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Comparison of Enzyme Immunoassays for Detection of Antibodies to Hepatitis D Virus in Serum Siu-Kei Chow,a* Ederlyn E. Atienza,a Linda Cook,a Harry Prince,b* Patricia Slev,c Mary Lapé-Nixon,b Keith R. Jeromea,d Department of Laboratory Medicine, University of Washington, Seattle, Washington, USAa; Focus Diagnostics Reference Laboratory, San Juan Capistrano, California, USAb; Department of Pathology, University of Utah, Salt Lake City, Utah, USAc; The Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington, USAd
Serology remains critical for diagnosing hepatitis D virus (HDV) infection, which affects 15 to 20 million people worldwide, but the literature on characterizing commercial enzyme immunoassays (EIAs) dates back to 15 years ago. We evaluated 2 commercial EIAs currently available for detecting anti-HDV antibodies. The DiaSorin assay demonstrated 100% sensitivity and specificity. Using a modified cutoff value, the Cusabio assay demonstrated a sensitivity of 81.3% and specificity of 90.9%. Our data show that recently developed EIAs are reliable for anti-HDV antibody detection.
H
epatitis D virus (HDV) is a small, defective RNA virus that requires hepatitis B virus (HBV) surface antigen (HBsAg) for replication and propagation. Among the estimated 240 million people worldwide with chronic HBV infection, 15 to 20 million are estimated to be coinfected with HDV (1). Individuals coinfected with both viruses have more severe liver disease, faster progression, and poorer prognosis than those with HBV infection alone (2, 3). While serology remains critical for diagnosing HDV infection, most studies in this area were performed in the late 1980s and early 1990s (4–7). The assays described in those studies have been discontinued or are no longer available in the United States. More recently developed HDV immunoassays have been mainly limited to research purposes or clinical use in local institutions (8–10). In this study, we evaluated two commercially available enzyme immunoassays (EIAs) for detecting anti-HDV antibodies and compared the data with those obtained in two reference laboratories. A total of 87 serum specimens initially submitted to ARUP Laboratories (ARUP) or Focus Diagnostics Reference Laboratory (Focus) between March 2014 and June 2014 for evaluation of HDV antibodies were randomly selected and analyzed with both reference enzyme-linked immunosorbent assays (ELISAs). All 87 deidentified specimens were kept at ⫺80°C before being sent to University of Washington and tested for HDV antibodies using commercially available kits from DiaSorin (Saluggia, Piedmont, Italy) and Cusabio (Wuhan, Hubei, China). Each of the kits used different HDV antigen preparations, as well as a variety of conjugate detection methods. The DiaSorin kit measures total Ig to HDV as a qualitative competitive ELISA, while the Cusabio ELISA kit qualitatively measures IgG. According to the manufacturer, the cutoff value for the DiaSorin kit is defined as (0.5 ⫻ mean negative
control) ⫹ (0.5 ⫻ mean positive control), whereas the cutoff for the Cusabio kit is set as 0.2 ⫹ mean negative control. Microtiter plates were read on the Epoch Microplate Spectrophotometer (BioTek, Winooski, VT) using the Gen5 data analysis software. HDV total antibodies were measured at Focus using a proprietary laboratory-developed assay. Briefly, serum samples diluted 1:101 in phosphate-buffered saline with 0.1% Tween 20 (PBST) containing 0.1% bovine serum albumin were added to microtiter wells coated with a proprietary recombinant HDV protein (GenScript, Piscataway, NJ). After incubation at room temperature (RT) for 1 h and 3 washes with PBST, wells received horseradish peroxidase (HRP)-conjugated F(ab=)2 fragment goat anti-human IgG⫹IgM⫹IgA (Jackson ImmunoResearch, West Grove, PA). After incubation at RT for 30 min, wells were washed and then received tetramethylbenzidine (Moss Inc., Pasadena, MD). The optical density at 450 nm (OD450) was measured using an ELISA
Received 17 January 2016 Returned for modification 23 February 2016 Accepted 30 May 2016 Accepted manuscript posted online 8 June 2016 Citation Chow S-K, Atienza EE, Cook L, Prince H, Slev P, Lapé-Nixon M, Jerome KR. 2016. Comparison of enzyme immunoassays for detection of antibodies to hepatitis D virus in serum. Clin Vaccine Immunol 23:732–734. doi:10.1128/CVI.00028-16. Editor: R. L. Hodinka, University of South Carolina School of Medicine Greenville Address correspondence to Keith R. Jerome, [email protected]. * Present address: Siu-Kei Chow, MultiCare Health System, Tacoma, Washington, USA; Harry Prince, Viracor-IBT Laboratories, Los Angeles, California, USA. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
TABLE 1 Performance of Cusabio and DiaSorin EIA kits compared to that of reference laboratory methods No. of samples giving the following results compared to reference laboratory methodsa ELISA kit c
Cusabio DiaSorin a b c
⫹/⫹
⫹/⫺
⫺/⫺
⫺/⫹
Sensitivity (% [95% CI])b
Specificity (% [95% CI])
26 32
5 0
50 55
6 0
81.3 (63.0–92.1) 100 (86.7–100)
90.9 (79.3–96.6) 100 (91.9–100)
⫹/⫹, true positive; ⫹/⫺, false positive; ⫺/⫺, true negative; ⫺/⫹, false negative. CI, confidence interval. A proposed cutoff value (0.14 ⫹ mean negative control) was used.
