Commercial Chemiluminescent Immunoassay Research Articles

Dual-label time-resolved fluoroimmunoassay for simultaneous measurement of human epidermal growth factor receptor 2 and human epididymis protein 4 in serum

In this study, a novel dual-label time-resolved fluoroimmunoassay (TRFIA) is described for simultaneous quantification of human epidermal growth factor receptor 2 (HER-2) and human epididymis protein 4 (HE4) in serum to screen gynecologic cancers. A double-antibody sandwich TRFIA was introduced with europium and samarium chelates to simultaneously detect the concentrations of HER-2 and HE4. Under optimal conditions, the proposed method exhibited wide linear ranges for HER-2 of 0.07–500 ng ml−1 and for HE4 of 0.32–1000 pmol l−1 with the average coefficient of variation below 10%. The specificity was satisfied through determining the other common tumor markers. The recovery rates were 94.5% and 96.6% on average for HER-2 and HE4, respectively. Good correlations were observed in clinical samples between developed method and commercial chemiluminescence immunoassay kits. The results demonstrated that dual-label TRFIA for HER-2 and HE4 was rapid and precise, and therefore could have a promising use in large sample detection for gynecological cancer screening.

The efficacy of addition of Tenofovir Disoproxil Fumarate to Peg-IFNα-2b is superior to the addition of Entecavir in HBeAg positive CHB patients with a poor response after 12 weeks of Peg-IFNα-2b treatment alone.

Background: There are limited data regarding the efficacy of addition of entecavir (ETV) or tenofovir disoproxil fumarate (TDF) to Peg-IFNα-2b in HBeAg positive chronic hepatitis B (CHB) patients without early response to Peg-IFNα-2b. In this study, we aimed to evaluate the efficacy of ETV and TDF in HBeAg positive CHB patients who had a poor response to Peg-INFα-2b at the end of 12 weeks of monotherapy.Methods: A total of 40 HBeAg-positive CHB patients who were naive to antiviral therapy were recruited. The patients received a subcutaneous injection of Peg-IFNα-2b (180 µg) once a week for 12 weeks. However, the patients had a poor response to Peg-INFα-2b at the end of the 12-week-period monotherapy. The patients were then divided into two therapeutic protocol groups: (1) Group A: Patients received Peg-IFNα-2b (180 µg) subcutaneously weekly and ETV (0.5 mg) orally once daily for 48 weeks; (2) Group B: Patients received Peg-IFNα-2b (180 µg) subcutaneously weekly and TDF (300 mg) orally once daily for 48 weeks. The therapeutic efficacy was evaluated. Blood samples were collected at baseline and every 12 weeks. Routine biochemical tests including ALT, AST, etc. were measured by automated biochemical technique. HBV DNA was quantified using the TaqMan PCR assay. The levels of HBsAg, HBsAb, HBeAg, HBeAb and HBcAb were measured using a commercial chemiluminescent microparticle immunoassay.Results: The HBsAg level declined rapidly in both two treatment groups during the first 12 weeks and declined gradually in the next 36 weeks. At week 48, the mean ΔHBsAg level in Peg-IFNα-2b+TDF group was significantly higher than that in Peg-IFNα-2b +ETV group (-1.799 ± 0.3063 vs. -1.078 ± 0.2028, P=0.0491). The HBeAg loss rate was significantly higher in TDF add-on group than that in ETV add-on group at week 48 (40% vs. 10%, P=0.028). At week 48, the proportions of patients with undetectable HBV DNA (<500 IU/mL) were 80% (16 out of 20) and 95% (19 out of 20) in Peg-IFNα-2b+ETV group and Peg-IFNα-2b+TDF group, respectively.Conclusions: This real world study demonstrated that the efficacy of addition of TDF to Peg-IFNα-2b is superior to the efficacy of addition of ETV to Peg-IFNα-2b in HBeAg positive CHB patients with a poor response after 12 weeks of Peg-IFNα-2b treatment alone. However, this present study also requires a larger sample size study to verify in the future.

