Evaluation of a fully automated glycoprotein G-2 based assay for the detection of HSV-2 specific IgG antibodies in serum and plasma

Fragments of glycoprotein G (gG-2 281-594His ), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1 t26-189His ), and glycoprotein D of HSV-1 (gD-1 1-313 ), were expressed in the baculovirus expression system to develop an assay for the detection of HSV-1 and HSV-2 type-specific antibodies. The expression of the gG-1 t26-189His and gG-2 281-594His fragments was analyzed by Western blotting using monoclonal antibodies LP10 and AP1, respectively. The molecular masses of the major products of gG-1 t26-189His and the fragment of gG-2 281-594His were 36 to 39 kDa and 64 to 72 kDa, respectively. Human sera positive for HSV-1 reacted with gG-1 t26-189His , sera positive for HSV-2 reacted with the gG-2 281-594His fragment, and sera positive for both types reacted with gG-1 t26-189His and gG-2 281-594His in Western blotting. The human sera recognized polypeptides of gG-2 281-594His with molecular masses of 57 to 67 and 120 to 150 kDa and additional faint bands of 21, 29, and 45 kDa. The recombinant gG-1 t26-189His and the recombinant gG-2 281-594His fragment were used as type-specific antigens for the detection of HSV-1-and HSV-2-specific antibody responses in human sera, respectively. As type-common antigens, an extract of HSV-1-infected Vero cells and recombinant gD-1 1-313 were used. An enzyme-linked immunosorbent assay to detect type-specific antibodies was developed, and the sensitivity and specificity were evaluated by comparison with commercial tests by using sera obtained from different sources. The sensitivity and specificity were 91.5 and 95.5%, respectively, compared to the Gull assay. The gG-2 281-594His fragment can be obtained in relatively large quantities at low cost.Herpes simplex virus type 1 (HSV-1) and HSV-2 are well known as similar subtypes of HSV (38). HSV-2 is the main cause of recurrent genital infections (8). Most primary infections are asymptomatic, and people silently shed virus (14,30,51,57,58). After primary infection, the virus establishes latent infections in the local ganglia and is reactivated frequently, and antibody titers against HSV become detectable in serum samples. Many of the HSV-2 reactivations are asymptomatic and clinically not recognized (15,30,39,40,57). Most HSV infections are transmitted in the absence of lesions (17,57,58). Severe, generalized infections are seen particularly in neonates and immunocompromised (human immunodeficiency virus-infected) patients. Epidemiological studies indicate that in developed countries there is an ongoing HSV-2 epidemic with a significant rise in HSV-2 prevalence. Risk factors for acquiring HSV-2 infections are related to sexual life style, gender, race, and socioeconomic status. HSV type-specific antibody testing may be considered in a number of at-risk situations. Thus, identification of HSV-2-infected individuals may be important to reduce the risk of transmission in these groups.Laboratory diagnosis of HSV infections is based on direct detection of the virus and on serology (2, 16). Viru...

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“…Twenty sera were from a study in which different commercially immunoassays had been compared (25,26). The sera VOL.…”

Section: Resultsmentioning

confidence: 99%

“…The sera from the GG/GD were collected in 1998. The sera from the Erasmus Medical Center were tested with three different HSV-2 typespecific tests (25) and gave discordant results with one of the three assays. The latter sera were tested in our laboratory in Groningen with our ELISA in a coded way (blind).…”

Section: Cells and Virusesmentioning

confidence: 99%

Use of a Fragment of Glycoprotein G-2 Produced in the Baculovirus Expression System for Detecting Herpes Simplex Virus Type 2-Specific Antibodies

Ikoma¹,

Liljeqvist

Groen

et al. 2002

J Clin Microbiol

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“…Several recombinant glycoprotein G (gG)-based immu-noassays for HSV type-specific serodiagnosis are commercially available (19,44). However, the use of recombinant antigens for B virus serodiagnosis has not been widely investigated.…”

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confidence: 99%

Production of Herpes B Virus Recombinant Glycoproteins and Evaluation of Their Diagnostic Potential

et al. 2005

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B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n ‫؍‬ 40) and -positive (n ‫؍‬ 75) macaque sera identified by a whole antigenbased ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG-and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys crossreacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)-and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.Human infection with B virus (also called cercopithecine herpesvirus 1, monkey B virus, and herpes B virus) is the most feared occupational hazard among individuals working with macaque monkeys, since fatality is often the outcome of infection, which proceeds in the absence of effective antiviral therapy (25,56). The use of macaques in research has been steadily growing over the last decade and is expected to rise quickly in the near future due to the increasing demands for these animals for use in HIV/AIDS investigations, vaccine trials, drug testing, and research into bioterrorism agents. As macaque usage increases, frequencies of human exposures to B virus are increasing as well. Rapid and accurate diagnostic tests are urgently needed to aid in the early identification of clinical cases, which is essential for a timely initiation of antiviral therapies in zoonotically infected humans. In addition to human diagnostics, enhanced assays are required for monitoring specific-pathogen-free (SPF) macaque colonies established by the National Institutes of Health for the breeding of B virusfree animals (55), as these animals often demonstrate only very low levels of antibody.Unfortunately, a direct diagnosis of infectio...

