Sensitive Enzyme Immunoassay Method Research Articles

The efficiency of using maternal markers in early detection and management of congenital anomalies in Majmaah city, KSA

Nowadays the serum analytic investigations have become the first step, noninvasive, detecting method of choice of foetal abnormalities specially for at-risk patients.This study volenteers were Saudi pregnant women in King Khalid General Hospital (KKGH) at Majmaah city, Riyadh, Saudi Arabia.The study intended to judge and evaluate using the concentrations of Beta Human Chorionic Gonadotropin (βHCG) in serum as efficient maternal markers for early detection as well as management of foetal abnormalities and pregnancy complications.A total of 201 serum samples were studied. 101 sera of pregnant women were taken in the first trimester, while the other 100 non-pregnant females were acted as control volenteers. The sensitive Enzyme immunoassay (ELISA) method was applied for quantitative scoring of beta HCG levels. Pregnant women were followed up by a consultant obstetrician till delivery. Trisomy 21 and blighted ovum disorders were selected as cases of interest.Serum βHCG concentrations multiple of median (MoM) for the mothers of affected babies compared with the healthy ones. Defrences were significant as p: value showed 0.001 for each appraisal.Non-pregnant serum beta HCG (2.1 IU/ml) compared with only three weeks of gestation (576 IU/ml) for early detection of pregnancy. Upon comparison a significant high level of β HCG was recorded for the three weeks gestation with (p,value:0.001) which clearly indicates the existence of pregnancy.The use of β HCG could be the method of choice for early detection of pregnancy. This will save the embryo life during the first trimester of gestation especially in the radiology departments.

Development of ochratoxin a in cereal by chemiluminescence enzyme immunoassay

A rapid, simple and sensitive chemiluminescence enzyme immunoassay method (CLEIA) was established to detect ochratoxin A (OTA) in cereal. Optimal conditions including antibody dilution ratio and enzyme conjugate, ionic strength, pH value and organic solvent. Established indirect competition inhibition curve to determine the linear working range, detection limit and recovery rate. Results: The 50% inhibitory concentration and the detection limit of the CLEIA were78.8pg/mL and 14.86 pg/mL, respectively, with a linear range of 0.015-0.4ng/mL. At 1∼4μpg/kg fortified levels in wheat, mean recoveries ranged from 67.47% to100.35%.

The Rapid Screening of Triazophos Residues in Agricultural Products by Chemiluminescent Enzyme Immunoassay.

A highly sensitive chemiluminescent enzyme immunoassay (CLEIA) method was developed in this study for efficient screening of triazophos residues in a large number of samples. Based on the maximum residue limits (MRLs) set by China and CAC for triazophos in different agro-products, the representative apple, orange, cabbage, zucchini, and rice samples were selected as spiked samples, and the triazophos at the concentrations of the MRL values were spiked to blank samples. Subsequently, the five samples with the spiked triazophos standard were measured by CLEIA 100 times, and the detection results indicated that the correction factors of the apple, orange, cabbage, zucchini, and rice were determined as 0.79, 0.66, 0.85, 0.76, and 0.91, respectively. In this experiment, 1500 real samples were detected by both the CLEIA and the GC-MS methods. With the GC-MS method, 1462 samples were identified as negative samples and 38 samples as positive samples. Based on the correction factors, the false positive rate of the CLEIA method was 0.13%, and false negative rate was 0. The results showed that the established CLEIA method could be used to screen a large number of real samples.

Development of a Highly Sensitive Chemiluminescence Enzyme Immunoassay for the Determination of Bisphenol A

A highly sensitive chemiluminescence enzyme immunoassay (CLEIA) method for the determination of bisphenol A (BPA) was developed, which used a secondary antibody labeled with horseradish peroxidase detected with a luminol-based substrate. Under the optimized conditions, the linear range was 0.01μg/mL~0.74μg/mL, and the detection limit was 0.008μg/mL. The average recovery for BPA in barreled water was 104%. This developed method could be applied for the selective, high-throughput, and rapid determination of BPA in barreled water.