Carbon fiber based electrochemical sensor for sweat cortisol measurement

This study examines the use of a conductive carbon fiber to construct a flexible biosensing platform for monitoring biomarkers in sweat. Cortisol was chosen as a model analyte. Functionalization of the conductive carbon yarn (CCY) with ellipsoidal Fe2O3 has been performed to immobilize the antibodies specific to cortisol. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) chemistry has been used to immobilize the antibodies onto the Fe2O3 modified CCY. Crystallinity, structure, morphology, flexibility, surface area, and elemental analysis were studied using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (FE-SEM/EDS) and Brunauer–Emmett–Teller (BET) analysis. Mechanical properties of the fiber such as tensile strength, young’s modulus have also been investigated. Under optimal parameters, the fabric sensor exhibited a good linearity (r2 = 0.998) for wide a linear range from 1 fg to 1 μg with a detection limit of 0.005 fg/mL for the sensitive detection of cortisol. Repeatability, reliability, reproducibility, and anti-interference properties of the current sensor have been investigated. Detection of cortisol levels in human sweat samples has also been investigated and the results were validated with commercial chemiluminescence immunoassay (CLIA) method.

Dual-mode fluorescent and colorimetric immunoassay for the ultrasensitive detection of alpha-fetoprotein in serum samples

We present a novel dual-mode fluorescent and colorimetric immunosensor based on conventional immunoassay platforms by utilizing a gold nanoflower (AuNF)-loaded fluorescein molecule (AuNF@Fluorescein) as signal output. The AuNFs were modified with thiolated carboxyl ligand, which consisted of a hydrophobic alkane chain as hydrophobic wallet for fluorescein encapsulation, a tetra (ethylene glycol) unit for biocompatibility and solubility, and a functional carboxyl group for the conjugation of biorecognition molecules for biosensing. The resultant AuNFs showed a high loading capacity of 3.74 × 106 fluorescein molecules per AuNF because of its flower-like shape with many complex branches. By adjusting the solution pH to 8.0, the fluorescein molecules can almost entirely be released from the hydrophobic wallet of AuNF@Fluorescein, which led to strong fluorescent-signal amplification. Under the optimal detection conditions, the proposed immunoassay based on fluorescent signal exhibited ultrahigh sensitivity for alpha-fetoprotein (AFP) detection, with a limit of detection (LOD) of 29 fg/mL. This value is approximately 9.3 × 103-fold lower than that of corresponding horseradish peroxidase (HRP)-based immunoassay (LOD = 270 pg/mL). The fluorescein molecule also had intrinsic peroxidase-like activity to catalyze 3,3′,5,5′-tetramethylbenzidine oxidation with hydrogen peroxide for colorimetric signal. The proposed method with colorimetric mode further exhibited a sensitivity with a LOD of 17.7 pg/mL, which is about 15-fold lower than that of conventional HRP-based immunoassay. The recoveries of the proposed dual-mode immunoassay for AFP spiked serum samples ranged within 89.85%–100.0%, with the coefficient of variations ranging from 0.5% to 2.4%, indicating acceptable accuracy and precision for AFP quantitative detection. The reliability of the developed dual-mode immunoassay was further compared with a commercial chemiluminescence immunoassay kit by analyzing 20 clinical serum samples, showing that the two methods well agreed with each other, with high correlation coefficients of 0.997 and 0.986 based on recorded fluorescence and colorimetric signals, respectively. In summary, the proposed method was highly suitable for the ultrasensitive analysis of biomarkers or infectious diseases by fluorescence mode and can be used for routine clinical diagnosis by colorimetric mode.

Low prevalence of HTLV1/2 infection in a population of immigrants living in southern Italy.

AimsTo assess the prevalence of HTLV-1 and HTLV-2 infections in a cohort of immigrants living in southern Italy.FindingsWe screened for antibody to HTLV-1/2 infection 1,498 consecutive immigrants born in endemic areas (sub-Saharan Africa or southern-Asia) by a commercial chemiluminescent microparticle immunoassay. If confirmed in a Western blot assay, which differentiates anti-HTLV-1 from anti-HTLV-2, the positive sera were tested for specific HTLV RNA by a home-made PCR. The immigrants investigated were more frequently males (89.05%), young (median age 26 years), with a low level of education (median schooling 6 years), born in sub-Saharan Africa (79.70%). They had been living in Italy for a median period of 5 months. Only one (0.07%) subject was anti-HTLV-1 -positive/HTLV-1 RNA-negative; he was an asymptomatic 27-year-old male from Nigeria with 6 years’ schooling who stated unsafe sexual habits and unsafe injection therapy.ConclusionsThe data suggest screening for HTLV1 and HTLV-2 infections all blood donors to Italy from endemic countries at least on their first donation; however, a cost-effectiveness study is needed to clarify this topic.

