Development of a Time-Resolved Fluorescence Immunoassay for the Diagnosis of Hepatocellular Carcinoma Based on the Detection of Glypican-3.

Glypican-3(GPC3), an oncofetal protein, is a potential novel marker for hepatocellular carcinoma (HCC). In this study, we attempted to establish a new method to detect serum GPC3 using the antibodies identified in our previous research, and then evaluated its clinical application for the diagnosis of HCC. Herein, a sandwich time-resolved fluorescence immunoassay (TRFIA) for detecting serum GPC3 was developed. The detection limit, analytical recovery, specificity and precision of the proposed TRFIA assay were satisfactory. A total of 415 patients were collected and divided into seven groups: hepatocellular carcinoma (101), colorectal cancer (67), gastric cancer (44), esophageal cancer (15), cirrhosis (55), hepatitis (61), normal liver (72). Using this proposed method, the concentration of serum GPC3 in these clinical samples was detected. The results demonstrated that the levels of GPC3 in serum from HCC patients were significantly higher than that in others. Compared with the results of chemiluminescence immunoassay (CLIA), a high consistency (Kappa =0.84) was observed. Thus, an effective, sensitive and reliable TRFIA-GPC3 kit for diagnosing HCC was successfully developed. It offers a suitable alternative to existed methods of determining GPC3 and is expected to be used in clinic in the future.

Dual-Labeled Time-Resolved Immunofluorometric Assay for the Simultaneous Quantitative Detection of Hepatitis B Virus Antigens in Human Serum.

In this paper, a novel time-resolved fluorescence immunoassay (TRFIA) is described that allows the simultaneous quantitative detection of hepatitis B virus surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in human serum to aid the diagnosis and monitoring of hepatitis B virus infection. The proposed method was developed based on a two-step sandwich immunoassay protocol in which monoclonal antibodies against HBsAg and HBeAg were co-coated in 96 microtitration wells, then tracer polyclonal antibodies against HBsAg labeled with samarium and tracer monoclonal antibodies against HBeAg labeled with europium chelates were used for detection. The detection range was 0.1-150 IU/mL for HBsAg and 0.5-160 PEIU/mL for HBeAg, and the detection limits were 0.03 IU/L and 0.09 PEIU/ml, respectively. The intra- and inter-assay coefficients of variation were below 8 % for both virus antigens. The dilution linearity and accuracy of the assay were satisfactory. No statistically significant differences were observed in sensitivity or specificity for the serum samples between the dual-label TRFIA and a commercial single-label TRFIA. These results demonstrate that an effective, reliable and convenient HBsAg/HBeAg dual-label TRFIA was successfully developed that may be clinically applicable for blood screening to monitor the course of hepatitis B virus infection and predict treatment responses.

A Fluorescence Immunochromatographic Assay Using Europium (III) Chelate Microparticles for Rapid, Quantitative and Sensitive Detection of Creatine Kinase MB.

The isoenzyme creatine kinase MB is very important for diagnosis of acute myocardial infarction (AMI). Some CK-MB immunoassays are sensitive, accurate and available for clinical application, but they are expensive and time-consuming procedures. Furthermore, conventional fluorescence immunochromatographic assays (FL-ICAs) have suffered from background fluorescence interference and low analytical sensitivity. A rapid and simple FL-ICA with Eu (III) chelate polystyrene microparticles was developed to determine CK-MB in 50uL serum samples using a portable test strip reader by measuring the fluorescence peak heights of the test line (HT) and the control line (HC) in 12 min. The assay was reliable with a good correlation coefficient between HT/HC ratio and CK-MB concentration in samples. A linear range was 0.85-100.29 ng/mL for CK-MB, and the LOD was 0.029 ng/mL. The intra- and inter-assay coefficients of variation (CV) were both <10 % and the average recoveries were from 90.17 % -112.63 % for CK-MB. The system performed well in interference experiments. Furthermore, a highly significant correlation (r = 0.9794, P < 0.001) between this method and the commercially available bioMérieux mini VIDAS system were attained for measuring 120 CK-MB samples. These results indicated that the Eu (III) chelate microparticles-based FL-ICA is simple, fast, highly sensitive, reliable, and reproducible for point-of-care testing of CK-MB concentrations in serum. Graphical Abstract ᅟ.