The Association Between C-Reactive Protein Levels and Depression: Results from the Northern Finland 1966 Birth Cohort Study

To investigate whether depressive episodes (previous, current single, and recurrent) are associated in both genders with highly sensitive C-reactive protein (hs-CRP) levels, earlier recommended for risk assessment of cardiovascular disease. The impact of the severity of current single and recurrent depressive episodes on this putative association was also investigated. The genetically homogeneous Northern Finland 1966 Birth Cohort was followed until age 31, when, in a cross-sectional setting (n = 5269), the highly sensitive enzyme immunoassay (hs-EIA) method was used to measure CRP concentration. Depressive episodes were defined through mailed questionnaires, including Hopkins Symptom Checklist-25 (HSCL-25) and information on self-reported, doctor-diagnosed depression. After adjusting for confounders, logistic regression analyses showed that in male subjects, elevated hs-CRP levels (> or =1.0 mg/L) increased the probability for severe current and recurrent depressive episodes 1.7-fold and 3.1-fold, respectively. Correspondingly, an hs-CRP level of >3.0 mg/L increased the probability for recurrent depression up to 4.1-fold. In female subjects, no statistically significant associations were found. Our results support the hypothesis that an activation of systemic inflammatory processes may contribute to the pathophysiology of severe depression in men. Further investigations are needed regarding the impact of our findings on diagnostic/treatment strategies concerning severe and, especially recurrent, depression in men.

Development of a highly sensitive, second antibody format chemiluminescence enzyme immunoassay for the determination of 17β-estradiol in wastewater

In the present work, a highly sensitive chemiluminescence enzyme immunoassay (CLEIA) method for the determination of 17β-estradiol (E2) in the wastewater was developed, which used the fluorescein- iso-thiocyanate (FITC)–anti-FITC system as solid phase and the biotin–streptavidin system as signal amplification system, with the highly sensitive 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD)–alkaline phosphatase (ALP) system as chemiluminescence detection system. The specific polyclonal antibody was produced against a conjugate of estradiol–bovine serum albumin (BSA), biotinoyl–diaminodioxaoctane–estradiol conjugate was synthesized. The specificity of anti-E2 antibody was also studied. The results showed that anti-E2 antibody has no cross-reactions for estriol, ethinyl E2, estrone commonly coexisting with E2 in the wastewater. Samples were prepared with solid-phase extraction using C 18 cartridges for the removal of matrix effects. The linear range was 2.5–1600 pg ml −1 and detection limit was 1.5 pg ml −1. As our known of knowledge, the method was more sensitive for the determination of E2 in water compared with other immunoassay methods. Recoveries were in the range of 80–110% in spiked tap water and wastewater. This method has been successfully used for the detection of E2 in wastewater, compared with the commercially radioimmunoassay (RIA) kit, the correlation was good.

