Competitive Chemiluminescent Immunoassay Research Articles

Determination of tetraiodo-l-thyronine in human serum by competitive indirect chemiluminescence immunoassay

Detection of T4 by the established CLEIA method.

Indirect Competitive Chemiluminescent Enzyme Immunoassay Method for Determination of Dimethyl Phthalate in Soy Sauce and Liquor

Abstract 4-Amino dimethyl phthalate (ADMP) as hapten was coupled to carrier protein and subsequently used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to dimethyl phthalate (DMP) was obtained, and on the basis of this, an indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) was developed. The experimental parameters for icCLEIA were optimized as follows: the concentration of coating antigen was 50 μg L −1 , the primary antibody concentration was 92.5 μg L −1 , the secondary antibody concentration was 1 μg mL −1 , ultrapure water (pH 6.0) was used as dilution solution and the time for competitive reaction was 40 min. Under the optimal conditions, the icCLEIA exhibited a linear working range from 0.74 μg L −1 to 30.32 μg L −1 , with the limit of detection of 0.29 μg L −1 . The cross-reactivity with thirteen structural analogues was less than 5%. The recoveries of DMP from spiked liquor and soy sauce samples were from 80.2% to 116% and the average RSD was less than 3.6%. The detection results of spiked liquor and soy sauce by icCLEIA were consistent with those by standard GC-MS method. The developed icCLEIA method exhibited a practical potential for detecting DMP residue in food samples.

Cod liver oil consumption at different periods of life and bone mineral density in old age.

Cod liver oil is a traditional source of vitamin D in Iceland, and regular intake is recommended partly for the sake of bone health. However, the association between lifelong consumption of cod liver oil and bone mineral density (BMD) in old age is unclear. The present study attempted to assess the associations between intake of cod liver oil in adolescence, midlife, and old age, and hip BMD in old age, as well as associations between cod liver oil intake in old age and serum 25-hydroxyvitamin D (25(OH)D) concentration. Participants of the Age, Gene/Environment Susceptibility-Reykjavik Study (age 66-96 years; n 4798), reported retrospectively cod liver oil intake during adolescence and midlife, as well as the one now in old age, using a validated FFQ. BMD of femoral neck and trochanteric region was measured by volumetric quantitative computed tomography, and serum 25(OH)D concentration was measured by means of a direct, competitive chemiluminescence immunoassay. Associations were assessed using linear regression models. No significant association was seen between retrospective cod liver oil intake and hip BMD in old age. Current intake of aged men was also not associated with hip BMD, while aged women with daily intakes had z-scores on average 0.1 higher, compared with those with an intake of < once/week. Although significant, this difference is small, and its clinical relevance is questionable. Intake of aged participants was positively associated with serum 25(OH)D: individuals with intakes of < once/week, one to six time(s)/week and daily intake had concentrations of approximately 40, 50 and 60 nmol/l respectively (P for trend < 0.001).

Abstract P3-07-01: Circulating vitamin D concentrations and breast cancer risk: A pooled analysis of 17 cohorts

