High-sensitivity Enzyme-linked Immunosorbent Assay Research Articles

Development of ELISA on microplate for serum C-reactive protein and establishment of age-dependent normal reference range

Background: C-reactive protein (CRP), a marker of systemic inflammation, has been proposed to predict outcome in patients with unstable angina; and elevated levels of CRP were found to be associated with an increased risk of coronary events. Methods: Two enzyme-linked immunosorbent assays (ELISA) of different sensitivities were developed on microplate for CRP. Both ELISA established used Dako polyclonal anti-CRP antibody for coating and Dako horse radish peroxidase (HRP)-conjugated polyclonal anti-CRP antibody for detection. Results: The sensitivity of the high and regular sensitivity ELISA was 0.16 and 0.6 mg/l, respectively. Our assays demonstrated an excellent correlation with commercial CRP assays performed on a Behring Nephelometer Analyzer II (BNII) at both regular and ultrasensitive levels, with both correlation coefficients above 0.98 and slopes of approximately 1. Using our microplate assays, we established normal reference value for serum CRP. Based on ANOVA statistical test, we found that the mean±S.D. was 1.3±1.27 mg/l ( n=202) for normal individuals of 50–80 years and 0.43±0.42 mg/l ( n=148) for the group of 20–50 years. Conclusions: The normal serum CRP mean concentrations for two age groups were distinctively different ( p value<0.001). Our study suggests two different normal cutoffs of serum CRP to be employed for individuals in different age groups.

Technical prerequisites for in vivo microdialysis determination of interleukin-6 in human dermis

Cutaneous microdialysis in vivo in human skin is demonstrably of use in the study of skin metabolism, percutaneous absorption and skin inflammation. A promising area for cutaneous microdialysis is the measurement of cytokines. This requires catheters equipped with membranes permeable to molecules of high molecular weight. To address technical problems of poor sample volume retrieval and analysis sensitivity in the simplest model of provocation, namely the insertion of the catheter itself in vivo into human dermis. Use of a polyethylenesulphone membrane, with a cut-off value of 100,000 Da, allowed measurement of target molecules of large molecular weight. Using an adaptation of a commercially available high sensitivity enzyme-linked immunosorbent assay, the ubiquitous proinflammatory cytokine interleukin (IL)-6 was measured in the normal skin of six healthy volunteers after insertion of the microdialysis catheter. Reliable sample volumes and high analyte recovery were achieved either by push-pull pumping or by standard pumping using a perfusate consisting of Ringers Dextran 60 Braun. No IL-6 was detected in 25 of 26 samples taken during the first 100 min after catheter insertion. The IL-6 concentration then increased and remained elevated for the duration of the experiments. Technical and analytical modifications in the microdialysis technique have allowed the measurement of IL-6 in vivo in human dermis. It is suggested that the cytokine production is the result of the dermal trauma caused by catheter insertion, but the cellular source of the IL-6 is at present unknown.

Carotid and femoral atherosclerosis, cardiovascular risk factors and C-reactive protein in relation to smokeless tobacco use or smoking in 58-year-old men.

Objectives. To examine the associations between smokeless tobacco use, smoking, cardiovascular risk factors, inflammation and ultrasound-assessed measures of atherosclerosis in the carotid and femoral arteries. Subjects. The study was performed in a population-based sample of clinically healthy men (n = 391) all 58 years old. Exclusion criteria were cardiovascular or other clinically overt diseases or continuous medication with cardiovascular drugs. Methods. The habits of smoking and oral moist snuff use were assessed by questionnaires. C-reactive protein (CRP) was assessed by high sensitive enzyme-linked immunosorbent assay (ELISA). Intima-media thickness (IMT) in the carotid bulb, the common carotid artery and the common femoral artery and plaque occurrence were measured by ultrasound. Results. The use of oral moist snuff was associated with serum triglycerides and waist-hip ratio (WHR), but not with CRP or ultrasound-assessed measures of subclinical atherosclerosis. Smoking, on the other hand, was associated with CRP, the components in the metabolic syndrome and IMT as well as plaques in the carotid and femoral arteries. In comparison to never-smokers the current smokers had higher values of WHR, triglycerides, C-reactive protein and IMT in carotid bulb and femoral artery. Ex-smokers were in general more obese and had a femoral IMT that was in-between that of never-smokers and current smokers. Conclusions. Tobacco smoking, but not oral moist snuff use, was associated with carotid and femoral artery IMT, and increased levels of CRP. Current smoking was also associated with abdominal obesity. Ex-smokers though, are generally more obese. Smoking was also associated with hyperinsulinaemia, dyslipidaemia and high blood pressure, i.e. the metabolic syndrome. The inhaled smoke from the combustion of tobacco seems to be an important aetiological factor in the atherosclerotic process.

