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5. Takehara N, Iwahara Y, Uemura Y, et al. Effect of immunization on HTLV-I infection in rabbits. Int J Cancer 1989;44:332-6. 6. Kwok S, Ehrlich G, Poiesz B, Kalish R, Sninsky JJ. Enzymatic amplification of HTLV-I viral sequences from peripheral blood mononuclear cells and infected tissues. Blood 1988;72: II 17-23. 7. Uemura Y, Uriyu K, Hirao Y. et al. Inactivation and elimination of viruses during the fractionation of an intravenous immunoglobulin preparation: liquid heat treatment and polyethylene glycol fractionation. Vox Sang 1989;56: 155-61. 8. Hoshino H, Clapham PR, Weiss RA, Miyoshi I, Yoshida M, Miwa M. Human T-cell leukemia virus type I: pseudotype neutralization of Japanese and American isolates with human and rabbit sera. Int J Cancer 1985;36:671-5. 9. Hino S, Doi H. Mechanisms ofHTLV-I transmission. In: Roman GC, Vernant .IC, Osame M, eds. HTLV-I and the nervous system. New York: Alan R. Liss, 1989:495-501.
JID 1991;164 (December)
10. Ando Y. Kakimoto K, Tanigawa T, et al. Effect of freeze-thawing breast milk on vertical HTLV-I transmission from seropositive mothers to children. Jpn J Cancer Res 1989;80:405-7. II. Hino S, Yamaguchi K. Katamine S, et al. Mother-to-child transmission of human T-cell leukemia virus type-I. Gann 1985;76:474-80. 12. Nakano S, Ando Y, Ichijo M, et al. Search for possible routes of vertical and horizontal transmission of adult T-cell leukemia virus. Gann 1984;75: 1044-5. 13. Kinoshita K, Hino S, Amagasaki T, et al. Demonstration ofadult Tvcell leukemia virus antigen in milk from 3 seropositive mothers. Gann 1984;75:103-5. 14. Iwahara Y, Takehara N, Kataoka R, et al. Transmission ofHTLV-I to rabbits via semen and breast milk from seropositive healthy persons. Int J Cancer 1990;45:980-3. 15. Narita M, Shibata M, Togashi T. Koga Y. Vertical transmission ofhuman T cell leukemia virus type I. J Infect Dis 1991; 163:204.
Demetrio Sanchez-Martinez, D. Scott Schmid, William Whittington, * Denise Brown, William C. Reeves, Subhendra Chatterjee, Richard J. Whitley, and Philip E. Pellett
Division of Viral and Rickettsial Diseases, Centerfor Infectious Diseases, and Division ofSexuallv Transmitted Diseases and HIV Prevention, Centerfor Prevention Services, Centers for Disease Control. Atlanta. Georgia; Molecular Biology Department. Biokit S.A., Barcelona. Spain; Department of Pediatrics and Microbiology. University ofAlabama at Birmingham
An immunoblot assay for discrimination of antibodies to herpes simplex virus (HSV) types 1 and 2 was devised using extracts of recombinant-baculovirus-infected insect cells expressing HSV-l or -2 glycoprotein G (gGI or gG2). The assay was evaluated by comparing its results with those obtained by using an immunodot assay based on gG immunopurified from HSV-1- and HSV-2-infected cells. Each of 110 human serum specimens was tested blindly and independently three times. At a serum dilution of 1:20, the maximum specificities were 96% and 100% and the maximum sensitivities were 100% and 92% for gG 1 and gG2, respectively. Reproducibility was 99% among readers and 95% among individually tested samples of each specimen. Results obtained in two laboratories from a different set of 15 serum specimens were in complete agreement, indicating the assay is accurate and reproducible. The ease of antigen production should allow the test to become widely available. Infection with herpes simplex virus (HSV) type I or 2 can produce acute local or systemic disease, either during primary infection or during reactivation oflatent infections. Serologic testing is often the only means to identify people with
Received 22 May 1991; revised 13 August 1991. Grant support: National Institutes of Health (AF-52187 to S.c. and AI62554, -635733, and -523117 to R.J.W.). D.S.M. and P.E.P. have filed a patent application for the baculovirus-expressed glycoprotein G I and 2. Reprints or correspondence: Dr. Philip E. Pellett, Centers for Disease Control, Mailstop G-18, 1600 Clifton Rd .. Atlanta. GA 30333. * Present address: Department of Epidemiology, University of Washington, Seattle. The Journal of Infectious Diseases 1991;164:1196-9 © \99\ by The University of Chicago. All rights reserved. 0022-1899/9\ /6406-0023$0 \.00