Abstract Optimizing vitamin D status through the use of supplementation and/or judicious sun exposure has been proposed as a BC risk reduction strategy. This simple and non-invasive approach to BC prevention is extremely appealing given that vitamin D concentrations in circulation are low in many individuals around the world. However, an Institute of Medicine (IOM) report found the evidence on vitamin D and breast cancer to be inconsistent. To clarify the relationship between vitamin D and breast cancer, data was pooled on approximately 25,000 women from 17 prospective cohorts worldwide. The association between pre-diagnostic circulating 25(OH)D levels, the accepted measure of vitamin D status, and breast cancer incidence was examined. For five cohorts, vitamin D status was assessed at a central laboratory (Heartland Assays, Inc.) using a direct, competitive chemiluminescence immunoassay that measures 25(OH)D2 and 25(OH)D3 equivalently. In 12 cohorts with previously measured 25(OH)D levels, a stratified sample of 29 controls was re-assayed at Heartland Assays and used to calibrate existing levels to a central assay using robust linear regression analyses. We standardized 25(OH) D levels for season using a periodic sine/cosine function. Conditional logistic regression analyses were performed in each study and were then pooled to generate pooled odds ratios by study-specific quantiles, consortium wide-quantiles, absolute cut points based on IOM guidance. Our preliminary analyses included 10,353 cases of incident invasive breast cancer (5305 estrogen receptor (ER) positive cases and 1311 (ER) negative cases and 12,313 matched controls. Median calibrated 25(OH)D levels in controls varied from 33 to 70 nmol/L across the cohorts. The consortium-wide median 25(OH)D among controls was 22% higher in summer as compared to winter months. Across all studies, median age at blood draw was 41 to 70 years; and median elapsed time from blood draw to diagnosis ranged from 2 to 13 years. The pooled odds ratio of breast cancer, comparing the highest to lowest study-specific 25(OH)D quintile, was 0.99 (95% confidence interval 0.90-1.08) after adjusting for body mass index, physical activity, menopausal status, menopausal hormone therapy use, parity/age at first birth, and family history of breast cancer. Results were not significantly different in analyses stratified by age of diagnosis (&amp;lt;50, 50-60, 60+ y).or by ER status. When calibrated circulating 25(OH)D levels were categorized based on the IOM definitions of "deficiency", "inadequacy", "adequacy", and "beyond adequacy", risk was similar across the categories. Further analyses are ongoing to examine especially low and high 25(OH)D concentrations, whether the vitamin D association varies according to tumor characteristics, the importance of elapsed time between blood draw and diagnosis. These will be completed before the meeting. In conclusion, preliminary results from the largest pooled analysis of prospective studies to date show no association between 25(OH)D levels and breast cancer risk and therefore suggest that increasing Vitamin D levels may not be an effective risk reduction strategy for breast cancer. Citation Format: Kala Visvanathan, Alison Mondul, Anne Zeleniuch-Jacquotte, Toqir K Mukhtar, Stephanie A Smith-Warner, Regina G Ziegler, On Behalf of Investigators in the Vitamin D Pooling Project of Breast and Colorectal Cancer. Circulating vitamin D concentrations and breast cancer risk: A pooled analysis of 17 cohorts [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-07-01.

A sensitive chemiluminescent immunoassay for point-of-care testing of repaglinide in natural dietary supplements and serum.

For point-of-care testing of the illegal fortification of repaglinide (Rep) in natural dietary supplements, a competitive chemiluminescent immunoassay (CLIA) was established, using a horseradish peroxidase (HRP)-luminol-H2O2 system for signal amplification. Polyclonal antibodies for Rep were produced via immunization technique. Following optimization of the enzyme reaction time and concentrations of antibody and coating antigen, the method showed a limit of quantification (LOQ) of 1.0ng/mL in PBS and limit of detection (LOD) of 8.3ng/mL in serum and 6.0ng/mL in blank tablets. When applied in natural dietary supplements, the method provided results consistent with those from HPLC, suggesting that the proposed method could be used for rapid screening of Rep in natural dietary supplements and detecting Rep in serum after administration.

Evaluate the Effect of Valproate Monotherapy on the Serum Homocysteine, Folate and Vitamin B12 Levels in Epileptic Children.

The data regarding Valproate and its influence on serum folate and homocysteine levels are conflicting. The aim of this study was to evaluate whether differences exist in homocysteine, folate, and vitamin B12 levels in children receiving Valproate. A total of 55 newly diagnosed epileptic children with ages ranging from 2 to 15 years were enrolled at the start of study but after 3 months follow up, the total sample size finally was only 50 epileptic children. 5 children dropped out of study due to poor follow up. 50 age and gender matched healthy control subjects were also studied on enrollment at the start of study. Serum homocysteine levels were analyzed by enzyme immunoassay method using the kits provided by Axis-Shield Diagnostics Ltd (Dundee DD2 1XA, United Kingdom). Serum folate and serum vitamin B12 were estimated by Competitive Chemiluminescent Enzyme Immunoassay method. The serum homocysteine level in epileptic children was found to be significantly increased after Valproate monotherapy as compared to before therapy. Moreover, a highly significant decrease was observed in the levels of serum folate in epileptic children after Valproate monotherapy as compared to before therapy. But a non significant difference was observed in serum vitamin B12 levels in epileptic children before and after Valproate monotherapy. Thus, we conclude that there is a significant increase in the levels of homocysteine and a significant decrease in the concentration of serum folate while vitamin B12 decreases non-significantly after Valproate monotherapy. The atherogenic effect of increased serum homocysteine level is well established; the patients under Valproate monotherapy should be monitored for possible atherogenic effects. Considering the above observation and results of children undergoing Valproate monotherapy, these children should be screened for levels of serum homocysteine, folate, and vitamin B12 and treated when their levels are found to be disturbed.