Simultaneous quantitation of carbohydrate antigen 125 and carcinoembryonic antigen in human serum via time-resolved fluoroimmunoassay

In clinical diagnosis of cancer, immunology assay with single tumor marker often lead to a false and missed inspection. A quantitative method with a high degree of accuracy, sensitivity, and effectiveness is required for its diagnosis. We developed a dual-label time-resolved fluoroimmunoassay (TRFIA) to simultaneously detect carbohydrate antigen 125 (CA125) and carcinoembryonic antigen (CEA) in human serum to aid the diagnosis and prognosis of gastric cancer. The method was based on a microplate sandwich immunoassay using europium-labeled anti-CA125 antibodies and samarium-labeled anti-CEA antibodies as fluorescent reporters. The assay detection range was widely, and the limit of detection was sufficiently for detecting clinical sample. The intra- and inter-assay coefficients of variation were below 6%, and recoveries ranged from 90% to 110%. No significant statistical difference in sensitivity or specificity was observed between dual label-TRFIA and commercial chemiluminescent immunoassays in serum samples. These results demonstrate the successful development of an effective, reliable, and convenient novel TRFIA method for the simultaneous detection of CA125 and CEA, which can be used for clinical blood screening to monitor the occurrence and development of tumors to facilitate early treatment.

CTAPIII/CXCL7: a novel biomarker for early diagnosis of lung cancer.

It is desirable to have a biomarker which can facilitate low‐dose CT in diagnosis of early stage lung cancer. CTAPIII/CXCL7 is reported to be a potential biomarker for diagnosis of early lung cancer. In this study, we investigated the serum level of CTAPIII/CXCL7 in patients at different stage of lung cancer and the diagnostic efficacy of CTAPIII/CXCL7 in NSCLC. The plasma level of CTAPIII/CXCL7 was assayed by ELISA. CEA, SCCAg, and Cyfra211 were measured using a commercial chemiluminescent microparticle immunoassay. A total of 419 subjects were recruited, including 265 NSCLC patients and 154 healthy individuals. The subjects were randomly assigned to a training set and a test set. Receiver operating characteristic (ROC) and binary logistic regression analyses were conducted to evaluate the diagnostic efficacy and establish diagnostic mathematical model. Plasma CTAPIII/CXCL7 levels were significantly higher in NSCLC patients than in controls, which was independent of the stage of NSCLC. The diagnostic efficiency of CTAPIII/CXCL7 in NSCLC (training set: area under ROC curve (AUC) 0.806, 95% CI: 0.748–0.863; test set: AUC 0.773, 95% CI: 0.711–0.835) was greater than that of SCCAg, Cyfra21‐1, or CEA. The model combining CTAPIII/CXCL7 with CEA, SCCAg, and Cyfra21‐1 was more effective for NSCLC diagnosis than CTAPIII/CXCL7 alone. In addition, plasma level of CTAPIII/CXCL7 may contribute to the early diagnosis of NSCLC. CTAPIII/CXCL7 can be used as a plasma biomarker for the diagnosis of NSCLCs, particularly early stage lung cancer, with relatively high sensitivity and specificity.

A nanocomposite containing Prussian Blue, platinum nanoparticles and polyaniline for multi-amplification of the signal of voltammetric immunosensors: highly sensitive detection of carcinoma antigen 125

The authors describe a method for signal amplification of label-free voltammetric immunosensors. A glassy carbon electrode (GCE) was modified with Prussian Blue-platinum nanoparticles (PB-PtNPs) as a redox-active species that gives a strong amperometric signal at 0.18 V (vs. Ag/AgCl). Benefitting from the excellent electrical conductivity and the strong catalytic activity to H2O2, the modified GCE gives a strongly enhanced signal. The PB-PtNPs were incorporated into a polyaniline (PANI) hydrogel to further enhance the signal. The signal response of the PB-PtNP-PANI/GCE is larger by a factor of 7.6 than that of PB-PtNP/GCE. In order to further improve electrical conductivity and immobilize antibody, gold nanoparticles (AuNPs) were deposited on the surface of the PB-PtNP-PANI hydrogel. The AuNP-PB-PtNP-PANI hydrogel nanocomposite on the GCE was used in an immunosensor for the model analyte carcinoma antigen 125 (CA125), a biomarker for epithelial ovarian cancer, by immobilizing the respective antibody on the modified GCE. A linear response found for the 0.01 to 5000 U mL−1 CA125 concentration range, with a detection limit of 4.4 mU mL−1 (at an S/N ratio of 3). The electrochemical sensitivity is as high as 119.76 μA·(U/mL)−1·cm−2. The detection of CA125 in human serum showed satisfactory accuracy compared to a commercial chemiluminescent microparticle immunoassay (CMIA).