12 Reduction of Urinary PGE2 Induced by Ibuprofen Treatment in Preterm Infants with Patent Ductus Arteriosus.

Background: Patent ductus arteriosus (PDA) is a common finding among preterm infants. Hemodynamically significant PDA requires medical treatment with cyclooxygenase inhibitors such as indomethacin or ibuprofen (IBU). IBU has been shown to be as effective as indomethacin in ductal closure, with fewer adverse effects.Aim: To investigate urinary excretion changes of PGE2 induced by IBU treatment in preterm infants with PDA.Methods: Twenty preterm infants (gestational age, 24 to 32 weeks; birth weight, 640 to 1820 g) with a significant PDA at 48–72 hours of age were enrolled. Two serial urine samples were noninvasively collected in each patient, by a cotton wool ball method, just before and 12–24 hours after a conventional course of IBU treatment, respectively. Urinary PGE2 concentrations were measured by a sensitive and accurate enzyme immunoassay (EIA) method (Cayman Chem, Ann Arbor, MI, USA). Furthermore, serum creatinine and other renal function parameters were assessed before and after IBU treatment.Results: Mean urinary PGE2 concentrations decreased significantly after IBU treatment (from baseline values of 66.95+/-16.78 to 27.15+/-17.92 pg/mL, P<0.001). Post-treatment urinary levels of PGE2 particularly low (<5 pg/mL) were found in two patients, who developed clinically relevant manifestations (intraventricular hemorrhage, acute renal failure, and intestinal perforation). Serum creatinine concentration increased from baseline value in 7 of 20 patients, 4 of them having an increase of >20%, after IBU treatment.Conclusions: The adverse effects of cyclooxygenase inhibitors may involve different organs of newborn, but particularly the kidney, which is considered prostaglandin-dependent. Although IBU represents the most safe drug in ductal closure, it induced a significant reduction of urinary PGE2 concentrations in our study population. In newborns with relevant side effects this reduction was dramatic. Further studies are needed to confirm these data and to determine whether urinary PGE2 may represent an index of toxicity in clinical settings.

Adrenomedullin release in the rat mesenteric resistance artery

Adrenomedullin (AM) is a potent vasodilator peptide whose major source is the vascular wall. In the present study, the mechanism of release of AM was investigated in the rat mesenteric resistance artery. The isolated mesenteric vascular bed was perfused with Krebs solution at a constant flow rate (5 ml/min) and AM in the perfusate was measured by a highly sensitive enzyme immunoassay (Immunoenzymometric assay; IEMA) method. In preparations without endothelium, spontaneous release of AM was detected in the perfusate (68.7 ± 5.8 fmol/ml, n = 45). Periarterial nerve stimulation (PNS, 4 and 8 Hz) caused 11.4 ± 3.9% (4 Hz) and 9.1 ± 3.5% (8 Hz) decreases in the spontaneous release of AM. Removal of Ca 2+ from the medium did not affect the spontaneous AM release, but abolished the PNS-induced inhibition of spontaneous AM release. Perfusion of 10 nM calcitonin gene-related peptide (CGRP) or 0.1 μM capsaicin (inducer of CGRP release) inhibited significantly the spontaneous AM release. PNS (8 Hz)-induced inhibition of spontaneous AM release was antagonized by CGRP(8–37) (CGRP receptor antagonist). These results suggest that AM is mainly released from vascular smooth muscle cells of the rat mesenteric artery and endogenous or exogenous CGRP inhibits AM release.

Effects of Sexually Activated Rams or Ewes on Pulsatile LH Secretion in Anoestrous Sheep

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CSF Aβ40 and Aβ42: Natural course and clinical usefulness

without CNS disease (CO) in whom total apolipoprotein E (ApoE), total tau protein (TT), Aβ42 peptide, interleukin-6 (IL-6), and substance P (SP) were measured in lumbar CSF samples using sensitive and specific enzyme immunoassay methods (ELISA). TT was highly significantly increased ( P< 0.0001) and Aβ42 was significantly decreased ( P< 0.001 vs OND/CO; p< 0.03 vs NAD) in the AD cohort compared with the other diagnostic groups. Significant increase in AD was found for ApoE ( p< 0.001) and IL-6 ( p< 0.03), but there was a considerable overlap between groups.

Risk of Occupational Exposure to &lt;I&gt;Cryptosporidium, Giardia,&lt;/I&gt; and &lt;I&gt;Campylobacter&lt;/I&gt; Associated with the Feces of Giant Canada Geese

Giant Canada geese (Branta canadensis maxima), which are nesting locally in Northwest Ohio and other parts of the state, are commonly perceived as a public nuisance when they inhabit urban areas. The feces of giant Canada geese litter both grass and pavement in many occupational and recreational sites in the Toledo area. The purpose of this study was to identify sites with fecal droppings of giant Canada geese that test positive for Cryptosporidium, Giardia, and Campylobacter, qualitatively assess the occupational risks of infections, and recommend protective measures. The fecal droppings of giant Canada geese were tested for Cryptosporidium, Giardia, and Campylobacter, using sensitive monoclonal enzyme immunoassay (EIA) methods. Fourteen out of 16 sites tested positive for at least one pathogen. None tested positive for all three. Cryptosporidium was the most common infectious organism found in the fecal droppings. It was detected in 14 out of 18 (77.8%) samples. Campylobacter was found in 7 out of 18 (3...

Risk of Occupational Exposure toCryptosporidium, Giardia,andCampylobacterAssociated with the Feces of Giant Canada Geese

Giant Canada geese (Branta canadensis maxima), which are nesting locally in Northwest Ohio and other parts of the state, are commonly perceived as a public nuisance when they inhabit urban areas. The feces of giant Canada geese litter both grass and pavement in many occupational and recreational sites in the Toledo area. The purpose of this study was to identify sites with fecal droppings of giant Canada geese that test positive for Cryptosporidium, Giardia, and Campylobacter, qualitatively assess the occupational risks of infections, and recommend protective measures. The fecal droppings of giant Canada geese were tested for Cryptosporidium, Giardia, and Campylobacter, using sensitive monoclonal enzyme immunoassay (EIA) methods. Fourteen out of 16 sites tested positive for at least one pathogen. None tested positive for all three. Cryptosporidium was the most common infectious organism found in the fecal droppings. It was detected in 14 out of 18 (77.8%) samples. Campylobacter was found in 7 out of 18 (38.9%) samples, and 3 out of 18 (16.7%) samples tested positive for Giardia. Since fecal droppings of giant Canada geese are dense in many sites, occupational exposure to Cryptosporidium is very plausible. In addition, fecal droppings from other carrier vertebrates are likely to be present in the same sites occupied by giant Canada geese, thereby increasing the likelihood of occupational exposure to one or more of these pathogens. It has also been suggested that houseflies and dung beetles may be mechanical carriers of Cryptosporidium. We recommend that work environments in close proximity to the nesting sites of giant Canada geese be maintained in a sanitary condition. Workers at risk for exposure should wear protective gloves while working, wash their hands after performing applicable activities and before touching their mouths, launder work clothes daily, and, ideally, shower at the end of the workday. We further recommend that potentially exposed workers who develop gastrointestinal infections have their stools tested for Cryptosporidium, Giardia, and Campylobacter.

Clinical significance of neurobiochemical profiles in the lumbar cerebrospinal fluid of Alzheimer's disease patients.

Immunoreactivities of total apolipoprotein E (ApoE-IR), amyloid beta peptide(1-42) (Abeta42-IR), interleukin-6 (IL-6-IR), substance P (SPIR) and total tau protein (TTIR) were measured in lumbar cerebrospinal fluid samples of patients with Alzheimer's disease (AD), non-Alzheimer's dementias (NAD), neurological disorders without cognitive impairment (OND) and controls without central nervous system disease using sensitive and specific enzyme immunoassay methods. TTIR was highly significantly increased (P < 0,001) and Abeta42-IR was significantly decreased (P < 0,001 vs. OND/CO, P < 0,03 vs. NAD) in the AD cohort compared with the other diagnostic groups. Significant increases in AD were also found for ApoE-IR (P < 0,001) and IL-6 (P < 0,03), but there was a considerable overlap between groups. In the total AD cohort, SPIR was not significantly changed, but AD patients with late disease onset (>65 years) showed significantly higher values than both early onset patients (<65 years) and controls (P < 0,05). Discriminant function analysis showed that Abeta42-IR (cut-off value 375pg/ml) and TTIR (cut-off value 440 pg/ml) levels contributed most to the group classification of patients. At 85% sensitivity for AD and 100% specificity for controls, the combined evaluation of Abeta42-IR and TTIR in this cross-sectional study resulted in a graph separating AD from non-AD patients with increased specificity of 91% and 75% for AD versus OND and NAD, respectively.

Higher concentrations of matrix metalloproteinases in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome.

This study was designed to investigate possible involvement of type IV collagenolytic matrix metalloproteinases (MMPs; 72-kDa type IV collagenase [MMP-2], 92-kDa type IV collagenase [MMP-9]), and the respective specific tissue inhibitors of these MMPs (TIMP-2 and TIMP-1) in the development of adult respiratory distress syndrome (ARDS). We determined the concentrations of these enzymes in the bronchoalveolar lavage fluid (BALF) from patients with ARDS using newly developed sensitive one-step sandwich enzyme immunoassay methods. BALF obtained from the 17 patients and eight healthy volunteer control subjects were also used for the analysis of the number of the cellular component. Concentrations of the 7S portion of type IV collagen and laminin in the BALF were measured as markers of basement membrane disruption. In the BALF from the ARDS patients, the concentrations of MMP-2 (66.7 +/- 57.0 ng/ml versus < 7.0 ng/ml for controls, p < 0.01) and MMP-9 (118.0 +/- 309.3 ng/ml versus 9.0 +/- 9.5 ng/ml for controls, p < 0.05), and the specific inhibitor of MMP-9 (TIMP-1) (161.0 +/- 145.0 ng/ml versus < 50 ng/ml for controls, p < 0.01) were significantly higher compared with those for healthy control subjects. In the ARDS patients, the concentrations of MMP-2 correlated both with those of 7S collagen and laminin; MMP-9 with the concentration of 7S collagen and the number of neutrophils. These findings suggest that the increased concentration of collagenolytic MMPs in lung plays a role in the pathogenesis of ARDS.

A reliable and sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F2α: a marker for lipid peroxidation after experimental brain injury

The objectives of this study were to determine (1) if levels of 8-isoprostaglandin F2α (8-isoPGF2α), a non-enzymatically produced prostaglandin, were increased after cortical contusion and (2) if enzyme immunoassay (EIA) could be used to quantify 8-isoPGF2α levels. 24 h after the contusion there was a significant rise in 8-isoPGF2α compared to control levels. The levels returned to baseline by 72 h postinjury. The results show that this EIA method is a reliable and sensitive technique for measuring brain lipid peroxidation.

A reliable and sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F 2α: a marker for lipid peroxidation after experimental brain injury

The objectives of this study were to determine (1) if levels of 8-isoprostaglandin F 2α (8-isoPGF 2α), a non-enzymatically produced prostaglandin, were increased after cortical contusion and (2) if enzyme immunoassay (EIA) could be used to quantify 8-isoPGF 2α levels. 24 h after the contusion there was a significant rise in 8-isoPGF 2α compared to control levels. The levels returned to baseline by 72 h postinjury. The results show that this EIA method is a reliable and sensitive technique for measuring brain lipid peroxidation.

Significance and Immunoassay of 9- and 13-Hydroxyoctadecadienoic Acids

Linoleic acid, the predominant polyunsaturated fatty acid in the diet, can be metabolized by cyclooxygenase, lipoxygenase and P450 enzymes. The monohydroxy lipoxygenation products of linoleic acid, 9- and 13-hydroxyoctadecadienoic acids (9(S)- and 13(S)-HODEs), are the most widely distributed of the known linoleic acid metabolites. These compounds exhibit interesting biological activities, including regulation of platelet function, maintenance of vascular thromboresistance and transduction of the cellular responses to certain growth factors. In view of their biological significance, we have produced polyclonal antibodies for the first time to these bioactive lipids to develop an easy, inexpensive, sensitive, specific and rapid enzyme immunoassay method for these bioactive lipids.