Lower serum 25-hydroxyvitamin D level is associated with impaired myocardial performance and left ventricle hypertrophy in newly diagnosed hypertensive patients.

Objective:Vitamin D deficiency is an independent risk factor for cardiovascular mortality. The relationship between vitamin D level and left ventricle (LV) myocardial performance index (MPI=Tei index), which incorporates both LV systolic function and diastolic function, was not investigated in previous studies. We hypothesized that vitamin D level may be associated with LV function and geometry. We aimed to investigate the association between serum 25-hydroxyvitamin D (25 [OH] D) levels and MPI and LV hypertrophy in hypertensive patients with newly diagnosed and preserved ejection fraction.Methods:We studied 151 sequential newly diagnosed hypertensive subjects who lived in the Çukurova region without known cardiovascular risk factors or overt heart disease (mean age: 62.8±10.4 years). Serum 25 (OH) D was measured using a direct competitive chemiluminescent immunoassay. The patients were divided into two groups according to serum 25 (OH) D level: vitamin D-non-deficient group (vitamin D> 20.00 ng/mL, n=53) and vitamin D-deficient group (vitamin D< 20.00 ng/mL, n=98). MPI was defined as the sum of isovolumic contraction and relaxation times divided by the ejection time. LV mass index (LVMI) was calculated by using the Devereux formula and body surface area.Results:MPI and LVMI values were lower and low-density lipoprotein (LDL) levels were higher in patients who were vitamin D-non-deficient than patients who were vitamin D-deficient (p<0.05 for all). Multivariate linear regression analysis showed that serum 25 (OH) D was indepen-dently associated with MPI (β=-0.426, p<0.001), LVMI (β=-0.345, p=<0.001), and LDL (β=0.140, p<0.026).Conclusion:Lower serum 25 (OH) D levels are significantly associated with impaired myocardial performance and LVMI.

Simultaneous detection of forbidden chemical residues in milk using dual-label time-resolved reverse competitive chemiluminescent immunoassay based on amine group functionalized surface.

In this study, a sensitive dual-label time-resolved reverse competitive chemiluminescent immunoassay was developed for simultaneous detection of chloramphenicol (CAP) and clenbuterol (CLE) in milk. The strategy was performed based on the distinction of the kinetic characteristics of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) in chemiluminesecence (CL) systems and different orders of magnitude in HRP CL value for CAP and ALP CL value for CLE in the chemiluminescent immunoassay. Capture antibodies were covalently bound to the amine group functionalized chemiluminescent microtiter plate (MTP) for efficient binding of detection antibodies for the enzymes labeled CAP (HRP-CAP) and CLE (ALP-CLE). The CL signals were recorded at different time points by the automatic luminometers with significant distinction in the dynamic curves. When we considered the ALP CL value (about 105) of CLE as background for HRP CL signal value (about 107) of CAP, there was no interaction from ALP CL background of CLE and the differentiation of CAP and CLE can be easily achieved. The 50% inhibition concentration (IC50) values of CAP and CLE in milk samples were 0.00501 µg L−1 and 0.0128 µg L−1, with the ranges from 0.0003 µg L−1 to 0.0912 µg L−1 and from 0.00385 µg L−1 to 0.125 µg L−1, respectively. The developed method is more sensitive and of less duration than the commercial ELISA kits, suitable for simultaneous screening of CAP and CLE.