previous HSV infections. Type-specific HSV serologic tests have been proposed for screening immunocompromised patients who may benefit from antiviral therapy, for identifying pregnant women at risk of transmitting HSV-2 to newborns, for examining patients in sexually transmitted disease clinics, and for conducting epidemiologic studies to assess the role of herpesviruses in human illness [I]. Historically, the extensive antigenic cross-reactivity between HSV -I and -2 made type-specific tests difficult to standardize and often inconclusive. Recently however, HSV-I and -2 glycoprotein G (gG I and gG2) were demonstrated to be type-specific antigens [2-5]. Two methods have been described and compared: an immunodot assay based on immunoaffinity-purified gG I and gG2 from HSV-l- and -2-infected cells [6, 7] and an immunoblot assay based on gG2
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Evaluation of a Test Based on Baculovirus-Expressed Glycoprotein G for Detection of Herpes Simplex Virus Type-Specific Antibodies
lID 1991; 164 (December)
Concise Communications
Materials and Methods Serum panels and test methodology. Two panels of serum specimens were used that had previously been tested by immunodot assay in the laboratory of A. .T. Nahmias (Emory University, Atlanta) in conjunction with other studies. Panel A, consisting of 110 serum specimens from a sexually transmitted disease clinic in Dekalb County, Georgia [11, 12], was used to determine the sensitivity, specificity, and intralaboratory reproducibility of our immunoblot assay. Specimens from panel A were each divided into three aliquots; the resulting 330 samples were tested blindly in random order. These specimens were tested at serum dilutions of 1:100, 1:50, and 1:20. Panel B, consisting of 26 serum specimens from individuals with known clinically recurrent genital herpesvirus infections, was used to confirm independently the results obtained with panel A and to assess interlaboratory variability. Immunoblots were independently read by three readers; in the event of disagreement the test was repeated. Sensitivity and specificity calculations. Due to the lack of an accepted standard for HSV type-specific serologic assays, in all of our analyses we considered the immunodot results to be the reference standard for comparison. To assess the effect of discrepancies among the three independently tested samples of some specimens from panel A, we calculated sensitivity and specificity of the results of this panel at low and high stringency. At low stringency, a specimen was considered to agree with the standard when at least one of the three aliquoted samples concurred with the standard. At high stringency, the concordance of all three samples with the standard was required. The former requirement served to define the maximum agreement with the standard, and the latter the minimum. Results were analyzed by using the following definitions and equations: %sensitivity = (100 X true positives)/(true positives + false negatives), and % specificity = (100 X true negatives)j (true negatives + false positives). Cells. viruses. and antigen preparation. Culture, infection, and preparation of the antigen were as described [10]. The amount of antigen to be used in the tests was titrated by using a
panel of human serum specimens ranging from weak to strong reactivity in an HSV type-specific indirect hemagglutination assay (IHA) [13]. The amount ofantigen loaded per gel was about double the amount needed to obtain a positive reaction from specimens weakly positive in the IHA. Immunoblot assay. Protein separation and transfer were as described [10] with the following modifications. Smaller gels (7.3 X 10.25 cm) were used (Mini-Protean II Electrophoresis apparatus; Bio-Rad Laboratories, Rockville Centre, NY), the polyacrylamide concentration was 8.5% in the separating gel, and electrophoresis was done at 100 V at room temperature for 80 min for AcDSM-gG I-infected cell extracts and for 3 h for AcDSM-gG2-infected cell extracts. Blots were placed in a manifold (Mini-Protean II Multiscreen apparatus; Bio-Rad) and incubated with blotto (5% skim milk, 0.01 M PBS, pH 7.4, and 0.05% Tween 20) for 1 h, then fresh blotto containing the appropriate dilution of human serum specimen for 1 h. After three 10-min washes in 0.05% Tween 20 in PBS, blots were incubated with alkaline phosphatase-conjugated goat anti-human IgG (Bio-Rad) in 0.05% Tween 20 in PBS for 2 h, washed three times for 10 min with the same buffer, and developed with p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Bio-Rad) according to the vendor's protocol. Criteria for definition of HSV-l- and -2-positive reactions in immunoblot. Serum specimens were considered to be HSVI-positive when they reacted with the gG 1 species of 37 and 42 kDa apparent molecular mass and to be HSV-2-positive when they reacted with the gG2 species of 118 kDa apparent molecular mass [10].