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reader (BioTek). Results were expressed as an index value, calculated by dividing the patient sample OD by the OD of a calibrator serum included in every run. Index values of ⬍0.90 were interpreted as negative, 0.90 to 1.10 as equivocal, and ⬎1.10 as positive. The reference assay at ARUP utilized a commercial kit that detects HDV total antibodies following the manufacturer’s instruction (International Immuno-Diagnostics, Foster City, CA). Briefly, proprietary HDV antigen was precoated to microtiter wells. A patient sample was added to the well in the presence of HRP-conjugated polyclonal antibodies for HDV. The plate was washed and then incubated with tetramethylbenzidine. The final OD was given by OD450 subtracted by OD620. The result was interpreted as the ratio between the cutoff value and the specimen value, where the cutoff is defined as 0.2 ⫻ (mean negative control ⫹ mean positive control). Ratios of ⬍0.90 were interpreted as negative, 0.90 to 1.10 as equivocal, and ⬎1.10 as positive. The HDV antigens used in the DiaSorin and Cusabio kits are proprietary. The DiaSorin kit has a competitive binding assay format, whereas the Cusabio kit is a direct binding assay. Each positive control was found to be specific to its own kit and showed no binding against the other (data not shown), suggesting that the HDV antigens in the kits are antigenically distinct. The lack of cross-reactivity between the two positive controls may be attributable to different types of HDV being used to raise antibodies. The genetic diversity of HDV, which confers different antigenic properties, has been shown to be related to the geographic origin of the isolates (11). The Cusabio and DiaSorin assays used in our study are manufactured in China and Italy, respectively. The linearity of each assay was assessed by serially diluting the positive control materials provided in the kits. For the DiaSorin kit, the OD-concentration curve generated by 8 data points using 1:2 serial dilutions of the positive control yielded an R2 value of 1 using the 4-parameter analysis of Gen5 data analysis software. For the Cusabio kit, 7 data points from 1:4 serial dilution of the positive control also yielded an R2 value of 1 using the 4-parameter analysis. To examine the limit of detection, a positive sample was tested in dilutions with both kits. The sample was 1:5 serially diluted, and the lowest positive titer was 1:3,125 for the DiaSorin kit; the same sample was serially diluted 1:4, and the lowest positive titer for the Cusabio kit was 1:16. Of 87 clinical samples tested at ARUP and Focus using their reference assays, 32 were positive and 55 were negative for antiHDV antibodies. The samples were then tested with the DiaSorin and Cusabio kits. The DiaSorin kit demonstrated sensitivity and specificity of 100% relative to the reference laboratory results (Table 1). Using the cutoff suggested by the manufacturer (0.2 ⫹ mean negative control), the overall sensitivity of the Cusabio kit was 53.1%. Because of the low sensitivity, we proposed a new cutoff defined as 0.14 ⫹ mean negative control based on the distribution of the false-negative readouts (Fig. 1). By doing so, the overall sensitivity of the Cusabio kit increased to 81.3% (Table 1). Of the 11 discrepant results between the Cusabio assay and the reference methods, 6 were false negatives and 5 were false positives. The discrepancies were attributable to the lower sensitivity for the Cusabio assay and a lack of specificity when the lower cutoff OD was used for analysis. The limitation of this study includes the poorly understood structural and antigenic property of HDV antigens in the commercial kits. In summary, results of this study show that the currently available commercial EIAs can effectively detect antibodies to
August 2016 Volume 23 Number 8
FIG 1 Distribution of EIA results for anti-HDV antibodies from clinical se-
rum specimens (n ⫽ 87) tested with Cusabio kit against Focus reference assay (A), Cusabio kit against ARUP reference assay (B), DiaSorin kit against Focus reference assay (C), and DiaSorin kit against ARUP assay (D).