Development of a time-resolved fluorescence immunoassay for Epstein-Barr virus nuclear antigen 1-immunoglobulin A in human serum.

Enzyme-linked immunosorbent assay (ELISAs) specific for Epstein-Barr virus nuclear antigen 1 (EBNA1)-immunoglobulin A (IgA) are most commonly used in the clinical diagnosis of EBV infection. But they have a low sensitivity and the enzyme-labeled antibodies are unstable. In this study, a novel immunoassay based on an indirect time-resolved fluoroimmunoassay (TRFIA) was developed. Microtiter plates were coated with recombinant EBNA1. We used Eu(3) (+)-labeled anti-human IgA as probe. The precision, sensitivity, specificity, and stability were evaluated, and comparison with traditional and commercially available ELISAs was also made. The cut-off value for our TRFIA was 2.7. Intra- and inter-assay coefficients of variation for the TRFIA were 1.56-4.99% and 3.92-6.95%, respectively; whereas those for the ELISA were 4.54-8.16% and 7.07-10.52%, respectively. Sensitivity was obviously better than traditional ELISA when diluted positive samples serially. Additionally, stability, specificity test and comparison of sensitivity and specificity between the TRFIA and commercial ELISAs all proved satisfactory. In conclusion, the results demonstrated that EBNA1 IgA TRFIA was a sensitive immunoassay and had potential value in large-scale screening of human serum samples in developing countries.

Development of a time-resolved fluorescence immunoassay for herpes simplex virus type 1 and type 2 IgG antibodies.

Enzyme-linked immunosorbent assays (ELISA) specific for anti-HSV glycoprotein G (gG) are most commonly used in the clinical diagnosis of HSV infection. But most of them are qualitative and with narrow detection ranges. A novel time-resolved fluoroimmunoassay (TRFIA) methodology was developed for the quantitative determination of HSV IgG in human serum. The assay was based on an indirect immunoassay format, and performed in 96-well microtiter plates. HSV-1 and HSV-2 were used as the coating antigens. Eu(3+)-labeled goat anti-(human IgG) polyclonal antibodies were used as tracers. The fluorescence intensity of each well was measured and serum HSV IgG levels quantified against a calibration curve. The detection range of the novel TRFIA was between 5 and 500 AU/mL. Assay sensitivity was 0.568 AU/mL. The intra- and inter-assay coefficients of variation were 0.59-3.63% and 3.65-6.81%, respectively. Analytical recovery, dilution tests and serum panel tests were performed using TRFIA and the results proved satisfactory. There were no statistically significant differences in sensitivity and specificity between the TRFIA and commercial ELISAs. An effective, sensitive and accurate quantitative HSV type 1 and type 2 IgG TRFIA was successfully developed and provided diagnostic value in clinical use.

Evaluation of the analytical and clinical performances of time-resolved fluoroimmunoassay for detecting carcinoma antigen 50.

We developed a TR-FIA kit for quantitative detection of CA50. This study aims to evaluate the analytical and clinical performances of this kit. Precision, accuracy, specificity, sensitivity, stability, and endogenous interference of this kit are evaluated. Reference range is established. Coincidence rate and correlation between TR-FIA and RIA are evaluated. ROC is adopted to evaluate the diagnostic performance. This kit shows excellent precision with a coefficients of variation (CVs) ranged from 2.2-9.3%, accuracy (average recovery, 98.5%), sensitivity (minimum detectable concentration is 0.2 U/mL), specificity (all cross-reactivity is less than 0.1% except CA199, which is 0.175%), and storage stability (recoveries, 90.8-100.4%). Bilirubin, hemoglobin, and triglyceride dose not interfere with CA50 detection (recovery, 97.13-109.1%). The range from 0-25 U/mL is chosen as the reference range. There are good correlation (r = 0.804) and coincidence (p = 0.608, kappa = 0.924) between TR-FIA and RIA. Diagnostic performance of this kits, which based on RIA results, is perfect (AUC = 0.996), and the diagnostic accuracy for malignancy diagnosis is in moderate degree (AUC, 0.802-0.861). The TR-FIA (CA50) kit performs well in analytical and clinical performances, and can be employed in the clinical diagnosis of malignancy.