Results Sensitivity and specificity. The comparison of results for panel A between baculovirus-expressed gG-based immunoblot and gG immunodot assays at three serum dilutions is summarized in tables I and 2. Of 46 specimens negative for gG 1 in the immunodot assay, 3 scored positive in all three of their samples in the immunoblot assay at a dilution of 1:20. Of 38 specimens positive for gG2 in the immunodot assay, 3 scored negative by immunoblot in all three samples tested at a dilution of 1:20, and 3 more scored negative in two of the three samples at every serum dilution. Results obtained from panel B at a serum dilution of 1:20 were similar to those obtained from panel A (data not shown). The two specimens in panel B that were positive for gG2 by immunodot assay and negative by the immunoblot assay were tested using twice the concentration of antigen and at serum dilutions of I: 10 and 1:5 to determine whether heightened assay sensitivity would bring their results into concordance with the immunodot results. The results were still negative. Reproducibility. The reproducibility of the test was analyzed in three ways: (I) We determined the percentage of samples in which the three readers were in agreement the first time every sample was tested at each serum concentra-
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present in crude extracts of HSV-2-infected cells [8]. These studies have clearly demonstrated the utility ofgG I and gG2 as type-specific serologic reagents. The main obstacle to widespread use of these antigens is the difficulty in obtaining adequate quantities of standardized reagent. To eliminate the problem of antigen availability, a fragment of gG2 was expressed in Escherichia coli [9], and gG I and gG2 were expressed in the baculovirus system [10]. The expressed antigens retained the type-specific antigenic reactivity of their natural counterparts. Here we describe the development of an immunoblot assay using crude extracts of baculovirus-recombinant gG land gG2-infected cells as type-specific antigens. We compared the results obtained by this assay with those obtained on the same panels of serum specimens by using the gG immunodot assay and evaluated reader-to-reader, test-to-test, and laboratory-to-Iaboratory reproducibility of the assay.
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Table 1. Comparison of herpes simplex virus types land 2 glycoprotein G (gG) 1 and gG2 immunoblot and immunodot assays for panel A calculated at low and high stringency at three serum dilutions. Immunodot gGI
Immunoblot
gG2
1:100
1:50
1:20
1:100
1:50
1:20
(n = 110)
(n = 110)
(n = 102)
(n = 110)
(n= 109)
(n = 102)
+
+
+
+
+
+
Low stringency
+
I 49
58 2
I 49
56 0
2 44
34 6
0 70
36 4
0 69
35 3
0 64
53 7
8 42
53 7
3 47
54 2
3 43
31 9
4 66
31 9
I 68
31 7
63
High stringency
+
tion. (2) We calculated the percentage of specimens whose three samples were in concordance. These percentages are summarized separately for gG I and gG2 at each of the three serum dilutions (table 3). (3) The reproducibility of the test between laboratories was analyzed by blindly testing a subset of 15 of the human serum specimens from panel B by immunoblot, using the same preparation ofantigen, at both the Centers for Disease Control and the University of Alabama at Birmingham. At the serum concentration tested (I :50), the results were in complete agreement.
Discussion We describe the evaluation of an immunoblot test based on baculovirus-expressed gG 1 and gG2. Because there is no accepted reference standard for HSV type differentiation of antibodies in serum specimens, we compared our results with those obtained by using immunoaffinity-purified gG 1 and gG2 in an immunodot assay that has been well characterized [6, 7] and used in large-scale epidemiologic studies [14]. We adopted the immunoblot format to reduce the probability of misinterpreting nonspecific reactions obtained with some human serum specimens [10]. Such reactions could interfere with the interpretation of assays lacking the ability to discriminate between reactive species. The availability of the baculovirus-expressed gGs will facilitate development of simpler assays based on highly purified antigens. We found the sensitivity and specificity of the immunoblot test to be differentially affected by serum dilution. gG 1 sensitivity did not change when the serum dilution was decreased from I: 100 to I :50 but increased at a serum dilution of 1:20, resulting in total agreement with the standard when evaluated at low stringency. gG I specificity was similar at serum dilutions of I :50 and I :20 and was in nearly complete agreement with the standard. gG2 specificity was similar at serum dilutions of 1:50 and 1:20 and was in total agreement
I
with the standard at low stringency. gG2 sensmvity increased as the serum dilution decreased from I: 100 to 1:20. The gG2 immunoblot assay appeared to be less sensitive than the immunodot assay (82% at high stringency at a serum dilution of I:20). The difference between the two methods might be accounted for by any of the following: a lack of sensitivity of the gG2 immunoblot assay, a failure of some serum specimens to react with the recombinant antigen, and false positives in the immunodot assay. Doubling the amount of antigen loaded in the gel or testing serum specimens at lower dilutions made no difference in the result. Under our assay conditions, we obtained the best sensitivity for both gG 1 and gG2 at a serum dilution of I:20. At this serum dilution, the intralaboratory reproducibility among readers was 99% and was also high among samples of a given specimen (97% for gG 1, 95% for gG2). Although the numbers were small (n = 15), we found the reproducibility ofthe assay between laboratories at a serum dilution of 1:50 to be 100%. These results demonstrate the reliability of the assay. There are some potential drawbacks to the use of gG 1 and gG2 as the only antigens for the detection of prior HSV-l
Table 2. Sensitivity and specificity percentages of immunoblot compared with immunodot assay for panel A for herpes simplex virus types land 2 glycoprotein 0 (gG) I and gG2, calculated at low and high stringency at three serum dilutions. gGI
Sensitivity Low stringency High stringency Specificity Low stringency High stringency
gG2
1:100
1:50
1:20
1:100
1:50
1:20
97 88
97 88
100 96
85 78
90 78
92 82
98 84
98 94
96 94
100 94
100 99
100 98
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Table 3.