SEASONAL VARIATION IN INTERLEUKIN 4 IN PATIENTS WITH HAYFEVER

This study was performed to investigate the value of interleukin 4 as a marker of activity in mild atopic disease. We compared IL-4 levels to eosinophil cationic protein (ECP), a suggested inflammatory marker in allergic disease, in patients with hayfever. Patients with hayfever were assessed during January and then in late June at the height of the grass pollen season, and their levels of serum ECP and IL-4 compared. Serum ECP was determined by radio-immunoassay and serum IL-4 by a high-sensitivity enzyme-linked immunosorbent assay. ECP was found to increase significantly in patients with hayfever during the grass pollen season (P<0.01). Conversely, serum levels of IL-4 were found to decrease significantly over the same period when compared with winter values. ECP and IL-4 were not seen to correlate significantly with each other. The fall in serum IL-4 seen during the grass pollen season in the hayfever patients may reflect allergen driven upregulation of membrane IL-4 receptor expression or sequestration of cytokine producing cells to inflammatory sites. These findings suggest that serum IL-4 is a poor indicator of inflammatory status in allergic disease.

Role of tumor necrosis factor-α in the differential diagnosis of parapneumonic effusion

Objective: This study was undertaken to investigate the role of tumor necrosis factor-α (TNF-α) in discriminating between uncomplicated parapneumonic effusion (UCPPE) and complicated parapneumonic effusion (CPPE). Method: Using a commercially available high sensitivity enzyme-linked immunosorbent assay (ELISA) kit, concentrations of TNF were measured in the serum (TNFs) and pleural fluid (TNFpf) of 21 patients with parapneumonic effusion (PPE), 13 patients with UCPPE, and 8 patients with nonempyemic CPPE. Results: No significant difference in values of TNF concentration was found between the group with UCPPE and that with CPPE (P > 0.05). Concentration levels of TNFpf were significantly higher in the group with CPPE than in that with UCPPE (P = 0.0008). Levels of TNF in pleural fluid were significantly higher than in serum in both groups (P < 0.001). The ratio of TNF in pleural fluid to that in serum (TNFr) was significantly higher in the CPPE group than in the UCPPE group (P=0.0002). At an optimal cutoff point of 10.7 pg/mL for TNFpf, the sensitivity was 87.5%, specificity was 92.3%, positive predictive value was 87.5%, negative predictive value was 93.3%, and total accuracy was 90.5% (P < 0.001). At an optimal cut-off point of 3.0 for TNFr, all values were 100% (P < 0.00001). Conclusions: The results of this study indicate that TNFpf, and particularly TNFr, may be helpful in discriminating between UCPPE and CPPE. However, further studies are needed in a larger population to confirm these findings.

High sensitivity ELISA determination of taxol in various human biological fluids

Taxol (paclitaxel)-the natural product isolated from Pacific yew ( Taxus brevifolia) is a novel agent with high activity in the treatment of patients with several malignant tumors including those resistant to other cytotoxic drugs. The therapeutic index of this promising anticancer drug could be further increased by the exploration of its pharmacokinetic–pharmacodynamic relationship in cancer patients. Since taxol is highly protein bound, a very specific and highly sensitive analytical method is required in order to determine free, protein unbound and biologically active taxol species in human physiological fluids: plasma; plasma ultrafiltrate; and salivary fluids. In order to accomplish this, a new indirect competitive enzyme-linked immunosorbent assay (ELISA), for quantitating such a low bioactive taxol concentration level, has been developed in our laboratories. This method uses taxol competitive inhibition of mouse anti-taxol antibodies binding to the solid phase coated antigen 7-succinyltaxol-bovine serum albumin. This indicates recognition of the active taxol in the solution phase, where a diluted horseradish peroxidase labeled goat anti-mouse enzyme conjugate is used. While employing this technique, after systematic optimization of the experimental conditions, we are able to detect the anticipated taxol in plasma ultrafiltrate and salivary fluids at the concentration level of subpicogram per milliliter. The working range of the assay is approximately five orders in magnitude, i.e. from pg ml −1 to 100 ng ml −1. The clinical part of this study verified the working range of the ELISA method using samples of physiological fluids from a cancer patient treated with 3 h intravenous (IV) infusion of this drug. Our results of taxol determination in plasma, plasma ultrafiltrate and saliva demonstrate the applicability of the newly developed ELISA method for further pharmacokinetic studies of free, biologically active taxol species in cancer patients.

Serum levels of tumor necrosis factor-alpha are increased in obese patients with noninsulin-dependent diabetes mellitus.