Evaluation and validation of biochip multi-array technology for the screening of six families of antibiotics in honey according to the European guideline for the validation of screening methods for residues of veterinary medicines

The main chemicals used against varoa are acaricides, and the antibiotics used for the control of bee bacterial diseases are mainly tetracyclines, streptomycins, sulfonamides and chloramphenicol. No maximum residue limits (MRLs) have been set for any antibiotics in honey. Therefore, in the European Union, minimum recommended concentrations (RC) for the analytical performance of methods to control a certain set of these non-authorised chemicals in honey were published by the European Union Reference Laboratory (EU-RL) in 2007. Concerning the strategy for the control for antibiotic residues in honey, there is still a great need for a cheap and single multi-residue method. Biochip array technology is an innovative assay technology for the multi-analyte screening of biological samples in a rapid and easy-to-use format. A multi-array system, called Evidence Investigator™ (Randox, Crumlin, Co., Antrim, UK), was evaluated in our laboratory. It is a semi-automated biochip system designed for research, clinical applications and veterinary use. A competitive chemiluminescent immunoassay is employed for the detection of antimicrobials. The MicroArray II kit (AM II) dedicated to the screening of six different families of antibiotic residues was validated according to the European guideline for the validation of screening methods for residues of veterinary medicines. The specificity was proven to be very satisfactory, and applicability to different kinds of honey was demonstrated. The detection capabilities (CCβ) of six antibiotic residues were determined and were below the RCs when exist. The AM II kit could detect at least six quinolones, four tetracyclines and three epimers, three aminoglycosides, three macrolides, thiamphenicol, florfenicol and ceftiofur along with one of its stabilised metabolites, the desfuroylceftiofurcysteine disulfide (DCCD).

Serum 25-Hydroxyvitamin D Level and Aortic Intima-Media Thickness in Patients Without Clinical Manifestation of Atherosclerotic Cardiovascular Disease.

Existing evidence suggests that impaired vitamin D metabolism contribute to the development of atherosclerosis. Aortic intima-media thickness (IMT) is an earlier marker than carotid IMT of preclinical atherosclerosis. However, there is a lack of researches on direct investigation of relevance between serum 25-hydroxyvitamin D (25(OH)D) and thoracic aortic IMT. In this study, we aimed to assess the relationship between thoracic aortic IMT and 25(OH)D. We studied 117 patients (mean age: 45.5 ± 8.4 years) who underwent transesophageal echocardiography (TEE) for various indications. Serum 25(OH)D was measured using a direct competitive chemiluminescent immunoassay. The patients were divided into three groups according to the their serum 25(OH)D levels (VitDdeficiency , VitDinsufficient and VitDnormal groups). TEE was performed in all subjects. High sensitive C-reactive protein (hsCRP) and other biochemical markers were measured using an automated chemistry analyzer. Only 24.8% (29 patients) of patients had normal levels of 25(OH)D. The highest aortic IMT values were observed in VitDdeficiency group compared with VitDinsufficient and VitDnormal groups (P < 0.05, for all). Also aortic IMT values of VitDinsufficient group were higher than VitDnormal group (P < 0.05). 25(OH)D was independently associated with hs-CRP (β = -0.442, P < 0.001) and aortic IMT (β = -0.499, P < 0.001). The lower 25(OH)D level was independently associated with higher aortic IMT values. Therefore, hypovitaminosis D may have a role on pathogenesis of subclinical thoracic atherosclerosis.

Detection of 3,5-Diiodothyronine in Sera of Patients with Altered Thyroid Status Using a New Monoclonal Antibody-Based Chemiluminescence Immunoassay