Reproducibility of the immunoblot assay, among readers and samples for panel A for herpes simplex virus types I and 2 glycoprotein g (gG) I and gG2. Reproducibility, % Among readers Serum Dilution 1:100 1:50 1:20
Among samples
gGI
gG2
gGI
gG2
93 99 99
94 99 99
89 94 97
93 95 95
Acknowledgments
We thank Jodi Black, James Dobbins, John Stewart, and Harold Wetmore for their contributions to this work. References 1. Whitley Rl. Herpes simplex viruses. In: Fields BN, Knipe DM, Chan-
ock RM, et al., eds. Virology. New York: Raven Press, 1990:184387.
2. Ackermann M, Longnecker R, Roizman B, Pereira L. Identification, properties, and gene location of a novel glycoprotein specified by herpes simplex virus 1. Virology 1986; 150:207-20. 3. Richman DD, Buckmaster A, Bell S, Hodgman C, Minson AC. Identification of a new glycoprotein of herpes simplex virus type I and genetic mapping of the gene that codes for it. 1 ViroI1986;57:64755. 4. Marsden HS, Buckmaster A, Palfreyman lW, Hope RG, Minson AC. Characterization of the 92,000-dalton glycoprotein induced by herpes simplex virus type 2. 1 Virol 1984;50:547-54. 5. Roizman B, Norrild B, Chan C, Pereira L. Identification and preliminary mapping with monoclonal antibodies of a herpes simplex virus 2 glycoprotein lacking a known type I counterpart. Virology 1984;133:242-7. 6. Lee FK, Coleman RM, Pereira L, Bailey PD, Tatsuno M, Nahmias A. Detection of herpes simplex virus type 2-specific antibody with glycoprotein G. 1 Clin Microbiol 1985;22:641-4. 7. Lee FK, Pereira L, Griffin C, Reid E, Nahmias A. A novel glycoprotein for detection of herpes simplex virus type I-specific antibodies. 1 Virol Methods 1986; 14: 111-8. 8. Ashley RL, Militoni 1, Lee F, Nahmias A, Corey L. Comparison of Western blot (immunoblot) and glycoprotein G-specific immunodot enzyme assay for detecting antibodies to herpes simplex virus types I and 2 in human sera. 1 Clin Microbiol 1988;26:662-7. 9. Parkes DL, Smith CM, Rose 1M, Brandis J, Coates SR. Seroactive recombinant herpes simplex virus type 2-specific glycoprotein G. 1 Clin Microbiol 1991 ;29:778-81. 10. Sanchez-Martinez D, Pellett PE. Expression of HSV-I and HSV-2 glycoprotein G in insect cells by using a novel baculovirus expression vector. Virology 1991;182:229-38. I I. Pulgiese Pl, Whittington WL, Miller RM, Nahmias Al. Sexual behavior patterns and the incidence ofinfection with HSV-2 among heterosexual men. Presented at the 7th meeting of the International Society for Sexually Transmitted Disease Research, Atlanta, 1987. 12. Whittington WL, lacobs B, Lewis 1, Lee F, Edwards T, Nahmias Al. HSV-2 in patients with genital lesions attending a North American STD clinic: assessment of risk factors. In: Program and abstracts: V International Conference on AIDS (Montreal). Ottawa: International Development Research Centre, 1989. 13. Bernstein MT, Stewart lA. Method for typing antisera to herpesvirus hominis by indirect hemagglutination inhibition. Appl Microbiol 1971;21:680-4. 14. Johnson RE, Nahmias Al, Magder LS, Lee FK, Brooks CA, Snowden CB. A seroepidemiologic survey of the prevalence of herpes simplex virus type 2 infection in the United States. N Engl 1 Med 1989;321 :7-12. 15. Longnecker R, Roizman B. Clustering of genes dispensable for growth in culture in the S component of the HSV-I genome. Science 1987;236:573-6.
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and -2 infections. Antibodies to gG 1 and gG2 are first detectable 10-21 days after infection [8]. The failure to detect recent primary infections is a matter of little consequence in most epidemiologic studies but is potentially significant in clinical settings. The gG gene is known to be nonessential for HSV-l infectivity [15]. It is possible that natural strains not expressing gG might occur at low frequency, although such gG-deficient variants have not been described. Further work will be required to address these problems. Our results indicate that the immunoblot assay based on the baculovirus-expressed gGs gives results comparable in sensitivity and specificity to those obtained by using the immunodot assay. The expression of these antigens in the baculovirus system will facilitate their reliable and efficient largescale production, help make this assay widely available, and allow their immediate use in epidemiologic studies as well as investigations of possible clinical applications, such as screening of pregnant women.