Validation of a Commercial Chemiluminescence Immunoassay for the Simultaneous Measurement of Three Different Amyloid-β Peptides in Human Cerebrospinal Fluid and Application to a Clinical Cohort.

A comprehensive assay validation campaign of a commercially available chemiluminescence multiplex immunoassay for the simultaneous measurement of the amyloid-β peptides Aβ38, Aβ40, and Aβ42 in human cerebrospinal fluid (CSF) is presented. The assay quality parameters we addressed included impact of sample dilution, parallelism, lower limits of detection, lower limits of quantification, intra- and inter-assay repeatability, analytical spike recoveries, and between laboratory reproducibility of the measurements. The assay performed well in our hands and fulfilled a number of predefined acceptance criteria. The CSF levels of Aβ40 and Aβ42 determined in a clinical cohort (n = 203) were statistically significantly correlated with available ELISA data of Aβ1-40 (n = 158) and Aβ1-42 (n = 179) from a different laboratory. However, Bland-Altman method comparison indicated systematic differences between the assays. The data presented here furthermore indicate that the CSF concentration of Aβ40 can surrogate total CSF Aβ and support the hypothesis that the Aβ42/Aβ40 ratio outperforms CSF Aβ42 alone as a biomarker for Alzheimer's disease due to a normalization to total Aβ levels.

Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads

Hepatitis B virus infection is one of the major causes of hepatitis, liver cirrhosis and liver cancer. In this study, we used highly luminescent quantum dot-beads (QBs) as signal amplification probes in the sandwich immunochromatographic assay (ICA) for ultrasensitive and quantitative detection of hepatitis B virus surface antigen (HBsAg) in human serum. Various parameters that influenced the sensitivity and stability of the QB-based ICA (QB-ICA) sensor were investigated. Two linear independent regression equations for detection of serum HBsAg were expressed with Y=0.3361X−0.0059 (R2=0.9983) for low HBsAg concentrations between 75pgmL−1 and 4.8ngmL−1, and Y=0.8404 X−2.9364 (R2=0.9939) for high HBsAg concentrations in the range from 4.8ngmL−1 to 75ngmL−1. The detection limit of the proposed ICA sensor achieved was 75pgmL−1, which is much higher than that of the routinely-used gold nanoparticle based ICA. The intra- and inter-assays recovery rates for spiked serum samples at HBsAg concentrations of 75pgmL−1, 3.75ngmL−1 and 18.75ngmL−1 ranged from 90.14% to 97.6%, and coefficients of variation were all below 7%, indicating that the QB-ICA sensor has an acceptable accuracy for HBsAg detection. Additionally, the quantitative method developed showed no false positive results in an analysis of 49 real HBsAg-negative serum samples, and exhibited excellent agreement (R2=0.9209) with a commercial chemiluminescence immunoassay kit in identifying 47 HBsAg-positive serum samples. In summary, due to its high fluorescence intensity, the sandwich QB-ICA sensor is a very promising point-of-care test for rapid, simple and ultrasensitive detection of HBsAg, as well as other disease-related protein biomarkers.

Dual-labeled time-resolved immunofluorometric assay for the determination of IgM antibodies to rubella virus and cytomegalovirus in human serum

ObjectivesThis study established a novel time-resolved fluorescence immunoassay (TRFIA) that allows the simultaneous determination of rubella virus (RV) IgM and cytomegalovirus (CMV) IgM in human serum. Design and methodsLanthanum elements labeled antibody and streptavidin–biotin system were used in the “capture sandwich” format simultaneously. ResultsThe working range of TRFIA for RV IgM was 2–80AU/mL and for CMV IgM was 5–400AU/mL. Intra- and inter-assay coefficient of variation (CV) for RV IgM and CMV IgM were both less than 10% and recoveries were from 90% to 110%. No significant statistical difference in sensitivity or specificity was observed between dual-TRFIA and commercial chemiluminescent immunoassays (CLIA) in serum samples. ConclusionThe novel dual-TRFIA for RV IgM and CMV IgM detection might have valuable clinical application, with satisfactory sensitivity, specificity and accuracy.