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HDV. The DiaSorin kit appears to have better sensitivity and specificity and shows performance comparable to that of the reference methods. ACKNOWLEDGMENT We acknowledge the excellent support of personnel of the Molecular Virology Laboratory at the University of Washington, Seattle, Washington.
REFERENCES 1. Ott JJ, Stevens GA, Groeger J, Wiersma ST. 2012. Global epidemiology of hepatitis B virus infection: new estimates of age-specific HBsAg seroprevalence and endemicity. Vaccine 30:2212–2219. http://dx.doi.org/10 .1016/j.vaccine.2011.12.116. 2. Fattovich G, Giustina G, Christensen E, Pantalena M, Zagni I, Realdi G, Schalm SW. 2000. Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. The European Concerted Action on Viral Hepatitis (Eurohep). Gut 46:420 – 426. http://dx .doi.org/10.1136/gut.46.3.420. 3. Romeo R, Del Ninno E, Rumi M, Russo A, Sangiovanni A, de Franchis R, Ronchi G, Colombo M. 2009. A 28-year study of the course of hepatitis Delta infection: a risk factor for cirrhosis and hepatocellular carcinoma. Gastroenterology 136:1629 –1638. http://dx.doi.org/10.1053/j.gastro.2009.01.052. 4. Dubois F, Goudeau A. 1988. Kinetics of delta antigen and delta antibody in acute delta hepatitis: evaluation with different enzyme immunoassays. J Clin Microbiol 26:1339 –1342.
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5. Bezeaud A, Rosenswajg M, Guillin MC. 1989. Evaluation of five hepatitis delta virus marker assays for detection of antigen and antibody. J Clin Microbiol 27:2880. 6. Govindarajan S, Valinluck B, Lake-Bakkar G. 1991. Evaluation of a commercial anti-delta EIA kit for detection of antibodies to hepatitis delta virus. Am J Clin Pathol 95:240 –241. http://dx.doi.org/10.1093/ajcp/95.2 .240. 7. Shattock AG, Morris MC. 1991. Evaluation of commercial enzyme immunoassays for detection of hepatitis delta antigen and anti-hepatitis delta virus (HDV) and immunoglobulin M anti-HDV antibodies. J Clin Microbiol 29:1873–1876. 8. Kuo YB, Chao M, Lee YH, Yeh CT, Chan EC. 2012. New enzyme-linked immunosorbent assay for detection of antibodies against hepatitis delta virus using a hepatitis delta antigen derived from a Taiwanese clone and comparison to the Abbott radioimmunoassay. Clin Vaccine Immunol 19: 817– 819. http://dx.doi.org/10.1128/CVI.05687-11. 9. Shen L, Gu Y, Sun L, Yang Y, Wang F, Li Y, Bi S. 2012. Development of a hepatitis delta virus antibody assay for study of the prevalence of HDV among individuals infected with hepatitis B virus in China. J Med Virol 84:445– 449. http://dx.doi.org/10.1002/jmv.23212. 10. Xie L, Huang D-Z, He L-X, Luo Z-X, Zhou Y-S, Wu X-D. 2009. Detection of antibody to hepatitis delta virus in human serum by double antigen sandwich ELISA. Virol Sin 24:44 –51. 11. Le Gal F, Gault E, Ripault MP, Serpaggi J, Trinchet JC, Gordien E, Deny P. 2006. Eighth major clade for hepatitis delta virus. Emerg Infect Dis 12:1447–1450. http://dx.doi.org/10.3201/eid1209.060112.