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5. Takehara N, Iwahara Y, Uemura Y, et al. Effect of immunization on HTLV-I infection in rabbits. Int J Cancer 1989;44:332-6. 6. Kwok S, Ehrlich G, Poiesz B, Kalish R, Sninsky JJ. Enzymatic amplification of HTLV-I viral sequences from peripheral blood mononuclear cells and infected tissues. Blood 1988;72: II 17-23. 7. Uemura Y, Uriyu K, Hirao Y. et al. Inactivation and elimination of viruses during the fractionation of an intravenous immunoglobulin preparation: liquid heat treatment and polyethylene glycol fractionation. Vox Sang 1989;56: 155-61. 8. Hoshino H, Clapham PR, Weiss RA, Miyoshi I, Yoshida M, Miwa M. Human T-cell leukemia virus type I: pseudotype neutralization of Japanese and American isolates with human and rabbit sera. Int J Cancer 1985;36:671-5. 9. Hino S, Doi H. Mechanisms ofHTLV-I transmission. In: Roman GC, Vernant .IC, Osame M, eds. HTLV-I and the nervous system. New York: Alan R. Liss, 1989:495-501.
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10. Ando Y. Kakimoto K, Tanigawa T, et al. Effect of freeze-thawing breast milk on vertical HTLV-I transmission from seropositive mothers to children. Jpn J Cancer Res 1989;80:405-7. II. Hino S, Yamaguchi K. Katamine S, et al. Mother-to-child transmission of human T-cell leukemia virus type-I. Gann 1985;76:474-80. 12. Nakano S, Ando Y, Ichijo M, et al. Search for possible routes of vertical and horizontal transmission of adult T-cell leukemia virus. Gann 1984;75: 1044-5. 13. Kinoshita K, Hino S, Amagasaki T, et al. Demonstration ofadult Tvcell leukemia virus antigen in milk from 3 seropositive mothers. Gann 1984;75:103-5. 14. Iwahara Y, Takehara N, Kataoka R, et al. Transmission ofHTLV-I to rabbits via semen and breast milk from seropositive healthy persons. Int J Cancer 1990;45:980-3. 15. Narita M, Shibata M, Togashi T. Koga Y. Vertical transmission ofhuman T cell leukemia virus type I. J Infect Dis 1991; 163:204.
Demetrio Sanchez-Martinez, D. Scott Schmid, William Whittington, * Denise Brown, William C. Reeves, Subhendra Chatterjee, Richard J. Whitley, and Philip E. Pellett
Division of Viral and Rickettsial Diseases, Centerfor Infectious Diseases, and Division ofSexuallv Transmitted Diseases and HIV Prevention, Centerfor Prevention Services, Centers for Disease Control. Atlanta. Georgia; Molecular Biology Department. Biokit S.A., Barcelona. Spain; Department of Pediatrics and Microbiology. University ofAlabama at Birmingham
An immunoblot assay for discrimination of antibodies to herpes simplex virus (HSV) types 1 and 2 was devised using extracts of recombinant-baculovirus-infected insect cells expressing HSV-l or -2 glycoprotein G (gGI or gG2). The assay was evaluated by comparing its results with those obtained by using an immunodot assay based on gG immunopurified from HSV-1- and HSV-2-infected cells. Each of 110 human serum specimens was tested blindly and independently three times. At a serum dilution of 1:20, the maximum specificities were 96% and 100% and the maximum sensitivities were 100% and 92% for gG 1 and gG2, respectively. Reproducibility was 99% among readers and 95% among individually tested samples of each specimen. Results obtained in two laboratories from a different set of 15 serum specimens were in complete agreement, indicating the assay is accurate and reproducible. The ease of antigen production should allow the test to become widely available. Infection with herpes simplex virus (HSV) type I or 2 can produce acute local or systemic disease, either during primary infection or during reactivation oflatent infections. Serologic testing is often the only means to identify people with
Received 22 May 1991; revised 13 August 1991. Grant support: National Institutes of Health (AF-52187 to S.c. and AI62554, -635733, and -523117 to R.J.W.). D.S.M. and P.E.P. have filed a patent application for the baculovirus-expressed glycoprotein G I and 2. Reprints or correspondence: Dr. Philip E. Pellett, Centers for Disease Control, Mailstop G-18, 1600 Clifton Rd .. Atlanta. GA 30333. * Present address: Department of Epidemiology, University of Washington, Seattle. The Journal of Infectious Diseases 1991;164:1196-9 © \99\ by The University of Chicago. All rights reserved. 0022-1899/9\ /6406-0023$0 \.00
