations determined with the TR-IFMA correlated well with those obtained using a commercially available EIA. CONCLUSIONS: This report describes a new assay for monitoring the concentrations of GSTAs in human serum. The method may be useful in further evaluating the potential of monitoring serum GSTAs in the routine clinical setting.     

Long-term health outcomes associated with detectable troponin I concentrations

BACKGROUND: Recent data suggest that older men with detectable cardiac troponin I (cTnI) concentrations that remain below the 99th percentile concentration cutoff are at increased risk for subsequent cardiovascular events. We designed this study to extend this observation by examining risk prediction in both men and women presenting to an emergency department with chest discomfort. METHODS: We obtained data for all-cause mortality and hospital discharges associated with either acute myocardial infarction (AMI) or congestive heart failure (CHF) for up to 8 years after the initial presentation in 448 patients who originally presented in 1996 with acute coronary syndrome (ACS). We performed retrospective analysis for cTnI (AccuTnI; Beckman Coulter) in frozen plasma samples based on the patients' reported time from onset of symptoms. Peak cTnI concentration was used for risk assessment. RESULTS: Patients with cTnI concentrations > or =0.02 microg/L (i.e., limit of detection), including those whose peak values remained below the 99th percentile (0.04 microg/L), were at greater risk for death and AMI/CHF readmissions at 2, 5, and 8 years of follow-up compared with those with peak cTnI <0.02 microg/L. All results were statistically significant (P <0.05) except for death within 2 years among patients with normal but detectable cTnI (0.02 to 0.03 microg/L), relative to the group with values <0.02 microg/L. Kaplan-Meier analyses indicated that both men and women with cTnI > or =0.02 microg/L had worse outcomes (P <0.001). CONCLUSION: Both men and women who present with possible ACS with detectable cTnI concentrations that remain below the 99th percentile are at a greater risk for future adverse events.     

Quantitative analysis of circulating plasma DNA as a tumor marker in thoracic malignancies

BACKGROUND: Increased plasma DNA has been found in cancer patients and may have potential as a tumor marker. The objectives of this study were to develop a controlled, quantitative PCR (QPCR) assay to measure plasma DNA and then evaluate plasma DNA concentrations as a tumor marker in patients with thoracic malignancies. METHODS: We developed a QPCR assay for DNA, using the human beta-actin gene. Plasma samples were analyzed from 58 patients with esophageal cancer (EC; 20 banked samples and 38 prospectively collected samples) and 25 patients with lung cancer (LC; all prospectively collected). Control groups consisting of 51 patients with gastroesophageal reflux disease (GERD; 23 banked samples and 28 prospectively collected) and 11 healthy volunteers were also analyzed. RESULTS: The assay had an experimental variability <4%. In our banked samples, the mean concentration of plasma DNA in EC was 819.0 microg/L (range, 46.2-4738.0 microg/L) vs 432.0 microg/L (6.0-2888.0 microg/L) in GERD (P = 0.02). However, the prospectively collected samples had lower DNA concentrations, and there was no difference between cancer patients and controls. The mean DNA concentration was 10.6 microg/L (range, 7.0-14.0 microg/L) in healthy volunteers and 10.5 microg/L (range, 4.0-23.5 microg/L) in GERD controls vs 13.0 microg/L (range, 4.5-46.5 microg/L) in EC and 14.6 microg/L (range, 3.0-30.0 microg/L) in LC. CONCLUSIONS: Our data indicate that plasma DNA concentrations are of limited diagnostic value when samples are prospectively collected and uniformly handled. This is in contrast to previously published results. Qualitative analysis of DNA may be needed if plasma nucleic acids are to be used as a diagnostic tool in cancer screening.     

Effects of mode of delivery on maternal-neonatal plasma antioxidant status and on protein S100B serum concentrations