The Correlation between Hepatitis C Core Antigen and Hepatitis C Virus RNA Levels with Respect to Human Immunodeficiency Virus Status, Hepatitis C Virus Genotype and Interferon-Lambda-4 Polymorphism

Objectives: Serum hepatitis C virus (HCV) core antigen (HCVcAg) concentrations correlate with HCV RNA levels in HCV monoinfected patients. Data in HCV/HIV coinfected patients are still limited. We aim to compare the use of HCVcAg measurement with respect to HIV status, HCV genotypes, interferon-lambda-4 (IFNL4) polymorphism and clinical parameters. Methods: We analyzed an untreated cohort of 104 patients with HCV monoinfection and 85 patients with HCV/HIV coinfection. Serum HCVcAg was measured by a commercial chemiluminescent microparticle immunoassay. The presence of IFNL4 polymorphism ss469415590 was identified by real-time PCR. Results: log₁₀ HCVcAg levels were significantly correlated with corresponding log₁₀ HCV RNA levels (r = 0.889, p < 0.001), but not with ALT levels and liver stiffness. The correlation between HCV RNA and HCVcAg was particularly high in coinfected patients and those with high viremia. Mean log₁₀ HCVcAg concentration was significantly higher in coinfected patients than in monoinfected patients. Patients harboring the TT/TT genotype of ss469415590 had significantly higher levels of log₁₀ HCVcAg than those with the non-TT/TT genotype. HCVcAg levels were similar across HCV genotypes. Conclusions: HCVcAg concentrations had an excellent correlation with HCV RNA levels, particularly in HCV/HIV-coinfected individuals and might be associated with IFNL4 polymorphism. HCVcAg testing could be used as an alternative to HCV RNA assays in resource-limited settings.

Multicenter Analytical Evaluation of the Automated Electrochemiluminescence Immunoassay for Cyclosporine

Cyclosporine A (CsA) is used as a posttransplantation immunosuppressant drug, and careful monitoring of CsA concentration in whole blood is essential. A new automated electrochemiluminescence immunoassay (ECLIA) for CsA measurement has been assessed in a multicenter evaluation. Residual EDTA whole blood samples from patients undergoing CsA therapy after organ transplant were used in assay evaluation at 5 clinical laboratories in Europe. Experiments included imprecision according to CLSI EP5-A2 (within-run and intermediate), lower limit of quantification, linearity according to CLSI EP6-A, and recovery of commercial external quality control samples. In addition, comparisons to liquid chromatography-tandem mass spectrometry methods in routine use at each investigational site and to commercial chemiluminescent microparticle immunoassay and antibody-conjugated magnetic immunoassay methods were performed. Imprecision testing gave coefficients of variation of less than 9% in the 30-2000 mcg/L range for both within-run and intermediate imprecision. Lower limit of quantification of 6.8 mcg/L at one investigational site and 1.8 mcg/L at a second site at 20% coefficient of variation were observed. Linearity was measured over the concentration range 0-2000 mcg/L, yielding a deviation of less than ±12%. External quality control sample recovery by ECLIA was 93%-114% of LC-MS/MS sample recovery. Deming regression analysis of ECLIA method comparison to combined LC-MS/MS results yielded a slope of 1.04 [95% confidence interval (CI), 1.03-1.06] and intercept of 2.8 mcg/L (95% CI, 1.5-4.1 mcg/L). Comparison to chemiluminescent microparticle immunoassay yielded a slope of 0.87 (95% CI, 0.85-0.89) and intercept of 1.4 mcg/L (95% CI, -0.89 to 3.7 mcg/L); comparison to antibody-conjugated magnetic immunoassay yielded a slope of 0.96 (95% CI, 0.93-0.98) and intercept of -4.2 mcg/L (95% CI, -7.1 to -1.2 mcg/L). The data from this multicenter evaluation indicate that the new ECLIA-based cyclosporine assay is fit for its purpose, the therapeutic monitoring of CsA.