Clinical and Vaccine Immunology
August 2016 Volume 23 Number 8
Comparison of Enzyme Immunoassays for Detection of Antibodies to Hepatitis D Virus in Serum Siu-Kei Chow,a* Ederlyn E. Atienza,a Linda Cook,a Harry Prince,b* Patricia Slev,c Mary Lapé-Nixon,b Keith R. Jeromea,d Department of Laboratory Medicine, University of Washington, Seattle, Washington, USAa; Focus Diagnostics Reference Laboratory, San Juan Capistrano, California, USAb; Department of Pathology, University of Utah, Salt Lake City, Utah, USAc; The Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington, USAd
Serology remains critical for diagnosing hepatitis D virus (HDV) infection, which affects 15 to 20 million people worldwide, but the literature on characterizing commercial enzyme immunoassays (EIAs) dates back to 15 years ago. We evaluated 2 commercial EIAs currently available for detecting anti-HDV antibodies. The DiaSorin assay demonstrated 100% sensitivity and specificity. Using a modified cutoff value, the Cusabio assay demonstrated a sensitivity of 81.3% and specificity of 90.9%. Our data show that recently developed EIAs are reliable for anti-HDV antibody detection.
H
epatitis D virus (HDV) is a small, defective RNA virus that requires hepatitis B virus (HBV) surface antigen (HBsAg) for replication and propagation. Among the estimated 240 million people worldwide with chronic HBV infection, 15 to 20 million are estimated to be coinfected with HDV (1). Individuals coinfected with both viruses have more severe liver disease, faster progression, and poorer prognosis than those with HBV infection alone (2, 3). While serology remains critical for diagnosing HDV infection, most studies in this area were performed in the late 1980s and early 1990s (4–7). The assays described in those studies have been discontinued or are no longer available in the United States. More recently developed HDV immunoassays have been mainly limited to research purposes or clinical use in local institutions (8–10). In this study, we evaluated two commercially available enzyme immunoassays (EIAs) for detecting anti-HDV antibodies and compared the data with those obtained in two reference laboratories. A total of 87 serum specimens initially submitted to ARUP Laboratories (ARUP) or Focus Diagnostics Reference Laboratory (Focus) between March 2014 and June 2014 for evaluation of HDV antibodies were randomly selected and analyzed with both reference enzyme-linked immunosorbent assays (ELISAs). All 87 deidentified specimens were kept at ⫺80°C before being sent to University of Washington and tested for HDV antibodies using commercially available kits from DiaSorin (Saluggia, Piedmont, Italy) and Cusabio (Wuhan, Hubei, China). Each of the kits used different HDV antigen preparations, as well as a variety of conjugate detection methods. The DiaSorin kit measures total Ig to HDV as a qualitative competitive ELISA, while the Cusabio ELISA kit qualitatively measures IgG. According to the manufacturer, the cutoff value for the DiaSorin kit is defined as (0.5 ⫻ mean negative
control) ⫹ (0.5 ⫻ mean positive control), whereas the cutoff for the Cusabio kit is set as 0.2 ⫹ mean negative control. Microtiter plates were read on the Epoch Microplate Spectrophotometer (BioTek, Winooski, VT) using the Gen5 data analysis software. HDV total antibodies were measured at Focus using a proprietary laboratory-developed assay. Briefly, serum samples diluted 1:101 in phosphate-buffered saline with 0.1% Tween 20 (PBST) containing 0.1% bovine serum albumin were added to microtiter wells coated with a proprietary recombinant HDV protein (GenScript, Piscataway, NJ). After incubation at room temperature (RT) for 1 h and 3 washes with PBST, wells received horseradish peroxidase (HRP)-conjugated F(ab=)2 fragment goat anti-human IgG⫹IgM⫹IgA (Jackson ImmunoResearch, West Grove, PA). After incubation at RT for 30 min, wells were washed and then received tetramethylbenzidine (Moss Inc., Pasadena, MD). The optical density at 450 nm (OD450) was measured using an ELISA
Received 17 January 2016 Returned for modification 23 February 2016 Accepted 30 May 2016 Accepted manuscript posted online 8 June 2016 Citation Chow S-K, Atienza EE, Cook L, Prince H, Slev P, Lapé-Nixon M, Jerome KR. 2016. Comparison of enzyme immunoassays for detection of antibodies to hepatitis D virus in serum. Clin Vaccine Immunol 23:732–734. doi:10.1128/CVI.00028-16. Editor: R. L. Hodinka, University of South Carolina School of Medicine Greenville Address correspondence to Keith R. Jerome, [email protected]. * Present address: Siu-Kei Chow, MultiCare Health System, Tacoma, Washington, USA; Harry Prince, Viracor-IBT Laboratories, Los Angeles, California, USA. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
TABLE 1 Performance of Cusabio and DiaSorin EIA kits compared to that of reference laboratory methods No. of samples giving the following results compared to reference laboratory methodsa ELISA kit c
Cusabio DiaSorin a b c
⫹/⫹
⫹/⫺
⫺/⫺
⫺/⫹
Sensitivity (% [95% CI])b
Specificity (% [95% CI])
26 32
5 0
50 55
6 0
81.3 (63.0–92.1) 100 (86.7–100)
90.9 (79.3–96.6) 100 (91.9–100)
⫹/⫹, true positive; ⫹/⫺, false positive; ⫺/⫺, true negative; ⫺/⫹, false negative. CI, confidence interval. A proposed cutoff value (0.14 ⫹ mean negative control) was used.