OBJECTIVE: To investigate the effect of the mode of labour and delivery on total antioxidant status (TAS) and on the protein S100B serum concentrations in mothers and their newborns. MATERIAL AND METHODS: Sixty women with normal pregnancies were divided into three groups: Group A (n = 20) with normal labour and vaginal delivery (VG), group B (n = 18) with prolonged labour+VG and group C (n = 22) with scheduled caesarean section (CS). Blood was obtained at the beginning of the labour process and immediately after delivery (pre- and post-delivery) as well as from the umbilical cord (CB). TAS and creatine kinase (CK) were measured using commercial kits. Serum S100B levels were evaluated with the electrochemiluminescence immunoassay "ECLIA" on the ROCHE ELECSYS 2010 immunoassay analyser. RESULTS: Post-delivery, TAS levels were significantly decreased in group A and especially in group B. S100B levels were increased in group B (0.0712+/-0.02 microg/L) as compared with those of group A (0.0567+/-0.03 microg/L, p<0.01) and group C (0.038+/-0.03 microg/L, p<0.01), the levels in group C remaining practically unaltered (pre- versus post-delivery). In the newborns, S100B levels were almost 2-fold higher in group B (0.67+/-0.18 microg/L) than those in group A (0.40+/-0.05 microg/L p<0.001) and group C (0.31+/-0.04 microg/L p<0.001). A negative correlation was found between TAS and S100B protein (r = -0.61, p<0.001), the latter positively correlated to CK (r = 0.48, p<0.01). CONCLUSIONS: The increased S100B serum levels in the mothers of group B, post-delivery, may have been due to the long-lasting, oxidative and/or psychogenic stress. The observed remarkably high levels of S100B in the group B newborns may have been due to compressive conditions on the foetus brain during this mode of delivery.     

Clinical significance of immunoassays for type-5 tartrate-resistant acid phosphatase

BACKGROUND: Tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) is a product of osteoclasts and a biochemical marker of bone resorption rate. However, erythrocytes and platelets contribute to total TRAP activity in serum, reducing the specificity of direct biochemical assays in serum. Osteoclast TRAP is also known as type-5 TRAP and is antigenically unique. Immunoassays are sought to improve the specificity and sensitivity of TRAP as a bone marker. METHODS: We developed two colorimetric microplate assays for type-5 TRAP: an enzyme capture immunoassay to measure antibody-bound enzymatic activity, and a two-site immunoassay to measure bound enzyme protein. Both use the same monoclonal antibody (14G6) to capture type-5 TRAP, which permits determination of specific activity of serum TRAP in health and disease. RESULTS: Both TRAP assays were linear from one-tenth to fivefold the mean value in 18 healthy subjects. In these subjects, the mean (SD) TRAP activity was 3.2 (0.54) U/L for the enzyme capture assay and 37 (13) microg/L for the two-site assay. Mean TRAP activity was not significantly increased in 64 patients with endstage renal disease requiring hemodialysis (HD) or 99 unselected patients with rheumatic diseases. By contrast, TRAP protein was increased in both the HD and rheumatic disease groups. The specific activity of TRAP in the 17 of 64 HD sera that had increased TRAP activity (0.088 U/microg) was similar to that in healthy subjects (0.091 U/microg). By contrast, the specific activity of TRAP in the 31 of 99 rheumatic sera with increased TRAP protein (0.035 U/microg) was significantly decreased. CONCLUSIONS: Wide sample distributions for TRAP activity in HD patients and TRAP protein in rheumatic disease patients suggest the presence of subpopulations of HD patients with increased TRAP activity and of rheumatic patients with increased TRAP protein. Each assay for TRAP activity and protein may have its own biological significance and clinical applications in specific groups of patients.     

Does a high peritoneal transport rate reflect a state of chronic inflammation?