previous HSV infections. Type-specific HSV serologic tests have been proposed for screening immunocompromised patients who may benefit from antiviral therapy, for identifying pregnant women at risk of transmitting HSV-2 to newborns, for examining patients in sexually transmitted disease clinics, and for conducting epidemiologic studies to assess the role of herpesviruses in human illness [I]. Historically, the extensive antigenic cross-reactivity between HSV -I and -2 made type-specific tests difficult to standardize and often inconclusive. Recently however, HSV-I and -2 glycoprotein G (gG I and gG2) were demonstrated to be type-specific antigens [2-5]. Two methods have been described and compared: an immunodot assay based on immunoaffinity-purified gG I and gG2 from HSV-l- and -2-infected cells [6, 7] and an immunoblot assay based on gG2
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Evaluation of a Test Based on Baculovirus-Expressed Glycoprotein G for Detection of Herpes Simplex Virus Type-Specific Antibodies
lID 1991; 164 (December)
Concise Communications
Materials and Methods Serum panels and test methodology. Two panels of serum specimens were used that had previously been tested by immunodot assay in the laboratory of A. .T. Nahmias (Emory University, Atlanta) in conjunction with other studies. Panel A, consisting of 110 serum specimens from a sexually transmitted disease clinic in Dekalb County, Georgia [11, 12], was used to determine the sensitivity, specificity, and intralaboratory reproducibility of our immunoblot assay. Specimens from panel A were each divided into three aliquots; the resulting 330 samples were tested blindly in random order. These specimens were tested at serum dilutions of 1:100, 1:50, and 1:20. Panel B, consisting of 26 serum specimens from individuals with known clinically recurrent genital herpesvirus infections, was used to confirm independently the results obtained with panel A and to assess interlaboratory variability. Immunoblots were independently read by three readers; in the event of disagreement the test was repeated. Sensitivity and specificity calculations. Due to the lack of an accepted standard for HSV type-specific serologic assays, in all of our analyses we considered the immunodot results to be the reference standard for comparison. To assess the effect of discrepancies among the three independently tested samples of some specimens from panel A, we calculated sensitivity and specificity of the results of this panel at low and high stringency. At low stringency, a specimen was considered to agree with the standard when at least one of the three aliquoted samples concurred with the standard. At high stringency, the concordance of all three samples with the standard was required. The former requirement served to define the maximum agreement with the standard, and the latter the minimum. Results were analyzed by using the following definitions and equations: %sensitivity = (100 X true positives)/(true positives + false negatives), and % specificity = (100 X true negatives)j (true negatives + false positives). Cells. viruses. and antigen preparation. Culture, infection, and preparation of the antigen were as described [10]. The amount of antigen to be used in the tests was titrated by using a
panel of human serum specimens ranging from weak to strong reactivity in an HSV type-specific indirect hemagglutination assay (IHA) [13]. The amount ofantigen loaded per gel was about double the amount needed to obtain a positive reaction from specimens weakly positive in the IHA. Immunoblot assay. Protein separation and transfer were as described [10] with the following modifications. Smaller gels (7.3 X 10.25 cm) were used (Mini-Protean II Electrophoresis apparatus; Bio-Rad Laboratories, Rockville Centre, NY), the polyacrylamide concentration was 8.5% in the separating gel, and electrophoresis was done at 100 V at room temperature for 80 min for AcDSM-gG I-infected cell extracts and for 3 h for AcDSM-gG2-infected cell extracts. Blots were placed in a manifold (Mini-Protean II Multiscreen apparatus; Bio-Rad) and incubated with blotto (5% skim milk, 0.01 M PBS, pH 7.4, and 0.05% Tween 20) for 1 h, then fresh blotto containing the appropriate dilution of human serum specimen for 1 h. After three 10-min washes in 0.05% Tween 20 in PBS, blots were incubated with alkaline phosphatase-conjugated goat anti-human IgG (Bio-Rad) in 0.05% Tween 20 in PBS for 2 h, washed three times for 10 min with the same buffer, and developed with p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Bio-Rad) according to the vendor's protocol. Criteria for definition of HSV-l- and -2-positive reactions in immunoblot. Serum specimens were considered to be HSVI-positive when they reacted with the gG 1 species of 37 and 42 kDa apparent molecular mass and to be HSV-2-positive when they reacted with the gG2 species of 118 kDa apparent molecular mass [10].