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reader (BioTek). Results were expressed as an index value, calculated by dividing the patient sample OD by the OD of a calibrator serum included in every run. Index values of ⬍0.90 were interpreted as negative, 0.90 to 1.10 as equivocal, and ⬎1.10 as positive. The reference assay at ARUP utilized a commercial kit that detects HDV total antibodies following the manufacturer’s instruction (International Immuno-Diagnostics, Foster City, CA). Briefly, proprietary HDV antigen was precoated to microtiter wells. A patient sample was added to the well in the presence of HRP-conjugated polyclonal antibodies for HDV. The plate was washed and then incubated with tetramethylbenzidine. The final OD was given by OD450 subtracted by OD620. The result was interpreted as the ratio between the cutoff value and the specimen value, where the cutoff is defined as 0.2 ⫻ (mean negative control ⫹ mean positive control). Ratios of ⬍0.90 were interpreted as negative, 0.90 to 1.10 as equivocal, and ⬎1.10 as positive. The HDV antigens used in the DiaSorin and Cusabio kits are proprietary. The DiaSorin kit has a competitive binding assay format, whereas the Cusabio kit is a direct binding assay. Each positive control was found to be specific to its own kit and showed no binding against the other (data not shown), suggesting that the HDV antigens in the kits are antigenically distinct. The lack of cross-reactivity between the two positive controls may be attributable to different types of HDV being used to raise antibodies. The genetic diversity of HDV, which confers different antigenic properties, has been shown to be related to the geographic origin of the isolates (11). The Cusabio and DiaSorin assays used in our study are manufactured in China and Italy, respectively. The linearity of each assay was assessed by serially diluting the positive control materials provided in the kits. For the DiaSorin kit, the OD-concentration curve generated by 8 data points using 1:2 serial dilutions of the positive control yielded an R2 value of 1 using the 4-parameter analysis of Gen5 data analysis software. For the Cusabio kit, 7 data points from 1:4 serial dilution of the positive control also yielded an R2 value of 1 using the 4-parameter analysis. To examine the limit of detection, a positive sample was tested in dilutions with both kits. The sample was 1:5 serially diluted, and the lowest positive titer was 1:3,125 for the DiaSorin kit; the same sample was serially diluted 1:4, and the lowest positive titer for the Cusabio kit was 1:16. Of 87 clinical samples tested at ARUP and Focus using their reference assays, 32 were positive and 55 were negative for antiHDV antibodies. The samples were then tested with the DiaSorin and Cusabio kits. The DiaSorin kit demonstrated sensitivity and specificity of 100% relative to the reference laboratory results (Table 1). Using the cutoff suggested by the manufacturer (0.2 ⫹ mean negative control), the overall sensitivity of the Cusabio kit was 53.1%. Because of the low sensitivity, we proposed a new cutoff defined as 0.14 ⫹ mean negative control based on the distribution of the false-negative readouts (Fig. 1). By doing so, the overall sensitivity of the Cusabio kit increased to 81.3% (Table 1). Of the 11 discrepant results between the Cusabio assay and the reference methods, 6 were false negatives and 5 were false positives. The discrepancies were attributable to the lower sensitivity for the Cusabio assay and a lack of specificity when the lower cutoff OD was used for analysis. The limitation of this study includes the poorly understood structural and antigenic property of HDV antigens in the commercial kits. In summary, results of this study show that the currently available commercial EIAs can effectively detect antibodies to
August 2016 Volume 23 Number 8
FIG 1 Distribution of EIA results for anti-HDV antibodies from clinical se-
rum specimens (n ⫽ 87) tested with Cusabio kit against Focus reference assay (A), Cusabio kit against ARUP reference assay (B), DiaSorin kit against Focus reference assay (C), and DiaSorin kit against ARUP assay (D).