Systematic analysis of serum peptidase inhibitor 3 in psoriasis diagnosis and treatment.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease. To date, there are no serum biomarkers for psoriasis that have been validated to diagnose or treat psoriasis. METHODS: Peptidase inhibitor 3 (PI3) levels in serum were measured using chemiluminescence immunoassay (CLIA) in two independent cohorts including healthy controls (HC) and patients diagnosed with chronic urticaria (CU), chronic eczema (CE), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), psoriatic arthritis (PsA), or psoriasis vulgaris (PV). Receiver operating characteristic (ROC) curve analysis determined the diagnostic performance of PI3 in patients with psoriasis. The correlation between PI3 levels and the Psoriasis Area Severity Index (PASI) score was analyzed using the Spearman correlation method. Additionally, the study evaluated PI3 expression and treatment response of PV patients 12 weeks before and after topical treatment with calcipotriol betamethasone and calcipotriol ointment (T#1) or topical therapy plus PSORI-CM01 granules (T#2). RESULTS: In cohort #1, PI3 levels effectively discriminate PV patients from HC and CU patients, with AUCs of 0.909 and 0.840, respectively. In cohort #2, AUCs for detecting PV patients among HC, CU, CE, SLE, and RA patients were 0.940, 0.926, 0.802, 0.989, and 0.951, respectively. For PsA patients, AUCs were 0.989, 0.986, 0.910, 1.000, and 0.984 compared to HC, CU, CE, SLE, and RA patients, respectively. In both cohorts, PI3 levels correlated significantly with PASI scores in PV patients (cohort #1, r = 0.433; cohort #2, r = 0.634) and PsA patients (cohort #2, r = 0.718). Moreover, univariate logistic regression analyses revealed that PV patients with higher PI3 expression had a significantly higher risk of treatment resistance, with an odds ratio of 3.45 [95% confidence interval (CI) 1.54, 7.74, p = 0.003]. Finally, PI3 levels decreased nearly 35-fold more in the responder than in the non-responder group before and after treatment. CONCLUSIONS: Serological PI3 is a reliable biomarker for PV diagnosis and may have the potential to predict and monitor the progression of PV before and after treatment. Key Points • This study validated PI3's diagnostic performance in two independent psoriasis cohorts using CLIA. • PI3 expression is significantly correlated with the psoriasis severity and with patients who benefited from the treatments. • Serological PI3 is a reliable biomarker for psoriasis diagnosis and may have the potential to monitor the psoriasis progression with and without treatments.

A novel immunoassay for the quantization of CYFRA 21-1 in human serum.

BACKGROUND: Cytokeratin 19 fragment antigen (CYFRA 21-1) is used to diagnose and monitor neoplasms. However, the main disadvantages of the currently available CYFRA 21-1 assays include heterogenous technology, being time-consuming, and having low through-put with low insensitivity. This study investigated the use of amplified luminescent proximity homogeneous immunoassay (AlphaLISA) for the quantization of CYFRA 21-1 in human serum. METHODS: The AlphaLISA kit was developed based on AlphaScreen detection technology with two different anti-CYFRA 21-1 monoclonal antibodies. One was coated on AlphaLISA acceptor beads and the other was biotinylated. Donor beads were coated with streptavidin. The test conditions were optimized and analytical performance was studied. RESULTS: The measurement range of AlphaLISA CYFRA 21-1 kit was 0.08-500 ng/ml. Assay detection limit was 0.08 ng/ml. The intra- and interassay coefficients of variation were 3.00-9.00% and 4.00-10.00%, respectively. There was no cross-reaction to alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 19-9 (CA19-9), cytokeratins 8 (CK8), and cytokeratins 18 (CK18). The correlation coefficient of blood samples involved was 0.974 between CYFRA 21-1-AlphaLISA assay and a commercial electrochemiluminescence immunoassay (ECLIA) CYFRA 21-1 kit (Roche). CONCLUSIONS: The AlphaLISA CYFRA 21-1 kit developed in this study had favorable performance characteristics for clinical application with acceptable analytical sensitivity, specificity, and accuracy.