Vitamin D Decreases In Vitro Secretion of Matrix Metalloproteinase 2 and 9 from Nasal Polyp–Derived Fibroblasts

Objectives: In order to study the role of vitamin D in the pathogenesis and manifestation of chronic rhinosinusitis with nasal polyposis, we designed the following study. In vivo: We tried to determine if serum vitamin D level was lower in patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) and if low serum vitamin D level correlated with the severity of CRSwNP. In vitro: We used the cultured nasal polyp–derived fibroblasts to investigate the in vitro effect of vitamin derivatives (calcitriol and tacalcitol) on the production of matrix metalloproteinase (MMP)-2 and MMP-9 . Methods: Patients with latest diagnosis of CRSwNP undergoing elective endoscopic sinus surgery were recruited, and patients with malignancies or asthma were excluded. The severity of CRSwNP was assessed with the Lund-Mackay score and polyp grading system. Vitamin D status was assessed by measuring circulating 25-hydroxyvitamin D (25OHD) by using commercial chemiluminescence immunoassay. Resected polyps were used for primary fibroblast culture. The fourth to eighth passage of human fibroblasts were used for the experiments. Results: Serum 25OHD levels (ng/mL ± SD) were significantly lower in patients with CRSwNP (21.4 ± 5.7) than in those with CRSsNP (28.8 ± 6.2) ( P &lt; .001). A significantly negative relationship was found between serum 25OHD level and polyp grade ( r = –0.63, P = .001), indicating lower serum 25OHD was associated with higher polyp grade. Serum 25OHD was inversely related to both LM score and total IgE level as well. However, statistical significance was not found. In the in vitro study, we demonstrated that TNF can significantly induce the secretion of MMP-2 and-9 from nasal polyp fibroblasts and this effect was significantly suppressed by adding calcitriol and tacalcitol. Conclusions: A significantly lower vitamin D level was found in a group of Taiwanese CRSwNP patients, which revealed an association with greater nasal polyp size. The study on the influence of vitamin D on inflammatory processes in NP may shed a light not only on the mechanism of its etiology but also prove its potential use in the pharmacology of NP.

A point-of-care testing system with Love-wave sensor and immunogold staining enhancement for early detection of lung cancer.

It has been reported that detection of exhaled breath condensate (EBC) is available for studies of pulmonary diseases, especially lung disease. In order to detect lung cancer (LC) at early stage, a point-of-care testing system suitable for measurement of tumor markers in EBC is developed. The assay, based on gold nanoparticle sandwich immunoassay and subsequent gold staining, was performed on a Love-wave sensor packaged inside a chip cartridge. Benefit from high sensitivity of Love-wave sensor, oriented immobilization of coating antibodies and immunogold staining enhancement, the present immunosensor could provide a sensitive, specific and rapid measurement. Carcinoembryonic antigen (CEA), neuron specific enolase (NSE) and squamous cell carcinoma antigen (SCC) in EBC collected from 17 patients with LC and 13 healthy volunteers were detected by this system. Results were compared with commercial chemiluminescence immunoassay and showed high correlation between two methods. Additionally, it revealed significantly statistical differences existing between two groups of subjects. These results indicate that the present system is suitable for detection of tumor markers in EBC and could be used as assistant tools for early detection of LC.

Seroepidemiology of hepatitis B virus infection among adults in Singapore: A 12-year review