Results Sensitivity and specificity. The comparison of results for panel A between baculovirus-expressed gG-based immunoblot and gG immunodot assays at three serum dilutions is summarized in tables I and 2. Of 46 specimens negative for gG 1 in the immunodot assay, 3 scored positive in all three of their samples in the immunoblot assay at a dilution of 1:20. Of 38 specimens positive for gG2 in the immunodot assay, 3 scored negative by immunoblot in all three samples tested at a dilution of 1:20, and 3 more scored negative in two of the three samples at every serum dilution. Results obtained from panel B at a serum dilution of 1:20 were similar to those obtained from panel A (data not shown). The two specimens in panel B that were positive for gG2 by immunodot assay and negative by the immunoblot assay were tested using twice the concentration of antigen and at serum dilutions of I: 10 and 1:5 to determine whether heightened assay sensitivity would bring their results into concordance with the immunodot results. The results were still negative. Reproducibility. The reproducibility of the test was analyzed in three ways: (I) We determined the percentage of samples in which the three readers were in agreement the first time every sample was tested at each serum concentra-
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present in crude extracts of HSV-2-infected cells [8]. These studies have clearly demonstrated the utility ofgG I and gG2 as type-specific serologic reagents. The main obstacle to widespread use of these antigens is the difficulty in obtaining adequate quantities of standardized reagent. To eliminate the problem of antigen availability, a fragment of gG2 was expressed in Escherichia coli [9], and gG I and gG2 were expressed in the baculovirus system [10]. The expressed antigens retained the type-specific antigenic reactivity of their natural counterparts. Here we describe the development of an immunoblot assay using crude extracts of baculovirus-recombinant gG land gG2-infected cells as type-specific antigens. We compared the results obtained by this assay with those obtained on the same panels of serum specimens by using the gG immunodot assay and evaluated reader-to-reader, test-to-test, and laboratory-to-Iaboratory reproducibility of the assay.
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Table 1. Comparison of herpes simplex virus types land 2 glycoprotein G (gG) 1 and gG2 immunoblot and immunodot assays for panel A calculated at low and high stringency at three serum dilutions. Immunodot gGI
Immunoblot
gG2
1:100
1:50
1:20
1:100
1:50
1:20
(n = 110)
(n = 110)
(n = 102)
(n = 110)
(n= 109)
(n = 102)
+
+
+
+
+
+
Low stringency
+
I 49
58 2
I 49
56 0
2 44
34 6
0 70
36 4
0 69
35 3
0 64
53 7
8 42
53 7
3 47
54 2
3 43
31 9
4 66
31 9
I 68
31 7
63
High stringency
+
tion. (2) We calculated the percentage of specimens whose three samples were in concordance. These percentages are summarized separately for gG I and gG2 at each of the three serum dilutions (table 3). (3) The reproducibility of the test between laboratories was analyzed by blindly testing a subset of 15 of the human serum specimens from panel B by immunoblot, using the same preparation ofantigen, at both the Centers for Disease Control and the University of Alabama at Birmingham. At the serum concentration tested (I :50), the results were in complete agreement.
Discussion We describe the evaluation of an immunoblot test based on baculovirus-expressed gG 1 and gG2. Because there is no accepted reference standard for HSV type differentiation of antibodies in serum specimens, we compared our results with those obtained by using immunoaffinity-purified gG 1 and gG2 in an immunodot assay that has been well characterized [6, 7] and used in large-scale epidemiologic studies [14]. We adopted the immunoblot format to reduce the probability of misinterpreting nonspecific reactions obtained with some human serum specimens [10]. Such reactions could interfere with the interpretation of assays lacking the ability to discriminate between reactive species. The availability of the baculovirus-expressed gGs will facilitate development of simpler assays based on highly purified antigens. We found the sensitivity and specificity of the immunoblot test to be differentially affected by serum dilution. gG 1 sensitivity did not change when the serum dilution was decreased from I: 100 to I :50 but increased at a serum dilution of 1:20, resulting in total agreement with the standard when evaluated at low stringency. gG I specificity was similar at serum dilutions of I :50 and I :20 and was in nearly complete agreement with the standard. gG2 specificity was similar at serum dilutions of 1:50 and 1:20 and was in total agreement
I
with the standard at low stringency. gG2 sensmvity increased as the serum dilution decreased from I: 100 to 1:20. The gG2 immunoblot assay appeared to be less sensitive than the immunodot assay (82% at high stringency at a serum dilution of I:20). The difference between the two methods might be accounted for by any of the following: a lack of sensitivity of the gG2 immunoblot assay, a failure of some serum specimens to react with the recombinant antigen, and false positives in the immunodot assay. Doubling the amount of antigen loaded in the gel or testing serum specimens at lower dilutions made no difference in the result. Under our assay conditions, we obtained the best sensitivity for both gG 1 and gG2 at a serum dilution of I:20. At this serum dilution, the intralaboratory reproducibility among readers was 99% and was also high among samples of a given specimen (97% for gG 1, 95% for gG2). Although the numbers were small (n = 15), we found the reproducibility ofthe assay between laboratories at a serum dilution of 1:50 to be 100%. These results demonstrate the reliability of the assay. There are some potential drawbacks to the use of gG 1 and gG2 as the only antigens for the detection of prior HSV-l
Table 2. Sensitivity and specificity percentages of immunoblot compared with immunodot assay for panel A for herpes simplex virus types land 2 glycoprotein 0 (gG) I and gG2, calculated at low and high stringency at three serum dilutions. gGI
Sensitivity Low stringency High stringency Specificity Low stringency High stringency
gG2
1:100
1:50
1:20
1:100
1:50
1:20
97 88
97 88
100 96
85 78
90 78
92 82
98 84
98 94
96 94
100 94
100 99
100 98
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Table 3.