Effect of temperature on the photoproperties of luminescent terbium sensors for homogeneous bioassays.

We developed a luminescent terbium sensor (LTS) based on energy resonance transfer for homogeneous bioassays. The effect of temperature on photoluminescence and time-resolved fluorescence of the LTS was investigated. When the temperature was increased from 277 K to 369 K, the photoluminescence quantum yield decreased by up to 25 %, time-resolved fluorescence decreased by up to 54 %, and the lifetime shortened dramatically. Studies showed that both photoluminescence and time-resolved fluorescence quantum yields were largely recovered after samples were heated from 298 to 310, 333 or 369 K and subsequently cooled to 298 K. These results indicate that the homogeneous bioassay with LTS is sensitive to temperature and should be conducted at a constant temperature to ensure the temperature effect does not influence data and to increase the accuracy of the results. The results of this study are important for LTS applications in homogeneous bioassays.

AlphaLISA for the determination of median levels of the free ß subunit of human chorionic gonadotropin in the serum of pregnant women.

Measurement of the free ß subunit of human chorionic gonadotropin (free ß-hCG) in serum is useful for prenatal screening. Concentrations of free ß-hCG vary in different races. Conventional assays used for such measurements have limitations. We applied the AlphaLISA to measure levels of free ß-hCG in maternal serum during 8-20 weeks of gestation in women from southern China. Two anti-free ß-hCG antibodies were used: one was coated on AlphaLISA acceptor beads and the one was biotinylated. The assay also contained donor beads coated with streptavidin. The AlphaLISA assay detection limit was 0.11 ng/mL, and the analytical range was 0.11-200 ng/mL. The intra- and interassay coefficients of variation were 1.32%-2.50% and 3.44%-5.45%, respectively. The correlation with commercial Eu(3+)-labeled free ß-HCG-TRFIA assay was good (y = 1.045x + 1.580, r(2) = 0.978). Median levels of free ß-hCG in maternal serum at 8-20 weeks gestation were higher in women from southern China compared with those reported in women from other countries. The AlphaLISA for free ß-HCG could become the assay of choice for applications in clinical diagnostics. The established median value of free ß-HCG is helpful in clinical diagnosis specific for southern Chinese women.

Development of a novel chemiluminescence immunoassay for the detection of procalcitonin.

OBJECTIVE: To evaluate the analytical performance of our previously developed chemiluminescence immunoassay (CLIA) kit for the detection of procalcitonin (PCT) and compare with the results obtained using the Vidas B.R.A.H.M.S. PCT™ test (PCT-V). DESIGN AND METHODS: Our laboratory previously designed a novel CLIA kit and supporting instrument (AE-180) for the detection of PCT. We analyzed the clinical performance of this system, including the imprecision, limit of detection, and linearity of analyses of 305 serum specimens. The results were compared with measurements of the same serum samples obtained with PCT-V. RESULTS: The limit of detection and blank of our kit were 0.0075 and 0.0039 ng/mL, respectively. The intra- and inter-assay coefficient of variation of the kit were both between 0.8% and 3.9%. The equation of linearity was found to be y = 1.03× + 0.06 (r = 0.99) for concentrations in the range of 0.01-110 ng/mL. The correlation coefficient with the results of PCT-V was 0.995, and the equation obtained for Passing and Bablok regression analysis was 1.061 for our CLIA PCT kit and - 0.003 for PCT-V. Our kit slightly overestimated the concentration according to comparison with PCT-V results. CONCLUSION: The kit that was previously developed in our laboratory for the measurement of serum PCT concentration using CLIA technology shows excellent performance, just that the functional sensitivity is not as good as the PCT-V; therefore, we suggest that this kit is suitable for clinical use.

