Development of a recombinant cell-based indirect immunofluorescence assay (RC-IFA) for the determination of autoantibodies against "rings and rods"-associated inosine-5'-monophosphate dehydrogenase 2 in viral hepatitis C.

BACKGROUND AND AIMS: Autoantibodies against so-called "rings and rods" structures, as determined by indirect immunofluorescence assay (IFA) using the human cell line HEp-2, have been described in chronic hepatitis C virus (HCV) infected patients treated with interferon/ribavirin. Recently, cytidine triphosphate synthase (CTPS) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), the enzyme inhibited by ribavirin, were proposed as the target antigens. We wanted to confirm the identification and setup a robust system for autoantibody testing in routine laboratories. METHODS: CTPS and IMPDH2 were individually expressed in HEK293 cells and the recombinant cells were used in IFA (RC-IFA) to analyze sera from 33 anti-"rings and rods" antibody positive individuals with unknown diagnosis, 50 patients with chronic HCV infection, 100 with autoimmune hepatitis, 50 with primary biliary cirrhosis and 50 healthy blood donors. RESULTS: We found that all sera with anti-"rings and rods" reacted with recombinant IMPDH2 but none with CTPS. In western blot or ELISA, anti-IMPDH2 positive sera reacted only weakly, if at all, with Escherichia coli derived recombinant IMPDH2 indicating that the autoantibody reaction probably depends on the 3-dimensional conformation of the antigen. A rabbit hyperimmune serum raised against bacterially expressed IMPDH2 produced the ring/rods pattern in IFA using HEp-2. CONCLUSIONS: We conclude, that IMPDH2 is indeed the main target of anti-"rings and rods" while CTPS is an unlikely target. Moreover, the novel RC-IFA test system allows a standardized semi-quantitative determination of anti-IMPDH2 basing on a defined recombinant antigen.

New enzymatic assay for serum urea nitrogen using urea amidolyase.

We established an enzymatic assay for measurement of serum urea nitrogen using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on hydrolysis of urea by the enzyme. In this assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (EC 1.4.1.4). Then in the presence of urea amido-lyase, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion was produced proportionally to urea concentration in serum. The concentra-tion of ammonium ion formed was determined by adding GLDH to produce NADP(+) in the presence of 2-oxoglutarate and NADPH. We then monitored the change of absorbance at 340 nm. The inhibitory effect of calcium ion on this assay was eliminated by adding glyco-letherdiamine-N, N, N', N'-tetraacetic acid to the reaction system. The with-in-assay coefficient of variations (CVs) of the present method were 1.80-3.76% (n = 10) at 2.8-19.0 mmol/L, respectively. The day-to-day CVs were 2.23-4.59%. Analytical recovery was 92-115%. The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, ammo-nium ion, or calcium ion did not affect this assay system. The correlation be-tween values obtained with the present method (y) and those by another enzy-matic method (x) was 0.997 (y = 1.02x - 0.10 mmol/L, Sy/x = 0.841, n = 100), with a mean difference of -0.18 +/- 0.86 mmol/L [(values by reference method - that of present method) +/- SD] using the Bland-Altman technique. J. Clin. Lab. Anal. 17:52-56, 2003.

Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells.

Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood cells isolated from healthy volunteers by density centrifugation and elutriation centrifugation. We also investigated the mRNA expression of CTP synthase in lymphocytes and monocytes. The highest activity of CTP synthase was found in thrombocytes (6.48 nmol CTP x mg(-1) x h(-1)), followed by that of monocytes (2.23), lymphocytes (1.69), granulocytes (0.52) and erythrocytes (0.42). The activity of CTP synthase in whole blood samples was at an intermediate level (1.27). The mRNA expression of CTP synthase in monocytes was comparable to that observed in lymphocytes.