Creatine kinase isoenzyme BB is associated with the estrogen receptor in breast cancer

A sensitive sandwich-type enzyme immunoassay method for measurement of brain-type isozyme of human creatine kinase (CK-BB) was developed using purified antibodies specific to the B subunit. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labelled with β-D-galactosidase from Escherichia coli. The assay was highly sensitive and 1 pg of CK-BB was measurable. The assay was specific to the B subunit of creatine kinase (CK-B), and it cross-reacted about 25% with CK-MB, the heart-type isozyme. However, the assay showed no cross-reactivity with CK-MM, the muscle type-isozyme or with neuron-specific αα enolase. Coefficients of variation in withinrun and between-run precision studies for serum CK-B were < 8%. Serum CK-B levels in healthy adults of various ages (16–59 yr old) ranged from 0.25–1.44 ng/ml, whereas the CK-B concentrations in children (< 10 yr old) were relatively high, ranging from 1.3–7.4 ng/ml. The CK-B levels in the cerebrospinal fluids (CSF) could be determined by the present method, and they ranged from 0.10–0.76 ng/ml in the samples from patients with non-neuronal disorders. Determination of immunoreactive CK-B in the extracts of various human tissues confirmed previous reports that CK-B was distributed at high concentrations in the central nervous tissue, prostate, uterus, bladder, gastrointestinal tract and heart muscle.

Detection of prostate specific antigen in breast tumors

A sensitive sandwich-type enzyme immunoassay method for measurement of brain-type isozyme of human creatine kinase (CK-BB) was developed using purified antibodies specific to the B subunit. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labelled with β-D-galactosidase from Escherichia coli. The assay was highly sensitive and 1 pg of CK-BB was measurable. The assay was specific to the B subunit of creatine kinase (CK-B), and it cross-reacted about 25% with CK-MB, the heart-type isozyme. However, the assay showed no cross-reactivity with CK-MM, the muscle type-isozyme or with neuron-specific αα enolase. Coefficients of variation in withinrun and between-run precision studies for serum CK-B were < 8%. Serum CK-B levels in healthy adults of various ages (16–59 yr old) ranged from 0.25–1.44 ng/ml, whereas the CK-B concentrations in children (< 10 yr old) were relatively high, ranging from 1.3–7.4 ng/ml. The CK-B levels in the cerebrospinal fluids (CSF) could be determined by the present method, and they ranged from 0.10–0.76 ng/ml in the samples from patients with non-neuronal disorders. Determination of immunoreactive CK-B in the extracts of various human tissues confirmed previous reports that CK-B was distributed at high concentrations in the central nervous tissue, prostate, uterus, bladder, gastrointestinal tract and heart muscle.

Reduced concentrations of the alpha-subunit of GTP-binding protein Go in schizophrenic brain.

Concentrations of the alpha-subunits of GTP-binding protein, Go (Go alpha) and of Gi2 (Gi2 alpha) in 6 areas (the hippocampus, parahippocampus, putamen, caudate head, orbital frontal cortex, and lateral temporal cortex) of control and schizophrenic postmortem brains were investigated using the highly sensitive enzyme immunoassay method. There was a significant decrease in Go alpha in the hippocampus and caudate head of the right hemisphere in schizophrenic patients compared to controls; the ANOVA (a general linear model; SAS Type II) demonstrated a significant diagnosis x side interaction only in the hippocampus. In other areas of the brain, analysis by grouping under diagnosis, side, age, gender, and postmortem delay showed no significant deviations in Go alpha between controls and schizophrenics. The concentrations of Gi2 alpha did not differ significantly in any area. These findings contrasted with the results yielded by ADP-ribosylation, which showed decreased pertussis toxin ADP-ribosylated amounts in the hippocampus and putamen of the contralateral (left) hemisphere. Some abnormal receptor-Go or Gi 1 signalling in hippocampus, putamen or caudate head may be involved in the pathogenesis of schizophrenia.