Measurement of urinary matrix metalloproteinase-7 for early diagnosis of acute kidney injury based on an ultrasensitive immunomagnetic microparticle-based time-resolved fluoroimmunoassay.

The morbidity and mortality associated with acute kidney injury (AKI) remain obstinately high. Early diagnosis is urgently required and should be pursued in at-risk populations. Recently, a newly validated biomarker, matrix metalloproteinase-7 (MMP-7), was reported as a novel indicator for early AKI prediction and a noninvasive surrogate biomarker of kidney function. Monitoring urinary MMP-7 (uMMP-7) levels fills the gaps in early diagnosis of AKI at early onset. However, the lack of available reagents for its rapid detection limits its use. Herein, we established an ultrasensitive and rapid immunomagnetic microparticles-based time-resolved fluoroimmunoassay to measure urinary MMP-7 in AKI patients. The assay time is 30 min. The calibration curve showed high linear correlation (r = 0.9998) with a linearity of detection of 0.063-150 ng mL-1 and lower limit of detection of 0.039 ng mL-1. The coefficient variation of the intra- and inter-assay lower than 5.17%, and the analytical recovery was 99.06%-105.60%. Testing of clinical samples using the proposed assay and a DUOSET@ ELISA kit showed good correlations in the comparison of uMMP-7 levels (r = 0.9541) and uMMP-7/uCreatinine (r = 0.9595). The proposed assay has satisfactory analytical performance and may serve as a promising tool for early diagnosis of AKI.

Development of a novel immunoassay for the simple and fast quantitation of neutrophil gelatinase-associated lipocalin using europium(III) chelate microparticles and magnetic beads.

Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker for diagnosing acute kidney injury (AKI). Currently, there are few assays for determining NGAL and they are complex, time-consuming or expensive. We aimed to establish an efficient immunoassay to measure NGAL in human urine simply and rapidly. A novel immunoassay for NGAL determination was established by combining a dissociation-enhanced-free time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a "sandwich"-type immunoassay format, analytes in samples were captured by a pair of monoclonal antibodies (mAb) in which one mAb was coated in magnetic beads and the other mAb was labeled with europium(III) chelate microparticles (CM-EUs) as "fluorescent reporters". NGAL concentrations were determined in a linear range (10-1500 ng mL-1) with a limit of detection of 0.32 ng mL-1. The reproducibility, recovery, and specificity of our TRFIA were acceptable. Our method was compared with that of a chemiluminescence immunoassay (CMIA) using 115 urine samples, and the results showed good correlation (R2 = 0.8677). We expect our novel method to be useful for the early diagnosis of AKI.

Europium (III) chelate microparticle-based lateral flow immunoassay strips for rapid and quantitative detection of antibody to hepatitis B core antigen.

Quantitative hepatitis B core antigen (anti-HBc) measurements could play an important role in evaluating therapeutic outcomes and optimizing the antiviral therapy of chronic hepatitis B infection. In this study, we have developed a simple and rapid fluorescence point-of-care test based on a lateral flow immunoassay (LFIA) method integrated with Eu (III) chelate microparticles to quantitatively determine anti-HBc concentrations in serum. This assay is based on a direct competitive immunoassay performed on lateral flow test strips with an assay time of 15 min. The Eu (III) chelate microparticle-based LFIA assay could quantitatively detect anti-HBc levels with a limit of detection of 0.31 IU mL-1, and exhibited a wide linear range (0.63-640 IU mL-1). The intra- and inter-assay coefficients of variation for anti-HBc were both less than 10% and a satisfactory dilution test and accuracy were demonstrated. There were no statistically significant differences in sensitivity or specificity in serum samples between the Eu (III) chelate microparticle-based LFIA strips and the Abbott Architect kit. A simple, rapid and effective quantitative detection of anti-HBc was possible using the Eu (III) chelate microparticle-based LFIA strips. The strips will provide diagnostic value for clinical application.