OBJECTIVE: It has recently been reported that a high peritoneal transport rate was associated with increased mortality in continuous ambulatory peritoneal dialysis (CAPD) patients. One possible explanation is that a high peritoneal transport rate might be caused by a state of chronic inflammation, which also per se might result in increased mortality. Therefore, in this study we investigated whether high peritoneal transport rate patients are in a state of chronic inflammation. METHODS: The study included 39 clinically stable peritoneal dialysis patients (free of peritonitis) who had been on PD for more than 3 months (16.8+/-11.8 months). Seven patients were treated with continuous cycling peritoneal dialysis (CCPD) and the others were on CAPD. A 4-hour standard peritoneal equilibration test (PET) using 2.27% glucose solution was performed in each patient. Dialysate samples at 4 hours and blood samples at 2 hours were measured for interleukin-1beta (IL-1beta), tumor necrosis factor(alpha)(TNFalpha), C-reactive protein (CRP), and hyaluronan as markers of inflammation. RESULTS: There was no significant correlation between dialysate/plasma (DIP) creatinine (0.82+/-0.15, range 0.51 - 1.15) and blood concentrations of IL-1beta (11.2 ng/L, range <5 - 65.9 ng/L),TNFalpha (12.1 ng/L, range <5 - 85.4 ng/L), CRP (<10 mg/L, range <10 - 76 mg/L), nor with the blood hyaluronan concentration (165 microg/L, range 55 - 955 microg/L).The dialysate concentrations of IL-1beta and TNFalpha were below the detectable level in most of the samples. Although dialysate hyaluronan concentration (334 microg/L, range 89 - 1100 microg/L) was correlated with D/P creatinine (r= 0.36, p< 0.05), there was no correlation between the total amount of hyaluronan in the effluent and D/P creatinine. However, a significant correlation was found between serum hyaluronan concentration and glomerular filtration rate (GFR) (r = -0.49, p< 0.005); GFR also tended to be correlated with serum TNFalpha (r = -0.31, p = 0.058) but not with serum IL-1beta and serum CRP. CONCLUSION: Our results suggest that a high peritoneal transport rate is not necessarily related to a state of chronic inflammation in CAPD patients.The high mortality rate observed in high transporters may relate to other issues, such as fluid balance or abnormal nutrition and metabolism, rather than to chronic inflammation.     

Evaluation of a particle-enhanced turbidimetric immunoassay for the measurement of ferritin: application to patients participating in an autologous blood transfusion program

OBJECTIVES: To evaluate a turbidimetric immunoassay for the measurement of ferritin, and to assay this method in a group of patients undergoing an autologous blood transfusion program.DESIGN AND METHODS: We used an ILab 900 analyzer. This instrument automates a particle-enhanced immunoturbidimetric assay with an analysis time of 9 min. This technique was compared with a microparticle immunoassay. The turbidimetric assay was used to measure ferritin in a group of 30 patients undergoing an autologous blood transfusion program.RESULTS: The assay was linear in the range 3-1400 microg/L (r = 0.9999). The intra- and inter-assay imprecision (CV) at 20, 97 and 469 microg/L were <3.0 and <5.0%, respectively. Recovery was 88. 7 to 97.4%. The detection limit was 3 microg/L. Hemoglobin (相似文献   

Association of increased ferritin with premature coronary stenosis in men

BACKGROUND: Body iron status has been implicated in atherosclerotic cardiovascular disease. The main hypothesis is that high iron status is associated with increased oxidation of LDL. We investigated the potential role of ferritin as an additional risk factor promoting atherosclerosis among a young population with coronary artery disease (CAD). METHODS: Four hundred consecutive patients (218 males, 182 females) referred for diagnostic coronary angiography were examined, and risk factors for CAD, lipids, C-reactive protein (CRP), and ferritin concentrations were recorded for all participants. RESULTS: Ferritin was higher in the male patients with CAD (121 microg/L; range, 56-258 microg/L) than in the men without significant CAD (73 microg/L; range, 32-138 microg/L; P <0.002). Multiple logistic regression analysis, after adjustment for the established coronary risk factors, showed ferritin as an independent discriminating risk factor for CAD (P <0.01). Men in the highest quartile of ferritin had an odds ratio (OR) of 1.62 [95% confidence interval (95% CI), 1.12-2.42; P <0.01] compared with men in the lowest quartile of ferritin. The association between ferritin and CAD was more pronounced in male patients < or =50 years (OR = 2.65; 95% CI, 1.35-5.51; P <0.003). Ferritin was significantly higher in diabetic male patients in comparison with nondiabetic male patients [168 microg/L (range, 74-406 microg/L) vs 106 microg/L (range, 44-221 microg/L), respectively; P <0.002]. No association was observed between ferritin and CAD among the female patients. CONCLUSION: Our data suggest that increased ferritin might be an independent predictor of premature CAD in male Iranian patients.     

Measurement of the noncomplexed free fraction of tissue inhibitor of metalloproteinases 1 in plasma by immunoassay

BACKGROUND: We previously found differences in total concentrations of tissue inhibitor of metalloproteinases 1 (TIMP-1) in plasma from donors and cancer patients. Because TIMP-1 can exist in more than one molecular form, a new immunoassay to specifically detect free TIMP-1 was developed and concentrations were determined in plasma from healthy donors and colorectal cancer (CRC) patients. METHODS: We established and validated an immunoassay for the specific measurement of free TIMP-1 that uses a polyclonal anti-TIMP-1 antibody for capture and a monoclonal anti-TIMP-1 antibody that binds only free TIMP-1 for detection of antigen. Plasma samples from healthy donors and CRC patients were assayed for free TIMP-1. Total TIMP-1 was measured by our previously published assay. RESULTS: The mean (SD) concentrations of free TIMP-1 were similar in citrate [55.5 (11.5) microg/L] and EDTA plasma [58.9 (13.3) microg/L] from 76 donors (r(2) = 0.82). In 154 donors, the ratio of free TIMP-1 [mean (SD), 64.5 (18.0) microg/L] to total TIMP-1 [83.8 (19.8) microg/L] in EDTA plasma was 0.77. Plasma concentrations of free and total TIMP-1 correlated significantly to age (free, r(2) = 0.19; total, r(2) = 0.27; P <0.0001), increasing 50% over an age span of 45 years. Free and total TIMP-1 were significantly increased in CRC patients (P <0.0001), whereas the ratio of free to total TIMP-1 (mean, 0.58) was significantly lower than in donors. CONCLUSIONS: Most of the TIMP-1 in donor plasma is present in its free form, and free TIMP-1 increases with age. Free and total TIMP-1 are increased in CRC patient plasma, but the ratio of free to total TIMP-1 is significantly lower in these patients than in donors.     

Growth hormone (GH) receptor isoform in acromegaly: lower concentrations of GH but not insulin-like growth factor-1 in patients with a genomic deletion of exon 3 in the GH receptor gene

BACKGROUND: A genomic deletion of exon 3 (d3-GHR) of the growth hormone (GH) receptor (GHR) has been linked to the effectiveness of GH therapy in children with GH deficiency. Carriers of the d3-GHR genotype had higher GH-induced growth rates than children homozygous for the full-length (fl)-GHR. The aim of this study was to test whether the relationship between GH and insulin-like growth factor-1 (IGF-1) concentrations is influenced by the GHR genotype in patients with acromegaly. METHODS: Study participants were 44 adult patients with established diagnosis of acromegaly. The genotype of the GHR was determined in leukocyte DNA from peripheral blood. Clinical and biochemical findings at the time of diagnosis of acromegaly were obtained from the medical records of the patients. RESULTS: fl-GHR homozygosity was found in 22 (50%) of patients, and 22 (50%) of patients had at least 1 d3 allele (d3-GHR). Demographic and clinical characteristics (age, height, weight, estimated duration of disease, and mean tumor size) of the 2 groups were comparable. Median (range) serum IGF-1 concentrations at the time of diagnosis were 670 (447-1443) microg/L in the fl-GHR group and 840 (342-1494) microg/L in the d3-GHR group (P = not significant). Basal GH concentrations were higher in the fl-GHR group [29.7 (3.8-159) microg/L] than in the d3-GHR group [8.4 (2.6-74 microg/L), P = 0.002], and so were mean (30.4 vs 6.1 microg/L, P = 0.005) and nadir (20.5 vs 5.1 microg/L, P = 0.003) GH concentrations during an oral glucose tolerance test. CONCLUSIONS: The GHR fl/d3 genotype modulates the relationship between GH and IGF-1 concentrations in patients presenting with acromegaly.     

Homogenous enzyme immunoassay for cyclosporine in whole blood using the EMIT 2000 cyclosporine specific assay with the COBAS MIRA-plus analyzer.