Clinical and Vaccine Immunology
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HDV. The DiaSorin kit appears to have better sensitivity and specificity and shows performance comparable to that of the reference methods. ACKNOWLEDGMENT We acknowledge the excellent support of personnel of the Molecular Virology Laboratory at the University of Washington, Seattle, Washington.
REFERENCES 1. Ott JJ, Stevens GA, Groeger J, Wiersma ST. 2012. Global epidemiology of hepatitis B virus infection: new estimates of age-specific HBsAg seroprevalence and endemicity. Vaccine 30:2212–2219. http://dx.doi.org/10 .1016/j.vaccine.2011.12.116. 2. Fattovich G, Giustina G, Christensen E, Pantalena M, Zagni I, Realdi G, Schalm SW. 2000. Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. The European Concerted Action on Viral Hepatitis (Eurohep). Gut 46:420 – 426. http://dx .doi.org/10.1136/gut.46.3.420. 3. Romeo R, Del Ninno E, Rumi M, Russo A, Sangiovanni A, de Franchis R, Ronchi G, Colombo M. 2009. A 28-year study of the course of hepatitis Delta infection: a risk factor for cirrhosis and hepatocellular carcinoma. Gastroenterology 136:1629 –1638. http://dx.doi.org/10.1053/j.gastro.2009.01.052. 4. Dubois F, Goudeau A. 1988. Kinetics of delta antigen and delta antibody in acute delta hepatitis: evaluation with different enzyme immunoassays. J Clin Microbiol 26:1339 –1342.
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5. Bezeaud A, Rosenswajg M, Guillin MC. 1989. Evaluation of five hepatitis delta virus marker assays for detection of antigen and antibody. J Clin Microbiol 27:2880. 6. Govindarajan S, Valinluck B, Lake-Bakkar G. 1991. Evaluation of a commercial anti-delta EIA kit for detection of antibodies to hepatitis delta virus. Am J Clin Pathol 95:240 –241. http://dx.doi.org/10.1093/ajcp/95.2 .240. 7. Shattock AG, Morris MC. 1991. Evaluation of commercial enzyme immunoassays for detection of hepatitis delta antigen and anti-hepatitis delta virus (HDV) and immunoglobulin M anti-HDV antibodies. J Clin Microbiol 29:1873–1876. 8. Kuo YB, Chao M, Lee YH, Yeh CT, Chan EC. 2012. New enzyme-linked immunosorbent assay for detection of antibodies against hepatitis delta virus using a hepatitis delta antigen derived from a Taiwanese clone and comparison to the Abbott radioimmunoassay. Clin Vaccine Immunol 19: 817– 819. http://dx.doi.org/10.1128/CVI.05687-11. 9. Shen L, Gu Y, Sun L, Yang Y, Wang F, Li Y, Bi S. 2012. Development of a hepatitis delta virus antibody assay for study of the prevalence of HDV among individuals infected with hepatitis B virus in China. J Med Virol 84:445– 449. http://dx.doi.org/10.1002/jmv.23212. 10. Xie L, Huang D-Z, He L-X, Luo Z-X, Zhou Y-S, Wu X-D. 2009. Detection of antibody to hepatitis delta virus in human serum by double antigen sandwich ELISA. Virol Sin 24:44 –51. 11. Le Gal F, Gault E, Ripault MP, Serpaggi J, Trinchet JC, Gordien E, Deny P. 2006. Eighth major clade for hepatitis delta virus. Emerg Infect Dis 12:1447–1450. http://dx.doi.org/10.3201/eid1209.060112.
Clinical and Vaccine Immunology
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