Reproducibility of the immunoblot assay, among readers and samples for panel A for herpes simplex virus types I and 2 glycoprotein g (gG) I and gG2. Reproducibility, % Among readers Serum Dilution 1:100 1:50 1:20
Among samples
gGI
gG2
gGI
gG2
93 99 99
94 99 99
89 94 97
93 95 95
Acknowledgments
We thank Jodi Black, James Dobbins, John Stewart, and Harold Wetmore for their contributions to this work. References 1. Whitley Rl. Herpes simplex viruses. In: Fields BN, Knipe DM, Chan-
ock RM, et al., eds. Virology. New York: Raven Press, 1990:184387.
2. Ackermann M, Longnecker R, Roizman B, Pereira L. Identification, properties, and gene location of a novel glycoprotein specified by herpes simplex virus 1. Virology 1986; 150:207-20. 3. Richman DD, Buckmaster A, Bell S, Hodgman C, Minson AC. Identification of a new glycoprotein of herpes simplex virus type I and genetic mapping of the gene that codes for it. 1 ViroI1986;57:64755. 4. Marsden HS, Buckmaster A, Palfreyman lW, Hope RG, Minson AC. Characterization of the 92,000-dalton glycoprotein induced by herpes simplex virus type 2. 1 Virol 1984;50:547-54. 5. Roizman B, Norrild B, Chan C, Pereira L. Identification and preliminary mapping with monoclonal antibodies of a herpes simplex virus 2 glycoprotein lacking a known type I counterpart. Virology 1984;133:242-7. 6. Lee FK, Coleman RM, Pereira L, Bailey PD, Tatsuno M, Nahmias A. Detection of herpes simplex virus type 2-specific antibody with glycoprotein G. 1 Clin Microbiol 1985;22:641-4. 7. Lee FK, Pereira L, Griffin C, Reid E, Nahmias A. A novel glycoprotein for detection of herpes simplex virus type I-specific antibodies. 1 Virol Methods 1986; 14: 111-8. 8. Ashley RL, Militoni 1, Lee F, Nahmias A, Corey L. Comparison of Western blot (immunoblot) and glycoprotein G-specific immunodot enzyme assay for detecting antibodies to herpes simplex virus types I and 2 in human sera. 1 Clin Microbiol 1988;26:662-7. 9. Parkes DL, Smith CM, Rose 1M, Brandis J, Coates SR. Seroactive recombinant herpes simplex virus type 2-specific glycoprotein G. 1 Clin Microbiol 1991 ;29:778-81. 10. Sanchez-Martinez D, Pellett PE. Expression of HSV-I and HSV-2 glycoprotein G in insect cells by using a novel baculovirus expression vector. Virology 1991;182:229-38. I I. Pulgiese Pl, Whittington WL, Miller RM, Nahmias Al. Sexual behavior patterns and the incidence ofinfection with HSV-2 among heterosexual men. Presented at the 7th meeting of the International Society for Sexually Transmitted Disease Research, Atlanta, 1987. 12. Whittington WL, lacobs B, Lewis 1, Lee F, Edwards T, Nahmias Al. HSV-2 in patients with genital lesions attending a North American STD clinic: assessment of risk factors. In: Program and abstracts: V International Conference on AIDS (Montreal). Ottawa: International Development Research Centre, 1989. 13. Bernstein MT, Stewart lA. Method for typing antisera to herpesvirus hominis by indirect hemagglutination inhibition. Appl Microbiol 1971;21:680-4. 14. Johnson RE, Nahmias Al, Magder LS, Lee FK, Brooks CA, Snowden CB. A seroepidemiologic survey of the prevalence of herpes simplex virus type 2 infection in the United States. N Engl 1 Med 1989;321 :7-12. 15. Longnecker R, Roizman B. Clustering of genes dispensable for growth in culture in the S component of the HSV-I genome. Science 1987;236:573-6.
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and -2 infections. Antibodies to gG 1 and gG2 are first detectable 10-21 days after infection [8]. The failure to detect recent primary infections is a matter of little consequence in most epidemiologic studies but is potentially significant in clinical settings. The gG gene is known to be nonessential for HSV-l infectivity [15]. It is possible that natural strains not expressing gG might occur at low frequency, although such gG-deficient variants have not been described. Further work will be required to address these problems. Our results indicate that the immunoblot assay based on the baculovirus-expressed gGs gives results comparable in sensitivity and specificity to those obtained by using the immunodot assay. The expression of these antigens in the baculovirus system will facilitate their reliable and efficient largescale production, help make this assay widely available, and allow their immediate use in epidemiologic studies as well as investigations of possible clinical applications, such as screening of pregnant women.
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