We describe the evaluation of the EMIT 2000 cyclosporine specific assay kit, an enzyme-multiplied immunoassay for cyclosporine in whole blood, with a COBAS MIRA-plus analyzer. The enzyme used for the assay was glucose-6-phosphate dehydrogenase (EC 1.1.1.49 G6PDH) from Leuconostoc mesenteroides; the monoclonal antibody is fairly specific for cyclosporine, and is not reactive with most metabolites. The assay principle is based on competitive immunoassay with G6PDH-labeled cyclosporine and cyclosporine in sample to the anticyclosporine mouse monoclonal antibody binding site. The within-assay coefficient of variation (CV) of this method was 2.7-4.2% (n = 10) at the levels of 56.2-339.7 microg/L. Day-to-day CVs ranged from 4.2-8.1% at the levels of 47.2-350.2 microg/L. The within-day CVs ranged from 2.0-6.4% at the levels of 43.3-330.5 microg/L. The functional detection limit was 24.9 microg/L. Samples treated with pretreatment reagent were stable at least 5 hr. Calibration was stable at least 10 days. The analytical recovery was 81-109%. The correlation between values obtained with the EMIT 2000 cyclosporine specific assay kit (y) and fluorescence polarization immunoassay (FPIA) (TDxFLx) (x) was: y = 0.880x - 13.053 microg/L (r = 0.984, Sy/x = 15.968, n = 71) with a mean difference of 31.42 +/- 19.89 microg/L ((TDxFLx - EMIT 2000) +/- SD); for the FPIA (AxSYM) (x): y = 0.989 - 4.144 microg/L (r = 0.981, Sy/x = 17.478, n = 71) with a mean difference of 5.56 +/- 17.38 microg/L ((AxSYM - EMIT 2000) +/- SD); and for the radioimmunoassay (RIA, CYCLO-Trac SP) (x): y = 0.893 - 6.764 microg/L (r = 0.993, Sy/x = 10.582, n = 71) with a mean difference of 22.18 +/- 14.98 microg/L ((RIA - EMIT 2000) +/- SD) using the Bland-Altman technique.     

Homogeneous enzyme immunoassay for triiodothyronine in serum

BACKGROUND: The concentration of triiodothyronine (T3) in human serum is extremely low and can be determined only by very sensitive methods. We developed a homogeneous enzyme immunoassay for T3 analysis in unextracted serum. METHODS: A T(3) derivative was conjugated to the -SH groups of glycogen phosphorylase b (GPb) from rabbit muscle. Conjugation caused inhibition of enzyme activity, and the enzyme conjugate was reactivated upon binding of anti-T3 antibody. Activation was blocked by the presence of non-antibody-bound T3; this was the basis for the development of the homogeneous enzyme immunoassay for T3 by determining GPb activity fluorometrically. RESULTS: We used furosemide to block the interaction of T3 with serum proteins with T3-binding sites, avoiding any serum treatment step. T3 was measured in the range 0.3-8 microg/L. T3 values obtained by this assay correlated well with those obtained by a RIA (y = 0.97x - 0.07 microg/L; r = 0.96; n = 92). Within- and between-run imprecision (CV) was 5-9% for normal and high concentrations and 16-20% for low concentrations. CONCLUSIONS: Chemical modification of GPb with a T3 derivative allows the development of a simple homogeneous enzyme immunoassay for T3 in unextracted serum.     

A disposable multianalyte electrochemical immunosensor array for automated simultaneous determination of tumor markers

BACKGROUND: Automated and convenient multianalyte detection with high throughput is increasingly needed in clinical diagnosis. We developed a disposable 4-by-2 array for programmed simultaneous amperometric immunoassay of 4 tumor markers. METHODS: We used a screen-printed technique, 1-step immobilization method, and flow injection technique. We immobilized carcinoembryonic antigen, alpha-fetoprotein, beta-human choriogonadotropin, and carcinoma antigen 125 as model analytes in a redox mediator-grafted, biopolymer-modified, screen-printed carbon electrode array to capture corresponding horseradish peroxidase-labeled antibodies in competitive immunoreactions. The simultaneous multianalyte immunoassay was automatically carried out to amperometrically monitor the mediator-catalyzed enzymatic response to hydrogen peroxide, which decreased in proportion to the concentrations of analytes in samples. RESULTS: The multianalyte immunosensor array had a throughput of 60 samples/h and allowed simultaneous detection of carcinoembryonic antigen, alpha-fetoprotein, beta-human choriogonadotropin, and carcinoma antigen 125 in clinical serum samples with concentrations up to 188 microg/L, 250 microg/L, 266 IU/L, and 334 kIU/L, respectively. The detection limits (limits of the blank, mean of blank plus 3 SD) were 1.1 micro/L, 1.7 microg/L, 1.2 IU/L, and 1.7 kIU/L. The inter- and intraassay imprecision (CVs) of the immunosensor arrays were <7.8% and <9.0%, respectively. The immunosensor arrays were stable for 28 days. CONCLUSIONS: This newly constructed immunosensor array provides a simple, automated, simultaneous multianalyte immunoassay with high throughput, short analytical time, and sufficiently low detection limits for clinical application. This method offers the capability of miniaturizing the multianalyte detection device.