Validation of the novel GLAS algorithm as an aid in the detection of liver fibrosis and cirrhosis based on GP73, LG2m, age, and sex.

BACKGROUND: Diagnosis of liver disease at earlier stages can improve outcomes and reduce the risk of progression to malignancy. Liver biopsy is the gold standard for diagnosis of liver disease, but is invasive and sample acquisition errors are common. Serum biomarkers for liver function and fibrosis, combined with patient factors, may allow for noninvasive detection of liver disease. In this pilot study, we tested and validated the performance of an algorithm that combines GP73 and LG2m serum biomarkers with age and sex (GLAS) to differentiate between patients with liver disease and healthy individuals in two independent cohorts. METHODS: To develop the algorithm, prototype immunoassays were used to measure GP73 and LG2m in residual serum samples collected between 2003 and 2016 from patients with staged fibrosis and cirrhosis of viral or non-viral etiology (n = 260) and healthy subjects (n = 133). The performance of five predictive models using combinations of age, sex, GP73, and/or LG2m from the development cohort were tested. Residual samples from a separate cohort with liver disease (fibrosis, cirrhosis, or chronic liver disease; n = 395) and healthy subjects (n = 106) were used to validate the best performing model. RESULTS: GP73 and LG2m concentrations were higher in patients with liver disease than healthy controls and higher in those with cirrhosis than fibrosis in both the development and validation cohorts. The best performing model included both GP73 and LG2m plus age and sex (GLAS algorithm), which had an AUC of 0.92 (95% CI: 0.90-0.95), a sensitivity of 88.8%, and a specificity of 75.9%. In the validation cohort, the GLAS algorithm had an estimated an AUC of 0.93 (95% CI: 0.90-0.95), a sensitivity of 91.1%, and a specificity of 80.2%. In both cohorts, the GLAS algorithm had high predictive probability for distinguishing between patients with liver disease versus healthy controls. CONCLUSIONS: GP73 and LG2m serum biomarkers, when combined with age and sex (GLAS algorithm), showed high sensitivity and specificity for detection of liver disease in two independent cohorts. The GLAS algorithm will need to be validated and refined in larger cohorts and tested in longitudinal studies for differentiating between stable versus advancing liver disease over time.

Instantaneous derivatization technology for the simultaneous and homogeneous detection of multiple double-stranded PCR amplicons.

Herein we report on the development of instantaneous derivatization technology for the homogeneous and simultaneous detection of multiple PCR amplicons specific to the Hepatitis B Virus (HBV) by using three carriers: magnetic beads, polystyrene beads, and thermo-sensitive poly-N-isopropylacrylamide (PNIP). Briefly, PCR amplicons are labeled with digoxin, biotin or FITC via the modified up-stream primers respectively. After PCR amplification, the immunoreactions occur between a mixture of three target PCR amplicons and three modified carriers with anti-digoxin antibody, streptavidin or anti-FITC antibody in a single vessel, and then each carrier is separated from the others under different conditions based on their physio-chemical attributes. And then direct CL detection proceeds via the instantaneous derivatization reaction between intrinsic guanine nucleobases and 3,4,5-trimethoxylphenylglyoxal (TMPG). This new protocol directly measures the double-stranded DNA and therefore does not require a denaturing step, thus offering an enhanced sensitivity due to the absence of competitive hybridization, i.e., the detection limit had a 20-fold improvement on the conventional PCR measurement. Additionally, by comparison of previous guanine based detection formats, this protocol is easy to be used for the detection of any guanine containing targets without the use of guanine-free or inosine-substituted capture probes. Overall, the proposed technique takes the advantages of sensitivity, high-speed and cost-effectivity, which provides a promising alternative for the analysis of multiple PCR targets in a variety of clinical, environmental, and biodefense fields.

Homogeneous temperature- and substrate-resolved technology for a chemiluminescence multianalyte immunoassay.