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“…Other comparative studies of recently developed commercial HSV type-specific antibody assays have been described (14,18,19 serology would be more useful in diagnosing and managing recurrent disease than for primary HSV infection (25). Recurrent genital herpes is often asymptomatic or atypical (23,35).…”

Section: Discussionmentioning

confidence: 99%

“…However, these samples had previously been tested by the COBAS HSV-2-specific EIA (Roche Diagnostics, Basel, Switzerland), which uses lectin-purified HSV-2 gG-2 as the antigen (15,19).…”

Section: Methodsmentioning

confidence: 99%

Comparison of a Monoclonal Antibody-Blocking Enzyme-Linked Immunoassay and a Strip Immunoblot Assay for Identifying Type-Specific Herpes Simplex Virus Type 2 Serological Responses

Doornum

Slomka

Buimer

et al. 2000

Clin Diagn Lab Immunol

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Detection of herpes simplex virus type 2 (HSV-2)-specific antibodies by a monoclonal antibody (MAb)-blocking enzyme-linked immunoassay (EIA) was compared with detection by a strip immunoblot assay (SIA) in a sexually transmitted disease (STD) clinic population. The study population consisted of 1,683 genitourinary medicine clinic attendees (582 women and 1,101 men). Sera were tested for the presence of HSV-2 antibody by use of the blocking EIA, in which binding of the MAb AP-1 to HSV-2 glycoprotein G-2 (gG-2) is blocked by HSV-2-specific antibody. The Chiron RIBA HSV-1 and -2 strip immunoassay (SIA) utilizes HSV-1-and HSV-2-specific or cross-reactive antigens immobilized on nitrocellulose strips (HSV gB-1 and HSV gG-1 peptide bands specific for HSV-1 antibody, HSV-2 gG-2 band specific for HSV-2 antibody, and HSV gD-2 band cross-reactive for HSV-1 and HSV-2 antibodies). A total of 1,612 sera were tested by MAb-blocking EIA for HSV-2 antibody and by SIA for HSV-1 and HSV-2 antibodies. By EIA, 541 (33.6%) sera were positive for HSV-2 antibody and 1,068 sera were negative for HSV-2 antibody; 3 sera gave equivocal results. HSV-2 antibody was detected in 555 (34.4%) sera by SIA; 144 (26%) of these sera possessed only HSV-2 antibody, and 411 (74%) sera contained both HSV-1 and HSV-2 antibodies. SIA detected HSV-1 antibody in 1,155 (71.6%) sera; 744 (64%) of these sera contained HSV-1 antibody alone. Sixteen sera contained antibody against HSV but could not be typed by SIA. A total of 512 sera were positive for HSV-2 antibody by both the EIA and SIA. We concluded that the blocking EIA and SIA showed a high level of agreement in detecting HSV-2 antibody in this population. In contrast to the SIA, the blocking EIA is a useful tool for large epidemiological studies, though the SIA proved to be slightly more sensitive once sera with discrepant results were further tested.The diagnosis of primary or recurrent genital herpes simplex virus (HSV) infections, which are mainly caused by HSV type 2 (HSV-2), is based on clinical symptoms, culture of clinical specimens, viral detection by nucleic acid amplification, and HSV antigen detection assays (4,30,34). HSV-1 and HSV-2 are closely related (13), and for the study of humoral responses to HSV infection, complement fixation assays, enzyme-linked immunoassays (EIA) with crude antigens, immunofluorescence assays, and neutralization assays all lack specificity due to the cross-reactivity of antibodies against HSV-1 and HSV-2 (3, 4, 5). Assays using type-specific HSV antigens which can be used to differentiate between HSV-1-or HSV-2-specific antibodies have been described (2, 7, 8, 18, 21, 24, 28; D. Alexander et al., Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. C-101, p. 18, 1996, with the immunoblot assay (Western blotting [WB]) considered the "gold standard" because it has been most extensively validated (1, 4). An alternative to WB which does not require affinity-purified antigen is detection of type-specific antibody by blocking monoclonal antibody (MAb) (28). Serolog...

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Evaluation of a fully automated glycoprotein G-2 based assay for the detection of HSV-2 specific IgG antibodies in serum and plasma

Cited by 10 publications

References 14 publications

Use of a Fragment of Glycoprotein G-2 Produced in the Baculovirus Expression System for Detecting Herpes Simplex Virus Type 2-Specific Antibodies

Use of a Fragment of Glycoprotein G-2 Produced in the Baculovirus Expression System for Detecting Herpes Simplex Virus Type 2-Specific Antibodies

Production of Herpes B Virus Recombinant Glycoproteins and Evaluation of Their Diagnostic Potential

Comparison of a Monoclonal Antibody-Blocking Enzyme-Linked Immunoassay and a Strip Immunoblot Assay for Identifying Type-Specific Herpes Simplex Virus Type 2 Serological Responses

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