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Hepatitis C virus (HCV) is a single-stranded RNA virus whose genome contains 10,000 nucleotides. Serologic tests for the detection of HCV or HCV antibody (Ab) were not available until recently, when the viral genome was cloned and expressed in the yeast Saccharomyces cerevisiae (3). The enzyme-linked immunosorbent assays ELISA-2, incorporating both structural (core) and nonstructural (NS3 and NS4) antigens (7, 10), and ELISA-3, incorporating recombinant antigens from the core, NS3, NS4, and NS5 regions of the HCV genome (4, 9), have been used worldwide for the routine screening of sera for the presence of anti-HCV Ab (1, 2, 5, 6).

Although these ELISAs are widely used, they require complex instrumentation. Therefore, a simpler method for detecting anti-HCV Ab is desired. Recently, a simple system based on the membrane assay system Ortho HCV Ab Quik Pack (hereinafter termed Quik Pack; Ortho Diagnostic Systems Inc., Tokyo, Japan) has been developed for detecting anti-HCV Ab. In the present study, we evaluated the clinical utility of this system.

A total of 600 serum samples obtained from 600 patients admitted to Nagoya University Hospital, Nagoya, Japan, between September 1996 and November 1996 were analyzed for anti-HCV Ab. Of the 600 samples, 298 were collected from patients whose sera were determined to be HCV positive by at least one ELISA and 302 were from patients with no history of HCV positivity or any liver diseases. After being tested by ELISA, these sera were immediately stored at 80°C until use with the Quik Pack system.

The Quik Pack system is based on enzyme immunoassay (EIA) technology and utilizes a membrane filter disk with a compact housing. The HCV recombinant antigens are blotted in the center of the filter disk (antigen dot), and human immunoglobulin G (as a control) is blotted around the portion of antigen (reference line). The recombinant antigens employed are as follows: c22-3, corresponding to the HCV structural (core) protein; c200, corresponding to the NS3 and NS4 nonstructural regions; and NS5 nonstructural protein. The procedure requires no reagent preparation, washing, or instrumentation and can be completed in 25 min. Each diluted specimen was prepared by adding 25 µl of serum to 400 µl of specimen diluent in a test tube and incubated 5 min. The whole specimen was added to the filter disk (reaction chamber) and allowed to absorb for 5 min. Thereafter, 400 µl of alkaline phosphatase-conjugated anti-human immunoglobulin G monoclonal antibody was added and allowed to absorb for 5 min. Then, 400 µl of 5-bromo-4-chloro-3-indolyl phosphate was added and allowed to absorb for 5 min. Finally, 400 µl of 0.2 N HCl (stop reagent) was added, after which the presence or absence of a colored dot in the center of the filter disk was determined. Samples in which both the HCV antigen dot and the reference line turned deep blue were interpreted as positive. Samples in which only the reference line turned blue were interpreted as negative. These procedures were performed at 15 to 30°C.

The ELISAs used for the detection of anti-HCV Ab were the second-generation Abbott HCV EIA (ELISA-2) (Abbott Diagnostics Division, North Chicago, Ill.) and the third-generation Ortho HCV 3.0 (ELISA-3; Ortho Diagnostic Systems). A third-generation strip immunoblot assay (RIBA-3; Chiron Corporation, Emeryville, Calif.) was utilized for the detection of antibodies which react with the recombinant proteins c33 and NS5, with the synthetic peptide c22, and with a mixture of c100 synthetic peptides. HCV RNA in the seropositive samples was detected with a commercial PCR kit (AMPLICOR-HCV; Roche Diagnostic Systems, Inc., Branchburg, N.J.) (11). To detect small amounts of HCV RNA in the specimens, we used a specific DNA probe-coating latex reagent (AMPLITEX-HCV; Nippon Roche, Co., Tokyo, Japan) (8).

Of the 297 ELISA-2-positive samples, 285 were positive by the Quik Pack assay (sensitivity, 96.0%), and of the 303 ELISA-2-negative samples, 302 were negative by the Quik Pack assay (specificity, 99.7%). The agreement between the results of the two methods was 97.8%. On the other hand, of the 285 ELISA-3-positive samples, 284 were positive by the Quik Pack assay (sensitivity, 99.7%), and of the 315 ELISA-3-negative samples, 313 were negative by the Quik Pack assay (specificity, 99.4%). The percent agreement between the results of the two methods was 99.5%.

Among all of the samples tested, 285 were positive and 298 were negative consistently by all three assays. All of the positive samples were confirmed to be HCV RNA positive by subsequent PCR. All 15 samples which showed discordant results among the assays were further tested for determination of their antibody spectra and the presence of HCV RNA by the RIBA-3 and PCR, respectively. Alanine aminotransferase values from the charts of these patients were also reviewed. Table 1 summarizes the results for the 15 samples that were discordant and the clinical features of these patients. Five (patient no. 1 and 6 through 9) of the 15 samples were nonreactive with all four HCV antigens included in the RIBA-3. Therefore, the positive results for these five samples obtained by any one of the three assays are probably false positives. All of the patients, including the remaining 10 patients whose sera gave indeterminate results, had no clinical history of hepatitis at the time of the test. None of the 15 samples was found to be positive for HCV RNA by PCR.

Satisfactory sensitivity and specificity are achievable with the ELISA-2 and ELISA-3 assays, and these methods are used for screening in clinical laboratories. The Quik Pack exhibited a sensitivity almost identical to those of the ELISA-2 and ELISA-3 for clinical samples. Among 15 samples with different degrees of discordance, the Quik Pack showed false-positive (Quik Pack-positive but PCR-negative) results for only three (patient no. 11, 14, and 15) (Table 1).

In summary, the Quik Pack system is a simple and reliable method for detecting anti-HCV Ab in human serum and requires neither complex reagent preparation nor expensive instrumentation.