Development of an automated chemiluminescent immunoassay for cancer antigen 72-4 and the evaluation of its analytical performance.

Objectives: Cancer antigen (CA) 72-4 assay is widely used for monitoring gastric and ovarian cancers. The antigen is a mucin-like, tumor-associated glycoprotein known as TAG-72. It has been identified and characterized using two different monoclonal antibodies, CC49 and B72.3, which recognize its glycochain epitopes, Galß(1-3) sialyl-Tn and sialyl-Tn antigens, respectively. This study describes the quantitative analytical performance of a newly developed CA 72-4 assay, ARCHITECT CA 72-4. Design: and Methods: The ARCHITECT CA 72-4 assay was developed using the ARCHITECT i2000SRs and three ARCHITECT i1000SRs. The assay performance was evaluated based on guidance from CLSI (Clinical and Laboratory Standards Institute) and correlation against Elecsys CA 72-4. Results: In the total precision study, the minimum coefficient of variation (CV) for Control/Panel samples over 4 U/mL was 1.1%. The measuring interval was from 0.95 to 200 U/mL with good linearity; and limits of blank (LoB), detection (LoD), and quantitation (LoQ) were 0.09, 0.18, and 0.95 U/mL, respectively. High dose hook effect; differences among specimen tube types; and interference of common drugs, potential cross-reactants, and endogenous substances were not observed. Significantly, this assay has high biotin tolerance at 4875 mg/mL and correlates well with the Elecys CA 72-4 assay (correlation coefficient: 0.95). Conclusions: ARCHITECT CA 72-4 is a highly sensitive and precise assay for CA 72-4 measurement in human sera and plasma.

Synthesis and electronic properties of carbazole-based core-modified diporphyrins showing near infrared absorption.

Directly linked carbazole-based core-modified diporphyrin D2 and fused diporphyrin F2 were synthesized. These diporphyrins showed significant electronic interactions and conjugation allowing for redshifted near infrared (NIR) absorption and small HOMO-LUMO gaps as confirmed by NIR absorption spectroscopy, cyclic voltammetry (CV) measurements, and DFT calculations.

Azahelicene-Fused BODIPY Analogues Showing Circularly Polarized Luminescence.

Helical carbazole-based BODIPY analogues were readily synthesized via aza[7]helicenes. The structures of azahelicene-incorporated BF2 dyes were elucidated by x-ray diffraction analysis. DFT calculations revealed that the π-conjugated system expanded from the helicene moiety to the BODIPY framework. The azahelicene-fused boron complexes showed the Cotton effects and the circularly polarized luminescence (CPL) in the visible region. Furthermore, an axially chiral binaphthyl group was attached to the helically chiral dyes, which enhanced the chiroptical properties.

Synthesis and electronic properties of π-expanded carbazole-based porphyrins.

Peripheral π-expansion of carbazole-based porphyrins was achieved for the first time by the Pt-catalyzed cyclization and the incorporation of a benzocarbazole unit. These two types of fused porphyrins showed red-shifted and broad NIR absorption due to the expanded π-conjugation as confirmed by NIR absorption spectroscopy and DFT calculations.

Measurement of microRNA with isothermal DNA amplification on fully automated immunoassay analyzers.