We undertook a national hepatitis B seroprevalence study to assess the seroprevalence of hepatitis B virus (HBV) markers in the adult population in Singapore in 2010 and make comparisons with the seroprevalence in 1998 and 2004. The study involved residual sera from national health surveys conducted every six years since 1998. The tests for HBV markers were carried out using commercial chemiluminescent microparticle immunoassay. In 2010, the prevalence of hepatitis B surface antigen (HBsAg) among 3293 Singapore residents aged 18–79 years was 3.6% (95% confidence interval [CI] 2.9–4.2%). Hepatitis B e antigen (HBeAg) was detected in 4.2% of those who were HBsAg positive. About 22.5% (95% CI 21.1–23.9%) were positive for antibody to hepatitis B core antigen (anti-HBc). The overall population immunity to HBV, as determined by antibody to hepatitis B surface antigen (anti-HBs)≥10mIU/mL, was 43.9% (95% CI 42.2–45.6%). Among young adults below 30 years of age, HBsAg prevalence (1.1%) was half that in 1998 and 2004, and in those positive for HBsAg, none was positive for HBeAg in 2010, compared to 20.8% in 1998 and 15.8% in 2004. In this age group, anti-HBc prevalence also decreased significantly from 22.1% in 2004 to 4.4% in 2010, while anti-HBs (≥10mIU/mL) prevalence increased significantly from 27.9% in 1998 to 43.3% in 2010 (p<0.001). The national childhood HBV immunisation and catch-up programmes implemented in 1987 and 2001–2004, respectively, had a significant impact in reducing HBV infection and in raising the immunity of the adult population 18–29 years of age.

Serum 25-hydroxyvitamin D Levels are Lower in Chronic Rhinosinusitis with Nasal Polyposis and are Correlated with Disease Severity in Taiwanese Patients

Vitamin D deficiency is reported to be associated with increased incidence of allergic airway diseases and is correlated with the severity of asthma. This study was designed to determine if serum Vitamin D level is lower in chronic rhinosinusitis with nasal polyposis (CRSwNP) patients and if low serum Vitamin D level is correlated with the severity of CRSwNP. New CRSwNP patients undergoing elective endoscopic sinus surgery were recruited. Patients with malignancies or asthma were excluded. Twenty chronic rhinosinusitis without nasal polyposis (CRSsNP) patients were used as control. Demographic characteristic information was collected. The severity of CRSwNP was assessed with the Lund-Mackay (LM) score and polyp grading system. Vitamin D status was assessed by measuring circulating 25-hydroxyvitamin D (25OHD) by using commercial chemiluminescence immunoassay. Data were stratified by factors known to affect serum 25OHD, including sex, race, and body mass index. Serum 25OHD levels (ng/mL ± SD) were significantly lower in patients with CRSwNP (21.4 ± 5.7) than in those with CRSsNP (28.8 ± 6.2; p < 0.001). The incidences of vitamin D deficiency (<20 ng/mL) in CRSwNP and CRSsNP patients were 45.5 and 6.3%; however, the incidences of vitamin D insufficiency (20-30 ng/mL) in these patients were 50.0 and 62.5%, respectively. A significantly negative relationship was found between serum 25OHD level and polyp grade (r = -0.63; p = 0.001), indicating lower serum 25OHD was associated with higher polyp grade. Serum 25OHD was inversely related to both LM score and total IgE level as well; however, statistical significance was not found. A significantly lower vitamin D level was found in a group of Taiwanese CRSwNP patients, which revealed an association with greater nasal polyp size. Serum vitamin D levels could be added to the routine workup of patients suffering from CRS and these data could be used to potentially help determine the disease severity.

A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum

Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02-700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7-8.7% and 3.8-7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X - 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus.

Clinico‐pathological significance of hepatitis C virus core antigen levels in chronic infection

Hepatitis C virus core antigen (HCVcoreAg) may be measured in serum with a sensitive, recently validated assay. Beyond its value as a marker of viral infection, there are little data on its relation with clinical, histological, and virological parameters. In this study, the significance of HCVcoreAg levels was studied in a prospective cohort of 114 patients with chronic hepatitis C. HCVcoreAg was measured by a commercial chemiluminescent microparticle immunoassay. Clinical and virological data included quantitative HCV-RNA, HCV genotype, ALT, GGT, IL28B rs12979860 polymorphism as well as liver histology parameters. HCVcoreAg levels were correlated significantly with HCV-RNA (r=0.56; P<0.0001) but also with ALT levels (r=0.258; P<0.01) and liver necroinflammatory activity (r=0.205; P<0.04). Patients harbouring HCV genotype 3 showed lower levels of HCVcoreAg than both genotype 1 and two patients. In genotype 3, a direct correlation between steatosis and HCVcoreAg was found. Levels of HCVcoreAg also varied according to the IL28B genotype. These data suggest that the evaluation of HCVcoreAg serum levels may provide relevant data for the baseline clinical evaluation of chronic hepatitis C patients. HCVcoreAg serum levels may be a useful tool to further the understanding of chronic hepatitis C pathobiology.

Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals.