A new sensitive and automated chemiluminescent microparticle immunoassay for quantitative determination of hepatitis C virus core antigen.

A new sensitive and automated chemiluminescent assay was developed for the quantitative determination of hepatitis C virus (HCV) core antigen (Ag) in human sera or plasma: the Abbott ARCHITECT HCV Ag test. The assay sensitivity was determined by testing 10 commercial HCV seroconversion panels. Without exception, a positive result for HCV core Ag was observed before anti-HCV detection, resulting in an average reduction in the period between exposure and detection of 35.8 days. Both HCV core Ag and HCV RNA were detected in the panels at the same time, indicating equivalent sensitivity and detectability. A total of 197 HCV specimens comprising genotypes 1a, 1b, 2a, 2b, 3a, 3k, 4a, 5a and 6a were evaluated. Among these, 196 (99.5%), 191 (97%) and 193 (98%) were reactive using the HCV Ag, the immunoradiometric HCV Ag and the Amplicor HCV Monitor 2 assays, respectively. A comparison with the Amplicor HCV Monitor 2 showed a correlation coefficient (r) of 0.74. The specificity of the assay was established at 99.8% by testing 5403 specimens from US volunteer blood donors, hospitalized patients and individuals with medical conditions unrelated to HCV infection, in addition to specimens containing potentially interfering substances.

Development and analytical performance evaluation of an automated chemiluminescent immunoassay for pro-gastrin releasing peptide (ProGRP).

BACKGROUND: Pro-gastrin releasing peptide (ProGRP) concentrations in blood play an important role in the diagnosis and treatment of patients with small cell lung cancer (SCLC). The automated quantitative ARCHITECT ProGRP assay was developed to aid in the differential diagnosis and in the management of SCLC. The purpose of this study was to evaluate the analytical performance of this chemiluminescent microparticle immunoassay at multiple sites. METHODS: ARCHITECT ProGRP measures ProGRP using a two-step sandwich using monoclonal anti-ProGRP antibodies coated on paramagnetic microparticles and labeled with acridinium. Analytical performance of the assay was evaluated at four sites: Abbott Japan, Denka Seiken, the Johns Hopkins University, and the University of Munich. RESULTS: Total precision (%CV) for nine analyte concentrations was between 2.2 and 5.7. The analytical sensitivity of the assay was between 0.20 pg/mL and 0.88 pg/mL. The functional sensitivity at 20% CV was between 0.66 pg/mL and 1.73 pg/mL. The assay was linear up to 50,000 pg/mL using a 1:10 autodilution protocol. The calibration curve was stable for 30 days. Comparison with the Fujirebio microtiter plate enzyme-linked immunosorbent assay (EIA) ProGRP assay gave a slope of 0.93 and a correlation coefficient (r) of 0.99. CONCLUSIONS: These results demonstrate that the ARCHITECT ProGRP assay has excellent sensitivity, precision, and correlation to a reference method. This assay provides a convenient automated method for ProGRP measurement in serum and plasma in hospitals and clinical laboratories.

Clinical significance of small dense low-density lipoprotein cholesterol levels determined by the simple precipitation method.

OBJECTIVE: Recently, we established a simple method for the quantification of small dense LDL cholesterol (C) using heparin-magnesium precipitation. The small dense LDL-C level was identical to cholesterol in the denser LDL fraction with a density of 1.044 to 1.063 g/mL. The aim of this study was to examine clinical significance of this precipitation method for small dense LDL-C. METHODS AND RESULTS: Small dense LDL-C was measured by a direct homogenous LDL-C assay in the supernatant that remained after heparin-magnesium precipitation with density <1.044 lipoproteins. In 313 normolipidemic subjects, the mean value of small dense LDL-C was 31+/-13 mg/dL. In 462 healthy subjects, small dense LDL-C levels were positively correlated with serum triglyceride and LDL-C and were inversely correlated with high-density lipoprotein cholesterol (HDL-C). Combined hyperlipidemia showed the highest small dense LDL-C level among the various types of hyperlipidemia. Patients with type 2 diabetes had an increased small dense LDL-C level (55+/-17). Patients with coronary heart disease also had increased small dense LDL-C levels (53+/-30) irrespective of the presence of diabetes, whereas their LDL-C levels were comparable to those of normolipidemic controls (111+/-31 versus 104+/-22). CONCLUSIONS: These results suggest that measurement of small dense LDL-C by the present precipitation method is useful to evaluate atherogenic risk and may be applicable to routine clinical examination.

Development and evaluation of analytical performance of a fully automated chemiluminescent immunoassay for protein induced by vitamin K absence or antagonist II.

OBJECTIVES: Protein induced by vitamin K absence or antagonist II (PIVKA-II), an abnormal form of prothrombin, has been used as an aid in the diagnosis of hepatocellular cancer (HCC) as a tumor marker. We developed a fully automated quantitative immunoassay for PIVKA-II on the ARCHITECT® i systems. The aim of this study was to evaluate the analytical performance of this assay. DESIGN AND METHOD: Assay imprecision, sensitivity, dilution linearity, high dose hook effect, sample type equivalency, assay interferences of potential interfering materials and correlation with Picolumi PIVKA-II (Eidia, Tokyo, Japan) were evaluated. RESULTS: The percentage coefficient of variation (%CV) of total imprecision ranged from 2.8% to 5.4% with 10 levels of samples. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) were less than 0.63 mAU/mL, 1.62 mAU/mL, and 8.25 mAU/mL, respectively. Linearity up to 30,000 mAU/mL, no high dose hook effect, no difference among sample types and no interference of common drugs and endogenous substances were observed. Correlation study with the Picolumi PIVKA-II gave a correlation coefficient of 0.93 and a regression slope of 1.07. CONCLUSIONS: The results demonstrate that the fully automated prototype ARCHITECT PIVKA-II assay is an accurate, highly sensitive and precise assay for the measurement of PIVKA-II levels in human sera and plasmas.

Cloning of an intracellular D(-)-3-hydroxybutyrate-oligomer hydrolase gene from Ralstonia eutropha H16 and identification of the active site serine residue by site-directed mutagenesis.

An intracellular D(-)-3-hydroxybutyrate (3HB)-oligomer hydrolase gene from Ralstonia eutropha (formerly Alcaligenes eutrophus) H16 was cloned, sequenced, and characterized. As a hybridization probe to screen restriction digests of chromosomal DNA, an extracellular 3HB-oligomer hydrolase gene from Ralstonia pickettii strain (formerly Pseudomonas sp. strain) A1 was used. A specific hybridization signal was obtained and a 6.5-kbp SmaI fragment was cloned in an Escherichia coli phagemid vector. The crude extract from E. coli with this plasmid showed 3HB-trimer hydrolase activity. The subcloned 3.2-kbp fragment still showed 3HB-trimer hydrolase activity in E. coli and expressed an approximately 78-kDa protein in an in vitro transcription-translation system. Nucleotide sequence analysis of the 3.2-kbp fragment showed an open reading frame that encodes a polypeptide with a deduced molecular weight of 78,510. The putative amino acid sequence showed 54% identity with that of the oligomer hydrolase from R. pickettii A1. By site-directed mutagenesis, a novel amino acid sequence (S-V-S*-N-G) containing an essential serine residue in the catalytic center of the enzyme was determined. The gene product was found in PHB-rich cells of R. eutropha by immunodetection. The expressed 3HB-oligomer hydrolase localized both in the supernatant fraction and the PHB granules of the cells.

A novel and simple method for quantification of small, dense LDL.

A preponderance of small, dense (sd) LDL is strongly associated with the development of coronary heart disease, but the method for the measurement of sd LDL is too laborious for clinical use. We report a simple method for the quantification of sd LDL that is applicable to an autoanalyzer. This method consists of two steps: first, to precipitate the lipoprotein of density (d) <1.044 g/ml using heparin-magnesium; and second, to measure LDL-cholesterol in the supernatant by the homogeneous method or apolipoprotein B (apoB) by an immunoturbidometric assay. The cholesterol and apoB values obtained by the precipitation method (45 +/- 26 and 33 +/- 20 mg/dl, respectively) were similar to those obtained in the lipoprotein (d = 1.044-1.063) separated by ultracentrifugation (42 +/- 22 and 31 +/- 17 mg/dl, respectively), and there was an excellent correlation between the two methods for sd LDL-cholesterol (y = 1.05X + 1, r = 0.88, n = 69) and apoB (y = 1.07X, r = 0.90). Sd LDL values had a significant inverse correlation with LDL size. A high correlation was found between sd LDL-cholesterol and apoB values (r = 0.94). Sd LDL value was related to triglyceride, apoB, and LDL-cholesterol, but not to the buoyant LDL level. These results suggest that this precipitation method is a simple and rapid method for the measurement of sd LDL concentration.