Zhi-Ning Dong

A Novel Immunoassay for the Quantization of CYFRA 21–1 in Human Serum

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.

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 assays 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.

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.

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

Measurement of the free β subunit of human chorionic gonadotropin (free β-hCG) in serum is useful for prenatal screening. Concentrations of free β-hCG vary in different races. Conventional assays used for such measurements have limitations. We applied the AlphaLISA to measure levels of free β-hCG in maternal serum during 8–20 weeks of gestation in women from southern China. Two anti-free β-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 Eu3+-labeled free β-HCG-TRFIA assay was good (y = 1.045x + 1.580, r2 = 0.978). Median levels of free β-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 β-HCG could become the assay of choice for applications in clinical diagnostics. The established median value of free β-HCG is helpful in clinical diagnosis specific for southern Chinese women.

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.

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 Eu3+‐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. J. Med. Virol. 87:1940–1945, 2015.

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.

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.