To clarify the significance of the serum levels of tumor necrosis factor-alpha (TNF-alpha) in the mechanism of insulin resistance, we studied 12 obese patients with noninsulin-dependent diabetes mellitus (NIDDM). We evaluated the relationship of TNF-alpha levels with the visceral, subcutaneous, and total fat areas measured by computed tomography (CT), and with insulin resistance evaluated by the glucose infusion rate (GIR) observed during an euglycemic hyperinsulinemic clamp study. Controls consisted of 12 normal subjects and 12 nonobese patients with NIDDM. TNF-alpha levels were measured using a high sensitivity enzyme-linked immunosorbent assay. Following admission, all patients with NIDDM participated in a 4-week program of diet and exercise. After this treatment, we evaluated the relationship of the serum levels of TNF-alpha with the area of body fat, the GIR, and the resultant change in the TNF-alpha level. Serum levels of TNF-alpha in the obese patients with NIDDM significantly exceeded those observed in normal subjects (P < 0.01) or in the nonobese patients with NIDDM (P < 0.01). Serum levels of TNF-alpha in obese NIDDM patients showed a significant positive correlation with the area of visceral fat before (r = 0.662, P < 0.03) and after (r = 0.508, P < 0.05) the treatment; similar correlation was observed in all patients with NIDDM before (r = 0.537, P < 0.02) and after (r = 0.430, P < 0.05) the treatment. Serum levels of TNF-alpha in obese NIDDM patients showed a significant negative correlation with GIR after the treatment (r = -0.595, P < 0.05). Serum levels of TNF-alpha were significantly reduced in the obese patients with NIDDM after the treatment (P < 0.01), while those in the nonobese NIDDM patients were unchanged. These results suggest that serum TNF-alpha levels may play an important role in mechanism of insulin resistance associated with obesity.

The peritoneal fluid levels of interleukin-12 in women with endometriosis.

Interleukin-12 (IL-12) is produced mainly by monocytes/macrophages, and it induces proliferation and cytotoxicity of T-cells and natural killer cells. In women with endometriosis, natural killer cell activity in the peritoneal fluid is significantly decreased. We aimed to measure the peritoneal fluid level of IL-12 in endometriosis. We measured IL-12 levels in peritoneal fluid samples from women with or without endometriosis and in supernatants from endometrial stromal, ovarian stromal, and mesothelial cell cultures, using a high-sensitivity enzyme-linked immunosorbent assay. The median concentration of IL-12 in the peritoneal fluid of women with endometriosis was 1.1 pg/ml (range, 0.2-5.5) and was 1.6 pg/ml (range, 0.4-2.8) in women without endometriosis, not a statistically significant difference. IL-12 was not detected in the supernatants of endometrial stromal, ovarian stromal, and mesothelial cell cultures. Concentrations of IL-12 in the peritoneal fluid of women with or without endometriosis are low, but they are detectable and are not affected significantly by the presence of endometriosis.

High glucose and hyperosmolarity increase secretion of interleukin-1 β in cultured human aortic endothelial cells

Interleukin-1 (IL-1) is secreted by endothelial cells (ECs) and smooth-muscle cells (SMCs), which are two major component cells of vessels and detected in atherosclerotic lesions. To evaluate the effect of hyperglycemia on the secretion of IL-1 β in endothelial cells in diabetic patients, we investigated the effects of high glucose and hyperosmolar conditions on the secretion of IL-1 β from cultured human aortic endothelial cells (HAECs). HAECs were treated with high concentration of glucose or hyperosmolar condition for 3 days. IL-1 β in the supernatant was measured by high sensitive enzyme-linked immunosorbent assay (ELISA). Under high concentration of glucose (16.6 mmol/L) and hyperosmolar condition (glucose 5.5 mmol/L + mannitol 11.1 mmol/L), the secretion of IL-1 β was significantly increased (41.0 ± 2.8 and 26.3 ± 5.9% increase, respectively, compared with that of 5.5 mmol/L glucose). In conclusion, high glucose and hyperosmolar condition increase the secretion of IL-1 β in HAECs. The results suggest that diabetic macroangiopathies might be accelerated partly through the increase of IL-1 β secretion in HAECs.

Interleukin-1β and interleukin-6 are elevated in the cerebrospinal fluid of Alzheimer's and de novo Parkinson's disease patients

Interleukin-1β (IL-1β), interleukin-2 (IL-2), and interleukin-6 (IL-6) were measured in the cerebrospinal fluid (CSF) and plasma of 12 control subjects, 11 sporadic Alzheimer's disease (AD) and 22 de novo Parkinson's disease (PD) patients using high sensitivity enzyme-linked immunosorbent assays (ELISA). IL-1β and IL-6 contents were significantly elevated in the CSF of de novo PD and AD patients in comparison to the control group. In contrast, the plasma levels were not significantly affected. IL-2 contents in the CSF and plasma samples were unchanged in the three groups compared. Because the two cytokines IL-1β and IL-6 are known to play a key role in the interaction between the nervous and immune system, e.g. in the so-called acute phase response, our results support the involvement of immunological events in the complex process of neurodegeneration in AD and PD.

Enzyme-linked immunosorbent assay for determination of plasma thyroglobulin and its application to postmortem diagnosis of mechanical asphyxia

A high sensitive enzyme-linked immunosorbent assay (ELISA) for determination of plasma thyroglobulin was devised and applied to the postmortem diagnosis of mechanical asphyxia. The sensitivity of the present ELISA was 5 ng/ml. All of the 15 victims who died from causes other than mechanical asphyxia showed plasma thyroglobulin levels lower than 200 ng/ml (99.8 ± 37.9 ng/ml), while 12 out of 14 victims of mechanical asphyxia showed the levels higher than 200 ng/ml (2100 ± 3450 ng/ml in 14 victims). Thus, the present ELISA appears to be useful for postmortem diagnosis of mechanical asphyxia.