3,5-Diiodo-L-thyronine (3,5-T2), a potential metabolite of 3,3',5-triiodothyronine (T3), exerts marked metabolic actions without the undesirable cardiac and central side effects of T3. So far the lack of reliable quantification methods for endogenous 3,5-T2 in human serum has limited further insight into its physiological and pathophysiological roles in endocrine homeostasis and disease status. Monoclonal anti-3,5-T2 antibodies (3,5-T2 mAbs) were produced in mice. We developed a competitive chemiluminescence immunoassay (CLIA) with one selected mAb and optimized it for high sensitivity, linearity, recovery, and low cross-reactivity to structurally related thyroid hormones (THs) and thyronamines. The CLIA was then used to investigate the origin and action of 3,5-T2 in humans under physiological and pathophysiological conditions in comparison with THs. Patient analysis included individuals with confirmed hypo- or hyperthyroidism and a separate population of thyroidectomized patients on L-thyroxine (T4) replacement therapy. 3,5-T2 is stable in human serum after storage at 4°C or room temperature as well as several freeze-thaw cycles. The immunoassay did not show any significant cross-reactivity with naturally occurring TH metabolites in physiological and pathophysiological concentrations. The assay shows a lower detection limit of 0.2 nM 3,5-T2 and an upper detection limit of 10.0 nM. The newly established CLIA generates reliable results after spiking exogenous 3,5-T2 or by linear dilution of sera. Intra-assay variation is between 4.1% and 9.0%. Overall mean of variation between different assays is 5.6%-12.9%. 3,5-T2 serum concentrations do not differ in hyperthyroid (0.31 ± 0.02 nM, n=24) compared to hypothyroid (0.43 ± 0.04 nM, n=31) individuals. 3,5-T2 was detectable and elevated in serum from thyroidectomized and T4-substituted patients (0.48 ± 0.03 nM, n=100) in comparison to a sex- and age-matched control group (0.29 ± 0.01 nM, n=99). The established CLIA is highly specific, sensitive, precise and accurate for 3,5-T2 detection in human serum. Because 3,5-T2 is not regulated in conditions of an altered thyroid state, it is most likely that serum 3,5-T2 concentrations are not directly dependent on feedback regulation via the hypothalamic-pituitary axis. In addition 3,5-T2 is present in thyroidectomized individuals on T4 substitution, and it is elevated after T4 substitution compared with healthy controls. We conclude that these data support extrathyroidal production of 3,5-T2 from T4.

Speed Of Bone Accretion Assessed by Dual X-Ray Absorptiometry (DXA) and Quantitative Ultrasound (QUS) in Mexican Health Workers Cohort Study

s 405 Advantage competitive binding chemiluminescence immunoassay (reference range 10-68 ng/mL, intra-assay precision 3.9% at 20.3 ng/mL, 3.0% at 49.9 ng/ mL, and 3.6% at 57.8 ng/mL, lower limit of sensitivity 7 ng/mL; Quest Diagnostics Clinical Trials Laboratory Test Development Department, Van Nuys, CA, USA) from a central laboratory. Lumbar spine (L2-L4) BMD was measured by DXA (GE Healthcare Lunar Prodigy). Lumbar spine TBS was derived from DXA lumbar spine examinations. Results: Mean serum (25OHD) level was 27.2 12.2 ng/ml and mean BMI was 26.9 5.2 kg/m. Age, weight, height and BMI were not correlated to serum (25OHD). Age, weight, height, BMI and serum (25OHD) were not correlated to TBS. Conclusion: These results suggest that serum (25OHD) is not a determinant of TBS in postmenopausal women. Further research is needed to identify the determinants of TBS in postmenopausal women. Disclosure of Interest: None Declared

AB0814 Cross Sectional Study of Hypovitaminosis D in among Rheumatology Patients in Brunei

BackgroundVitamin D deficiency is increasingly recognised as a global health problem. The important role of vitamin D in skeletal health is well established. Studies have shown vitamin D deficiency in...

An imbalance in serum concentrations of inflammatory and anti-inflammatory cytokines in hypertension

Hypertension is an important risk factor for cardiovascular disease and there is increasing evidence that inflammation and abnormal immune responses are involved in the pathogenesis of hypertension. However, the data on the association between specific cytokine concentrations and hypertension are inconsistent. We have evaluated the association between 12 cytokines/growth factors and the presence of different degrees of hypertension, comparing these concentrations to values in a healthy group of subjects. The concentrations of interleukin (IL)-1α, -1β, -2, -4, -6, -8, -10, tumor necrosis factor (TNF-α), interferon-γ (IFN-γ), monocyte chemoattractant protein (MCP-1), epidermal growth factor, and vascular endothelial growth factor were measured in 155 hypertensive patients and 148 healthy subjects, using EV-3513 cytokine biochip arrays, a competitive chemiluminescence immunoassay. Univariate and multivariate analyses were used to evaluate the association of specific cytokines and growth factors with systolic blood pressure (SBP) and diastolic blood pressure (DBP). Hypertensive subjects had higher serum concentrations of IL-1α, -2, -8, vascular endothelial growth factor, IFN-γ, TNF-α, MCP-1, and epidermal growth factor; and lower concentrations of anti-inflammatory cytokine, IL-10 (P < .05), compared with the healthy individuals. The serum concentrations of IL-4, -6, and -1β did not differ between the hypertensive subjects and control group. Univariate and multivariate analyses revealed that IL-1α and IFN-γ were independent predictors of a high SBP, while IFN-γ, IL-1α, TNF-α, and MCP-1 remained statistically significant for DBP after correction for age, gender, Body mass index, smoking, fasting blood glucose, and triglycerides. There was a significant association between the concentrations of several cytokines and hypertension. These associations may either be related to common underlying factors that cause hypertension and may also be proinflammatory or because these inflammatory cytokines might directly be involved in the etiology of hypertension.