MicroRNAs (miRNAs) in a blood sample are usually measured by quantitative reverse transcription PCR (qRT-PCR), microarray, and next-generation sequencing (NGS) which requires time-consuming pre-treatment, manual operation, and a stand-alone instrument. To overcome these disadvantages, miRNA testing has been developed using the automated analyzers routinely used in clinical laboratories. An isothermal DNA amplification reaction was adapted to a fully automated immunoassay analyzer that conducts extraction, amplification, and detection processes at 37 °C in 44 min. In a reaction vessel, a pre-designed single-stranded signal DNA was amplified in the presence of miRNA, using DNA templates, DNA polymerase, and nicking endonuclease. Then, the amplified signal DNA was hybridized by one DNA probe attached to a magnetic particle and another DNA probe labeled with acridinium ester. After the chemiluminescence reaction, luminescence intensity was automatically measured. The automated assays of cancer-related miRNAs were implemented on the analyzer with throughput of 66 tests per hour. In the assays with one-step amplification, three miRNAs (miR-21-5p, miR-18a-5p, and miR-500a-3p) at concentrations lower than 100 fM were automatically detected and the cross reactivity for miR-21-5p with fifteen similar miRNAs was not higher than 0.02%. In the assay with two-step amplification, detection sensitivity and amplification rate for miR-21-5p were 3 fM and 103-fold, respectively. The coefficient of variations (CVs) in the measurement at the target concentrations from 5 fM to 1000 pM were less than 8%. Furthermore, we also achieved automated nucleic acid detection in human serum. The proposed fully automated miRNA assays showed high sensitivity, low cross reactivity, and reproducibility suitable for clinical use. Graphical abstract.

Epitope characterization of an anti-PIVKA-II antibody and evaluation of a fully automated chemiluminescent immunoassay for PIVKA-II.

OBJECTIVES: Protein induced by vitamin K absence or antagonist-II (PIVKA-II) has been used as a tumor marker to aid in the diagnosis of hepatocellular carcinoma (HCC). We developed an anti-PIVKA-II monoclonal antibody, 3C10, and a fully automated quantitative immunoassay for PIVKA-II on the ARCHITECT® i-systems. The aim of this study was to characterize the epitope of 3C10 and to evaluate the reactivity to PIVKA-II of this assay. METHODS: The epitope characterization was examined by using prothrombin γ-carboxyglutamic acid residues (Gla) domain polypeptides which are amino acid residues 17-27 that include four Gla residues at positions 19, 20, 25 and 26. The correlation with Picolumi PIVKA-II MONO (Eidia, Tokyo, Japan) and tube type equivalency was evaluated by using the developed fully automated quantitative immunoassay. RESULTS: Peptides having glutamic acid residues (Glu) at Gla domains strongly reacted to 3C10 but lost reactivity when the Glu at positions 19 or 20 was changed to Gla. The results were equivalent with an existing in vitro diagnostics product for PIVKA-II using the MU-3 antibody. A correlation study with the Picolumi PIVKA-II MONO gave a correlation coefficient of 0.99 and a regression slope of 0.92. No difference between a plain serum tube and a rapid serum tube including thrombin (RST) was observed on ARCHITECT PIVKA-II. CONCLUSIONS: The results demonstrate that this anti-PIVKA-II antibody detects equivalent epitopes with MU-3 and has equivalent reactivity to PIVKA-II as MU-3. Moreover, the ARCHITECT PIVKA-II assay has good correlation with the existing PIVKA-II product, and is applicable for use with RST.

Production of free methylarginines via the proteasome and autophagy pathways in cultured cells.

ω-NG-monomethylarginine (MMA) and asymmetric ω-NG, ω-NG-dimethylarginine (ADMA), are endogenous competitive inhibitors for three isoforms of nitric oxide synthase (NOS). Although free methylarginines are thought to be liberated through the intracellular proteolysis of proteins methylated by protein arginine methyltransferases (PRMTs), the degradation pathways of the arginine-methylated proteins involved in the biosynthesis of free methylarginines have yet to be determined. In this study, the biosynthesis of free methylarginines with cultured cells was analyzed as follows: first, we established a method for quantifying trace amounts of free intracellular methylarginines by means of ultra high­performance liquid chromatography (UPLC). Second, we determined the type of methylation produced in the cultured cell lines using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry (MALDI-QIT-TOF/MS). Finally, we investigated whether methylarginines are generated via the proteasome and autophagy pathways, the primary intracellular protein degradation systems. By using specific inhibitors for each pathway, we found that the blockade of proteasome activity reduced the amount of free ADMA and symmetric ω-NG, ω-N'G-dimethylarginine (SDMA), while the inhibition of autophagy significantly reduced cellular ADMA only. These results suggest that both the proteasome and autophagy pathways play an essential role in the production of free methylarginines.