A monoclonal blocking EIA for herpes simplex virus type 2 antibody: validation for seroepidemiological studies in Africa

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Abstract

A competitive type-specific enzyme-linked immunosorbent assay (ELISA) for herpes simplex virus type 2 (HSV-2) antibody was developed using an infected cell antigen and a monoclonal antibody to glycoprotein G-2. This assay has been validated for use for epidemiological studies using a large panel of sera collected in rural Uganda and a panel of 143 sera characterised previously by Western blotting, the ‘gold standard’ for HSV type-specific serology. This evaluation was found to have a sensitivity of 96% and a specificity of 91% in comparison with Western blot on 143 sera from clinic patients. The ELISA had a sensitivity of 93% and a specificity of 91% in comparison with Western blot on 495 sera collected in Uganda. The assay showed good reproducibility and a low percentage of sera gave equivocal results, indicating its suitability for epidemiological studies.

Introduction

Herpes simplex virus type 2 (HSV-2) is a major cause of genital disease. While in many cases primary infection is accompanied by significant morbidity, in the majority of individuals infection is asymptomatic. Upon infection, the virus adopts a latent state in cervical ganglia with frequent reactivation and viral shedding. As with primary infection, on reactivation, a spectrum of disease is evident from severe to asymptomatic (Munday et al., 1998). Transmission of the virus is by sexual contact, and is facilitated (Whitley and Gnann, 1993) by the large burden of asymptomatic virus shedding.

HSV-2 is homologous genetically and antigenically to herpes simplex virus type 1 (HSV-1). It is well established that HSV-1 and HSV-2 share common epitopes, which elicit cross-reactive antibody responses in infected individuals (Ashley, 1993). This homology has limited the value of all commercial HSV antibody tests to determine if an individual is infected with one or both viruses (Ashley et al., 1991). However, virus type-specific antigenic epitopes have been identified, notably on HSV-2 glycoprotein G (gG2) (Marsden et al., 1984). As a result, several type-specific antibody assays have been described based on Western blot or using native or recombinant gG2 (Ashley and Wald, 1999). However, these assays have been used largely for clinical research and epidemiological studies (reviewed by Slomka, 1996).

Recently, there has been increasing interest in the development of practical diagnostic HSV type-specific antibody tests for use for individual patient management (Munday et al., 1998) and in the potential for developing screening programmes (Corey, 1998). Such assays would provide a measure of the burden of infection within a population and, in addition, allow large epidemiological studies of HSV. It has been proposed that the prevalence of HSV-2 infection in a population may be used as an objective marker of sexual behaviour because it is unaffected by treatment for other sexually transmitted diseases, e.g. HIV, syphilis (Cowan et al., 1994, Wagner et al., 1994, Van de Laar et al., 1998). This would be useful for monitoring behavioural changes over time and evaluating clinical interventions aimed at reducing transmission.

The present study describes the validation of an ELISA for HSV-2 antibody. This is based on the principles a radioimmunoassay described recently by Slomka et al. (1995). Validation of the ELISA is by comparison with the established ‘gold standard ’ Western blot assay and using samples from patients confirmed as HSV-2 infected by viral culture. The evaluation uses sera from both a London clinic and an African population based study.

Section snippets

Sera panels for assay development and evaluation

Two panels of sera were used for the evaluation of the HSV-2 type-specific enzyme-linked immunosorbent assay (ELISA); one panel of 68 sera that had been tested by Western blot previously for antibodies to HSV-1/2, 28 of which were from patients with confirmed infection by virus culture, generously provided by Dr F Cowan (University College Medical School London). A second panel of 75 sera also tested by Western blot were provided by Smith Kline Beecham, Vaccine Research Division. Initial

Design of the HSV-2-specific ELISA

The HSV-2-specific ELISA was based on the type-specific radioimmunoassay (RIA) described previously (Slomka et al., 1995). The epitope detected by the monoclonal antibody AP-1 and the gG-2 was used in a competitive format. To establish an effective dynamic range and highest sensitivity for the assay, without the need for an amplification detection system, the maximum amount of HSV-2 antigen possible was bound to the immunoassay plates. To block all AP-1 reactive epitopes on the plates using

Discussion

The lack of a simple, rapid, inexpensive assay to subtype HSV antibody has limited routine serodiagnosis and, more particularly, the development of epidemiological studies. Previous evaluations have shown that enzyme immunoassays (EIA) assays measuring the relative reactivity of sera against whole HSV-1 and HSV-2 antigen are unreliable (Ashley et al., 1991, Field et al., 1993). Of the methods available, work has focused on type-specific epitopes in HSV-1 and HSV-2 glycoproteins G (gG1 and gG2)

Acknowledgements

The authors wish to thank Dr J.Dobbins (Centers for Disease Control and Prevention, Atlanta, USA) who carried out the Western blots in the Ugandan study, and S Malamba (MRC Programme, Uganda) for data analysis in the early stages of this study.

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