Evaluation of tumor marker HE4 assay on the Elecsys 2010 analyzer

Introduction: Whey-acidic protein human epididymis protein 4 (HE4) is a new promising biomarker for epithelial ovarian cancer. The measured HE4 values may depend on the testing procedure used. The aim of this study was to evaluate theMethods: We evaluated a HE4 method on Elecsys 2010 analyzer. The method for quantitative determination of HE4 is direct, competitive chemiluminescent immunoassay. For quality control we use Elecsys PreciControl HE4 1 and 2. HE4 was measure on sera obtained from 56 women ( 20 healthy and 36 with epithelial ovarian cancer).Results: The Roche HE4 assays showed a good linearity (r=0.99) and precision (intrassayed total CV&lt;5%). The median HE4 serum concentrations was significantly higher among EOC patients than healthy females (p&lt;0,05). Elevated levels HE4 were found in 78 % patients with epithelial ovarian cancer.Conclusions: The presented results of the analytical evaluation methods for the determination of HE4 on the Elecsys 2010 analyzer showed an acceptable accuracy and precision.

Environmental influences on cardiovascular risk factors among Turkish immigrants in the Netherlands with type 2 diabetes (806.4)

The study examined the associations of serum cotinine (a biomarker of smoke exposure) and serum 25‐hydroxyvitamin D [25(OH)D] with blood pressure and serum lipids in Turkish immigrants with type 2 diabetes (T2D). A total of 110 participants, physician‐diagnosed with T2D, aged 30 years and older were recruited from multiple sources from The Hague, Netherlands in a cross‐sectional design. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using automated office blood pressure equipment. Serum cotinine levels were measured with a solid‐phase competitive chemiluminescent immunoassay. Serum 25(OH)D was determined by electrochemiluminescence immunoassay. Serum lipids were determined by enzymatic assay and included total cholesterol (CHOL), high‐density lipoprotein cholesterol (HDL), low‐density lipoprotein (LDL), and triglycerides (TG). Total cholesterol to HDL ratio (CHOL:HDL) and atherosclerosis‐index (AI) were calculated. High cotinine levels (蠅 25 ng/mL) were associated with high CHOL:HDL ratio (B= 0.59, SE= 0.28, P&lt;0.05) and high AI (B= 0.59, SE= 0.28, P&lt;0.05). Low 25(OH)D levels (&lt; 25 nmol/L) were associated with low DBP (B= ‐4.14, SE= 2.00, P&lt;0.05). These findings suggest that serum cotinine and 25(OH)D may be assessed as a standard of care for T2D management in the Turkish immigrant population. Prospective studies of diabetes outcomes are recommended to further verify these findings.

Serum Thymidine Kinase Activity: Analytical Performance, Age-Related Reference Ranges and Validation in Chronic Lymphocytic Leukemia

BackgroundTo date no age-related reference ranges are available for serum thymidine kinase (TK1) activity. Being a proliferation marker, it may be used as a prognostic marker in malignant diseases, including chronic lymphocytic leukemia (CLL). Our aim was to establish age-specific reference ranges for TK1 and examine its utility as a screening marker in CLL, a disease of the elderly.MethodsSerum TK1 activity was measured by a competitive chemiluminescent immunoassay in 369 healthy adults and 115 de novo CLL patients.ResultsWe observed a statistically significant decline in TK1 activity from young (18–35 years) to middle-aged (36–60 years) and further on to elderly (60–86 years) healthy individuals. Age-related reference range was: <30 U/L for young, <25 U/L for middle-aged and <19 U/L for elderly. There was no difference in TK1 activity between the studied healthy men and women. In CLL patients, TK1 activity was the highest in the advanced Rai stages. The area under the receiver operating characteristic curve (ROC-AUC) for TK1 was 0.840 (95% CI: 0.787–0.892), for differentiating CLL patients from age and sex matched healthy controls, with a cut-off value of 10.5 U/L (sensitivity: 80.9%, specificity: 73.4%). TK1 was significantly elevated in CD38+/Zap70+ CLL patients, and showed significant correlation with WBC and absolute B-cell count.ConclusionIn the healthy, serum TK1 activity does not differ in the two sexes but declines significantly with age. As such, use of age-related reference ranges is warranted, especially when evaluating CLL patients who generally belong to the elderly age group.