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

OBJECTIVES: This 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 METHODS: Lanthanum elements labeled antibody and streptavidin-biotin system were used in the "capture sandwich" format simultaneously. RESULTS: The working range of TRFIA for RV IgM was 2-80 AU/mL and for CMV IgM was 5-400 AU/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. CONCLUSION: The novel dual-TRFIA for RV IgM and CMV IgM detection might have valuable clinical application, with satisfactory sensitivity, specificity and accuracy.

Development of an amplified luminescent proximity homogeneous assay for quantitative determination of hepatitis B surface antigen in human serum.

BACKGROUND: Hepatitis B virus (HBV) poses a serious risk to human health and the hepatitis B surface antigen (HBsAg) is a popular biomarker in the diagnosis of HBV infection. A quantitative method with a high degree of accuracy, sensitivity and throughput is needed. METHODS: A novel amplified luminescent proximity homogeneous assay (AlphaLISA) was developed for HBsAg determination. A set of monoclonal antibodies was screened against the main subtypes of HBsAg (adr, ay) to confirm the assay's sensitivity to mutants. Technological processes and reaction conditions were optimized and the assay performance was evaluated. RESULTS: HBsAg concentrations were determined within a linear range of 0.04 to 100 IU ml(-1). The detection sensitivity was established as 0.01 IU ml(-1). Assay sensitivity to mutant HBsAg was achieved through antibody screening. The results demonstrate that the reproducibility, recovery, and specificity of this assay for HBsAg were better than acceptable. Compared with the commercial light-initiated chemiluminescence assay, the correlation coefficient of the novel assay was established as 0.921. CONCLUSIONS: The novel AlphaLISA developed in this study has shorter incubation time and easier protocol than the ones of conventional ELISA. It could be used for the clinical determination of HBsAg in human serum. We established a platform for further development of other biomarkers.

[Development of an amplified luminescent proximity homogeneous immunoassay kit for detecting human hepatitis B virus e antibody].

OBJECTIVE: To develop an amplified luminescent proximity homogeneous immunoassay (AlphaLISA) kit for the detection of human hepatitis B virus e antibody (HBeAb). METHODS: The neutralizing and competitive inhibition method was used to develop the AlphaLISA kit for detection of serum HBeAb. RESULTS: The working range of the kit was 0.003-16 NCU/ml with a sensitivity up to 0.003 NCU/ml. The intra- and inter-assay coefficient of variation was 5.3% and 6.8%, respectively. The kit showed no cross-reaction with HBcAb, and comparison of the detection results with those of a commercially available Elecsys HBeAb kit (Roche) for 136 samples showed a correlation coefficient of 0.961. CONCLUSION: The AlphaLISA kit for HBeAb detection meets the clinical requirements for detection HBeAb in human serum.

[Development of time-resolved fluoroimmunoassay kit for detection of IgM antibodies against hepatitis B core antigen].

OBJECTIVE: To prepare a time-resolved fluoroimmunoassay (TRFIA) kit for clinical detection of IgM antibodies to hepatitis B core antigen (HBc). METHODS: Immunocapture method was used to develop the TRFIA kit for detection of the anti-HBc IgM antibodies, and the precision, cross-reactivity and sensitivity of the kit were tested with the clinical serum samples. RESULTS: The intra- and inter-assay coefficients of variation of the TRFIA kit were 4.8%-7.2% and 7.5%-8.6%, respectively, and no cross-reactivity with anti-HBs, anti-HBc-IgG or anti-HBe was found. Comparison of the results of the TRFIA kit and enzyme-linked immunosorbent assay (ELISA) demonstrated greater sensitivity of the kit than ELISA in detecting the anti-HBc IgM antibodies in 584 serum samples. According to the detection results in 300 serum samples from healthy donors, the cutoff value of the TRFIA kit was 4.5 times of the fluorescence value of the negative control. CONCLUSION: This TRFIA kit for detecting anti-HBc IgM antibodies meets the demand for clinical application and can replace the ELISA kits.