Detection of hepatitis C after liver transplantation. Four serologic tests compared.

To determine the best method for detecting HCV infection in immunosuppressed patients, stored frozen serum from 101 liver transplant recipients was tested for hepatitis C virus. Each sample was tested by four assays. HCV RNA was detected by both polymerase chain reaction (PCR) and branched DNA signal amplification. Antibody to HCV was determined using second-generation enzyme-linked immunoassay (EIA) and recombinant immunoblot assay. Forty one transplant recipients met the working definition for true positives of HCV infection. Of these "true positives," 98% were positive by HCV RNA PCR assay, 88% by b-DNA signal amplification assay, 88% by anti-HCV EIA, and 63% demonstrated two or more reactive bands on recombinant immunoblot. Five of 57 (9%) HCV-antibody negative recipients had HCV RNA detected by both methods. Of 44 HCV enzyme-linked immunoassay (EIA) repeatedly reactive samples, the recombinant immunoblot was negative in 2 and indeterminate in 13. HCV RNA was present in 9 of 13 recombinant immunoblot indeterminate sera. Nine EIA repeatedly reactive sera were negative by both tests for HCV RNA. In liver transplant recipients, HCV infection is best determined by measurement of HCV RNA. Antibody formation may be delayed or suppressed in a minority of patients despite > 10(9) equivalents/L (> 10(6)/mL) of HCV RNA in serum. Recombinant immunoblots with a single reactive band pattern often indicate HCV infection in immunosuppressed patients.

New generation RIBA hepatitis C strip immunoblot assays.

Second generation hepatitis C virus (HCV) Elisas are currently in use in Europe and have been submitted for approval in the United States. These new assays contain additional antigens from the putative nucleocapsid and NS-3 regions of the HCV genome in addition to the c100-3 antigen present in first generation Elisas. A supplementary test, the second generation RIBA (Chiron Co trademark) HCV strip immunoblot assay (2-RIBA HCV SIA) has also been developed. The strip immuno-blot assay uses four recombinant HCV antigens (5-1-1 [NS-4], c100-3 [NS-4], c33c [NS-3], and c22-3 [NS-3 [nucleocapsid]) slot blotted on nitrocellulose. Screening of random volunteer blood donors with the Ortho (Ortho Diagnostic Systems trademark) second generation HCV Elisa (2-Ortho HCV Elisa) indicates that a substantial change in the repeat reactive donor population is observed with the new test. Two notable features of this change are: i) a large number of samples reactive in the 2-RIBA HCV SIA for the second generation antigens, c33c and c22-3, are detected by the 2-Ortho HCV Elisa; ii) the percentage of 2-Ortho HCV Elisa reactive specimens found indeterminate (reactive for only one HCV antigen) by the 2-RIBA HCV SIA is higher than in first generation HCV Elisas (approximately 25 versus 5%). In addition, 2-ortho HCV Elisa repeat reactive, 2-RIBA HCV SIA indeterminate samples are dominated by reactivity to c22-3 instead of c100-3 which is the case for first generation HCV Elisa repeat reactive samples.(ABSTRACT TRUNCATED AT 250 WORDS)

New-generation RIBA hepatitis C strip immunoblot assays.

Second-generation hepatitis C virus (HCV) ELISAs are currently in use in Europe and have been submitted for approval in the United States. These new assays contain additional antigens from the putative nucleocapsid and NS-3 regions of the HCV genome in addition to the c100-3 antigen present in first-generation ELISAs. A supplementary test, the second-generation RIBA HCV strip immunoblot assay (2-RIBA HCV SIA) has also been developed. The strip immunoblot assay uses four recombinant HCV antigens [5-1-1 (NS-4), c100-3 (NS-4), c33c (NS-3), and c22-3 (NS-3) (nucleocapsid)] slot blotted on nitrocellulose. Screening of random volunteer blood donors with the Ortho second-generation HCV ELISA (ORTHO HCV 2.0 ELISA) indicates that a substantial change in the repeat reactive donor population is observed with the new test. Two notable features of this change are: (1) A large number of samples reactive in the 2-RIBA HCV SIA for the second-generation antigens, c33c and c22-3, are detected by the ORTHO HCV 2.0 ELISA; (2) the percentage of ORTHO HCV 2.0 ELISA reactive specimens found indeterminate (reactive for only one HCV antigen) by the 2-RIBA HCV SIA is higher than in first-generation HCV ELISAs (approximately 25 vs. 5%). In addition, ORTHO HCV 2.0 ELISA repeat reactive, 2-RIBA HCV SIA indeterminate samples are dominated by reactivity to c22-3 instead of c100-3, which is the case for first-generation HCV ELISA repeat reactive samples. Resolution of 2-RIBA HCV SIA indeterminate samples as either containing anti-HCV antibodies or not, is important in both diagnostic and blood screening environments, especially where donor notification is required. Our approach to resolution of these troublesome samples evolved from initial work with HCV peptides. Early studies with an experimental strip immunoblot assay containing 5 peptides from the nucleocapsid, E2 (NS-1), NS-4, and NS-5 regions of the viral genome indicated that peptides from the nucleocapsid and NS-4 regions of the genome could provide additional evidence for the presence of anti-HCV antibodies with good specificity, but other peptides suffered from poor specificity. In addition, no immunoreactive peptide from the NS-3 (c33c) region of the virus is available, presumably because the major epitope(s) of this key second-generation antigen is a conformational determinant.(ABSTRACT TRUNCATED AT 400 WORDS)

Substituted hemins as probes for structure-function relationships in horseradish peroxidase.

Low temperature visible spectra of Compounds I from peroxidases reconstituted with protohemin, 2-formyl-4-vinyldeuterohemin, 2-vinyl-4-formyldeuterohemin, 2,4-dimethyldeuterohemin, and 2,4-diacetyldeuterohemin reveal that these Fe(IV) porphyrin pi-cation radicals take the 2A2u of peroxidase-type electronic ground state. Compound I of deuterohemin horseradish peroxidase, however, takes the 2A1u or catalase type pi-cation radical electronic ground state. Since deuterohemin horseradish peroxidase possesses no catalase activity, the structure of the peroxidase apoprotein (other than those factors which might influence the Compound I pi-cation radical ground state) is concluded to play the major role in determining the reactivity of Compound I toward hydrogen donors. Studies on peroxidases substituted with the hemins 2-formyl-4-vinyldeuterohemin, 2-vinyl-4-formyldeuterohemin, 2,4-dimethyldeuterohemin, and mesohemin reveal that isoelectronic hemins differentially interact with the peroxidase apoprotein. The hemin 2- and 4-substituents are therefore concluded to interact sterically with the horseradish peroxidase apoprotein. While a variety of 2- and 4-substituted hemins were observed to bind rapidly with apo horseradish peroxidase to form active substituted enzymes, small changes in the substituents in the 6- and 7-positions had drastic effects on the rates of binding to apoperoxidase and the activities of the reconstituted enzymes. Even addition of a single methylene to form butyrate instead of propionate side chains drastically altered the rate of binding of the hemin and the activity of the substituted enzyme. It therefore appears that while the 2-, 4-, 6-, and 7-substituents of the hemins in horseradish peroxidase all interact with the protein, the polypeptide chain possesses more conformational flexibility in the area which binds the 2- and 4-substituents.