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.

Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

Methods to evaluate the impact of SARS-CoV-2 nucleocapsid mutations on antigen detection by rapid diagnostic tests.

Mutations in the nucleocapsid of SARS-CoV-2 may interfere with antigen detection by diagnostic tests. We used several methods to evaluate the effect of various SARS-CoV-2 nucleocapsid mutations on the performance of the Panbio™ and BinaxNOW™ lateral flow rapid antigen tests and a prototype high-throughput immunoassay that utilizes Panbio antibodies. Variant detection was also evaluated by immunoblot and BIAcore™ assay. A panel of 23 recombinant nucleocapsid antigens (rAgs) were produced that included mutations found in circulating SARS-CoV-2 variants, including variants of concern. All mutant rAgs were detected by all assays, at a sensitivity equivalent to wild-type control (Wuhan strain). Thus, using a rAg approach, we found that the SARS-CoV-2 nucleocapsid mutations examined do not directly impact antigen detection or antigen assay performance.

Development of a new automated IL-6 immunoassay.

OBJECTIVES: Quantitative detection of interleukin-6 (IL-6) in serum and plasma can help monitor immune responses and the development of acute inflammation to guide patient management. We developed an IL-6 immunoassay for use with the automated ARCHITECT system for detecting an increase in the inflammatory response. METHODS: Immunized mouse sera were tested and selected B-cells were harvested for fusion with myeloma cells. A panel of monoclonal antibodies were produced, from which capture and detection monoclonal antibodies for the prototype IL-6 immunoassay were selected and screened on the ARCHITECT instrument. The antibody pair that most effectively captured and detected IL-6 was selected to develop a prototype IL-6 immunoassay. Calibrator and panel preparations using an internal recombinant IL-6 standard were compared to serum panels prepared with the IL-6 International Standard 89/548. Assay specificity and spike recovery were determined, and assay sensitivity was compared with the Roche EUA Elecsys IL-6 assay run on the cobas analyzer. RESULTS: Twenty-one antibodies in 441 antibody pairs were screened. The prototype IL-6 assay showed high sensitivity with an estimated limit of detection of 0.317 pg/mL and limit of quantitation of <1.27. Spike recovery was 90%-110% in serum and plasma. The internal recombinant human IL-6 calibrator showed excellent stability for 63 days at 2-8 °C. The prototype IL-6 immunoassay was specific for IL-6, exhibited no cross reactivity to related cytokines and interleukins, and was 10-fold more sensitive than the Elecsys IL-6 assay. CONCLUSIONS: The prototype ARCHITECT IL-6 automated immunoassay is a reliable and robust method for the quantitative determination of IL-6 in human serum and plasma.

Generation and characterization of chimeric antibodies against NS3, NS4, NS5, and core antigens of hepatitis C virus.

Mouse-human chimeric antibodies (cAbs) against hepatitis C virus (HCV) core, NS3 (nonstructural), NS4, and NS5 antigens were developed as quality control (QC) reagents to replace the use of human sera/plasma for Abbott HCV immunoassays. The cAb retains the mouse monoclonal antibody (MAb) specificity and affinity but still reacts in the existing HCV assay format, which measures human anti-HCV immunoglobulin. Mouse heavy-chain (V(H)) and light-chain (V(L)) variable regions of anti-HCV core, NS3, NS4, and NS5 antigens were PCR amplified from hybridoma lines and then cloned with human IgG1 heavy-chain (C(H)) and light-chain (C(L)) constant regions, respectively. A single mammalian expression plasmid containing both heavy-chain and light-chain immunoglobulin genes was constructed and transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells. The transfected CHO cells were selected using hypoxanthine- and thymidine-free medium and screened by an enzyme immunoassay (EIA). The clone secreting the highest level of antibody was isolated from the CHO transfectants and further subcloned. Each cAb-expressing CHO cell line was weaned into serum-free medium, and the cAb was purified by protein A affinity chromatography. The levels of cAb production for the various CHO cell lines varied from 10 to 20 mg/liter. Purified anti-HCV cAbs were tested with Abbott HCV immunoassays and showed reactivity. Moreover, yeast surface display combined with alanine-scanning mutagenesis was used to map the epitope at the individual amino acid level. Our results suggest that these HCV cAbs are ideal controls, calibrators, and/or QC reagents for HCV assay standardization.

Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology.

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.