Development of chemiluminescent enzyme immunoassay for the determination of malachite green in seafood

An indirect competitive chemiluminescent enzyme immunoassay method was developed for screening malachite green (MG). Assay conditions, including the concentration of coating, antibody dilution, incubation time, dilution buffer, ionic strength and pH, were optimised. Under the optimised conditions, coating antigen concentration was 125 ng/mL; and dilution fold of antibody was 40,000. The 50% inhibition values of 0.22 ng/mL for MG were achieved with a limit of detection of 0.01 ng/mL, the linear range was from 0.03 ng/mL to 3.27 ng/mL. The assay showed a little cross-reactivity of 3.4%, 2.7% and 1.0% with methylene blue, brilliant green and crystal violet, respectively, and negligible cross-reactivity with other analogues of MG. The developed method has been used to quantify MG in seafood samples. The recovery of MG ranged from 82.43% to 108.0% at four concentrations (0.1, 1, 3 and 5 ng/mL), and the coefficients of variation were less than 13%. A comparison results between the high-performance liquid chromatography and the developed assay showed good relativity. The results indicated that the assay was a sensitive and stable method for screening MG.

Development of chemiluminescent enzyme immunoassay for the determination of aflatoxin M1 in milk products

In this study, an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for determining aflatoxin M1 (AFM1) in milk products has been developed. A luminol–hydrogen peroxide chemiluminescence system catalysed by horseradish peroxidase (HRP) was used as the signal detecting system. The effects of several factors, including concentration and pH of phosphate buffer, dilution ratio of antibody and antigen and other relevant variables on the immunoassay, were studied and optimised by single-factor experiments. The developed method presented an IC50 of 0.05 ng/mL, a detection limit of 0.01 ng/mL and a linear range from 0.018 to 0.13 ng/mL. This method has been successfully applied to the evaluation of AFM1 in milk products, the recoveries ranging from 71.9% to 109.0%. A good correlation with the commercial available ELISA kit for AFM1 (r = 0.9978) was obtained, indicating that the ic-CLEIA method developed can be used to determine AFM1 in real samples.

A competitive immunoassay for sensitive detection of small molecules chloramphenicol based on luminol functionalized silver nanoprobe

Chloramphenicol (CHL) as a broad-spectrum antibiotic has a broad action spectrum against Gram-positive and Gram-negative bacteria, as well as anaerobes. The use of CHL is strictly restricted in poultry because of its toxic effect. However, CHL is still illegally used in animal farming because of its accessibility and low cost. Therefore, sensitive methods are highly desired for the determination of CHL in foodstuffs. The immunoassays based on labeling as an important tool have been reported for the detection of CHL residues in food-producing animals. However, most of the labeling procedures require multi-step reactions and purifications and thus they are complicated and time-consuming. Recently, in our previous work, luminol functionalized silver nanoparticles have been successfully synthesized, which exhibits higher CL efficiency than luminol functionalized gold nanoparticles. In this work, the new luminol functionalized silver nanoparticles have been used for the labeling of small molecules CHL for the first time and a competitive chemiluminescent immunoassay has been developed for the detection of CHL. Owing to the amplification of silver nanoparticles, high sensitivity for CHL could be achieved with a low detection limit of 7.6×10−9gmL−1 and a wide linear dynamic range of 1.0×10−8–1.0×10−6gmL−1. This method has also been successfully applied to determine CHL in milk and honey samples with a good recoveries (92% and 102%, 99% and 107% respectively), indicating that the method is feasible for the determination of CHL in real milk and honey samples. The labeling procedure is simple, convenient and fast, superior to previously reported labeling procedures. The immunoassay is also simple, fast, sensitive and selective. It is of application potential for the determination of CHL in foodstuffs.