A novel dual-resolution chemiluminescence (CL) immunoassay platform for the homogeneous determination of four proteins is proposed. The immunoassay is based on temperature- and substrate-resolved technologies. As a proof-of-concept, we evaluated our method for the simultaneous determination of four model proteins (i.e. immunoglobulin A (IgA), IgG, IgM and PEGylated recombinant growth hormone (GH, protein drug)) by using two homogeneous carriers (thermo-sensitive poly-N-isopropylacrylamide (PNIP) and magnetic beads (MB)), and two different CL substrates for alkaline phosphatase (ALP) and horseradish peroxidase (HRP). Briefly, one pair of capture antibodies was immobilized on the PNIP and another pair of capture antibodies was bound to the MB. The four carrier-antibody conjugates were mixed and reacted with the four proteins in a single vessel, and the mixture of two ALP- and two HRP-labeled tracer antibodies was subsequently added to form the four sandwich immunocomplexes. After washing, the two MB conjugates could be easily separated from the two PNIP conjugates by magnetic force while ensuring that the temperature was lower than the lower critical solution temperature (LCST). The CL substrates for ALP and HRP were delivered sequentially into the resultant MB and PNIP solutions. A simple CL setup was employed to perform our novel multiplexed protein assays in the measurement order of ALP and HRP. No obvious cross-reaction was observed. IgA, IgG, IgM and GH were found to be suitably assayed in the ranges of 1.0-50, 0.50-50, 0.50-50 and 0.50-50 ng/mL with limits of detection of 0.50, 0.25, 0.25 and 0.25 ng/mL, respectively. Overall, this simple and homogeneous technique will find applications in areas such as genomics, combinatorial chemistry, drug screening and clinical diagnosis.

Instantaneous derivatization technology for simultaneous and homogeneous determination of multiple DNA targets.

There are potential advantages, in terms of simplicity and speed, for detecting DNA hybridization steps directly without using any external labels, especially for the multiplexed assays. In the current paper, we describe the use of a carrier-resolved label-free multiplexed assay for the simultaneous detection of multiple DNA targets. Herein we demonstrate that this protocol, using three homogeneous carriers thermosensitive poly(N-isopropylacrylamide), polystyrene beads, and magnetic beads, respectively, for simultaneous determination of three short DNA fragments specific to hepatitis B virus. Briefly, one hybridization occurs between a mixture of three different capture probe DNAs immobilized onto three carriers and three targets in a single vessel, and then chemiluminescence (CL) detection proceeds via an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxylphenylglyoxal (TMPG) and the guanine nucleotide-rich regions within the target DNA. An excellent linearity is found within the range between 0.1 and 6.0 pmol with the lowest detection limit of 100 fmol. In contrast to current encoding strategies, every hybridization signal for the corresponding DNA target in our protocol is uniquely immobilized onto one carrier vehicle with a unique and intrinsic physical-chemical signature. Moreover, an instantaneous derivatization reaction is employed for the label-free determination of three targets in a single vessel. In addition, a simple CL setup is employed to read the carrier code instead of an expensive and complicated flow cytometer or imaging system commonly used for multiplexed assays. Further signal amplification is achieved by employing three amplified DNAs for second hybridization, which include a guanine nucleobase-rich sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with one of the target DNA as an amplification platform. Such simple amplified CL transduction allows detection of DNA targets down to the 15-fmol level. This new protocol also provided a good capability in discriminating perfectly complementary DNA from single-base mismatches and noncomplementary sequences. Overall, the protocol described here may have value in a variety of clinical, environmental, and biodefense applications for which the accurate quantitative analysis of multiple DNA targets is desired.

[Studies on the chemical constituents from Fructus Rubi].

Five compounds have been isolated from Fructus Rubi. On the basis of spectral analysis and physicochemical properties, their structures were establised as 4-hydroxy-3-methoxybenzoic acid (I), p-hydroxybenzoic acid (II), gallic acid (III), tiliroside (IV) and ellagic acid (V). I, II and III were found from the plant for the first time.

[Quantitative determination of morphine in opium powder by addition and correlation method using capillary electrophoresis].

The morphine in opium powder has been quantitatively determined by addition and correlation method (ACM), in which capillary zone electrophoresis was applied, and the average recovery was 100.6%. The relative standard deviation (RSD) of migration time was not more than 2.4%, the RSD of relative migration time was not more than 1.1%, and the RSD of the relative area was not more than 0.51%. Meanwhile, the contrast test has been done by the calibration curve method with an internal standard correlation. The content of morphine in opium powder determined by ACM was the same as that by using the calibration curve method with an internal standard correlated. The study shows that ACM is simple, quick and accurate.