ELISA for detection of IgG and IgM antibodies to HSV-1 and HSV-2 in human sera

A rapid, enzyme-linked immunoassay (ELISA) was applied to identify and measure specific IgG and IgM antibodies to herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2). Detergent solubilized infected cells and mock-infected cells were used as antigens in the assay. Identification of type-specific antibodies was achieved by a competition assay in which clinical sera mixed with HSV-1 or HSV-2 antigens were assayed for reactivity to identical antigens coating wells of polystyrene microtiter plates. Reactivity and the specificity of the reactive immunoglobulin class was quantitated using biotinylated goat anti-IgG and biotinylated goat anti-IgM. Five paired sera from patients with diagnosed herpes simplex genital infections and one human anti-HSV-1 reference serum were tested with this assay and results were compared to results previously obtained using a complement fixation test and micro-SPRIA. The results indicate that the ELISA is a specific, sensitive and simple test which confirms the herpes simplex virus infection history of patients.     

Novel recombinant ELISA assays for determination of type-specific IgG antibodies against HSV-1 and HSV-2

The two novel Novagnost enzyme-linked immunosorbent assays (ELISA, Dade Behring) based on recombinant viral glycoprotein G were evaluated for determination of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) IgG. ELISA and immunoblot procedures approved by the US Food and Drug Administration served as the reference for selecting different serum panels to be tested. The sensitivity and specificity of the novel assay for HSV-1 IgG were 93.1-98.0% and 99.3-100%, respectively, while the sensitivity and specificity of the novel assay for HSV-2 IgG were 100% and 94.6-97.6%, respectively. Using Kappa statistics, the Kappa indices were computed at 0.93-0.98 for the HSV-1 assays and 0.92-0.96 for the HSV-2 assays suggesting an almost perfect agreement between the novel ELISAs and the reference tests. In conclusion, the novel immunoassays for determination of HSV-1 or HSV-2 IgG can be recommended for the reliable type-specific diagnosis of HSV infections.     

Detection and typing of HSV-1, HSV-2, CMV and EBV by quadruplex PCR

The development of a multiplex polymerase chain reaction (PCR) method for rapid and accurate detection and typing of herpes simplex virus type 1 (HSV-1), and type-2 (HSV-2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) is very important for clinical diagnosis to allow the deliver of therapy as early as possible. Large scale amplifications by multiplex PCR of viral DNA can lower the cost and time for viral diagnosis. In this study, therefore sensitive quadruplex PCR was achieved by optimizing parameters such as primers, and 1.5 mM magnesium and 200 uM dNTPs concentrations. The concentrations of HSV-1, HSV-2, CMV and EBV primers were 0.5, 0.3, 0.25 and 0.25 pmoles, respectively. Optimal annealing temperature was 54 degrees C. Employing these conditions, we could detect 10 copies of reconstructed template plasmid DNA, which were cloned to vectors containing target sequences of viral DNA. PCR products of 271 bp for HSV-1, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 5.0% polyacrylamide gel electrophoresis and confirmed by direct sequencing. The present study showed that the quadruplex PCR assay described herein has potential application in clinical diagnosis, when rapid, accurate detection and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.     

Evaluation of a new general primer pair for rapid detection and differentiation of HSV-1, HSV-2, and VZV by polymerase chain reaction

The polymerase chain reaction (PCR) enables rapid and sensitive detection of VZV and HSV DNA and its efficiency depends mainly on the choice of the primers. Primers should hybridize to conserved DNA sequences within the viral genomes in order to avoid unreliable amplification due to DNA sequence variation between different strains. The aim of the study was to design and to evaluate a general primer pair which permits fast and reliable detection of HSV and VZV. The genes UL 15 of HSV and UL 42 of VZV share the highest degree of homology within the two genomes. We designed a primer pair (GPHV-RU) which hybridizes to these genes. The genetic variability of amplified sequences from clinical specimens was analyzed by restriction enzyme cleavage analysis and by temperature gradient SSCP analysis (TG-SSCP). PCR with GPHV-RU amplified viral sequences from all analyzed specimens (25×VZV, 10×HSV-1, 5×HSV-2) obtained from patients with clinical evidence of HSV or VZV infection. Restriction enzyme cleavage analysis with Hpa II further permitted reliable distinction between VZV, HSV-1, and HSV-2. Analysis of the heterogeneity of the amplified sequences by restriction enzyme cleavage and by TG-SSCP demonstrated no variability between the analyzed clinical specimens of VZV and of HSV-2 and only one differing TG-SSCP-pattern within the HSV-1 isolates. The results suggest that detection of HSV and VZV using the new primer pair GPHV-RU should give reliable results as the amplified sequences show little genetic variability within clinical isolates of HSV-1/2 and VZV. © 1996 Wiley-Liss, Inc.     

Seroprevalence of HSV-1 and HSV-2 in Barbados

Type-specific enzyme-linked immunosorbent assays, based upon recombinant glycoprotein G (gG), were used to detect antibodies to HSV-1 and HSV-2, in a small Caribbean island population. A blinded serosurvey was performed on samples from 184 blood donors, 122 pregnant women, and 120 HIV-positive patients. The seroprevalence of HSV-1 and HSV-2 was 81% and 34%, respectively, in blood donors, 84% and 40% in the antenatal population and 89% and 77% in the HIV-positive group. As expected the majority of adults were seropositive against HSV-1. However, the HSV-2 seroprevalence was significantly higher in HIV-infected adults than in the other groups. These findings support the need for prospective epidemiological studies in this population.     

The changing epidemiology of HSV-1 and HSV-2 and implications for serological testing

An increased prevalence of genital herpes simplex virus (HSV) infection has been documented worldwide. In the USA, the National Health and Nutrition Examination Survey (NHANES), conducted from 1988 to 1994, revealed the seroprevalence of HSV-2 in persons 12 years of age or older to be 21.9%, an increase of 30% in age-adjusted seroprevalence of HSV-2 since the previous survey that was conducted from 1976 to 1980. Several European studies have documented a high prevalence of HSV-2 in antenatal populations. The advent of highly sensitive virological tests has confirmed that HSV-2 is the most common cause of genital ulceration in the developing world. People with a high standard of living may escape oral HSV-1 infection in childhood. Since asymptomatic oral shedding of HSV-1 is common, adults without immunity to HSV-1 who practise oral sex are especially at risk for genital HSV-1 infection. In some European cohorts, HSV-1 has been a more common aetiological agent of primary genital herpes than HSV-2. These patient groups may benefit from the use of HSV-1 in addition to HSV-2 type-specific serology.     

Comparative Evaluation of AmpliVue HSV 1+2 Assay with ELVIS Culture for Detecting Herpes Simplex Virus 1 (HSV-1) and HSV-2 in Clinical Specimens

The AmpliVue HSV 1+2 assay was compared to the ELVIS HSV ID and D³ Typing Culture System for the qualitative detection and differentiation of herpes simplex virus 1 (HSV-1) and HSV-2 DNA in 1,351 cutaneous and mucocutaneous specimens. Compared to ELVIS, AmpliVue had sensitivities of 95.7 and 97.6% for detecting HSV-1 and HSV-2, respectively. Following arbitration of discordant results by an independent molecular method, the AmpliVue assay had a resolved sensitivity and specificity of 99.2 and 99.7%, respectively, for both HSV-1 and HSV-2, whereas ELVIS had a resolved sensitivity of 87.1% for HSV-1 and 84.5% for HSV-2.     

A transforming plasmid from HSV-2 transformed cells contains rat DNA homologous to the HSV-1 and HSV-2 genomes

Morphologically transformed rat (3Y1) cell lines were established following transfection with HSV-2 mtrII DNA sequences (0.585 to 0.601 map units). The mtrII sequences were cloned in plasmids containing the neor gene. Cells resistant to the antibiotic G-418 were passed into soft agarose, and clonal lines were established from individual colonies. The DNAs from two cell lines examined by Southern blot hybridization were shown to contain the original transfected viral DNA sequences in a fashion consistent with a multiple and complex pattern of integration. From one cell line, an approximately 20-kbp plasmid was isolated after transformation of bacteria with Hirt supernatant DNA. This plasmid was capable of rapidly transforming rat cells at a greater than 1000-fold higher frequency than the mtrII DNA. This plasmid consists mainly of unique sequence rat DNA with two copies of the HSV-2 mtrII region DNA (HSV-2 genomic map unit location of ca. 0.595) present at sites distant from each other. The rat DNA in the rescued plasmid is homologous to the putative focus-forming sequences present in the HSV-2 mtrIII (0.53 to 0.58 map units) and the colinear HSV-1 DNA. The genomic copy of these rat sequences in four HSV-2 mtrII transformed cell lines appears to have undergone rearrangement. These data provide evidence that the HSV-2 mtrII sequences are involved in transformation, and that the HSV-2 mtrII region may affect transformation by rearranging the cellular sequences that are homologous to mtrIII.     

An ELISA technique to detect IgG antibody to the early herpes simplex virus type 2 (HSV-2) antigen AG-4 in HSV-2 patients

An ELISA system, based on urease activity was used for the detection and titration of IgG to the immediate early AG-4 antigen in sera from HSV-2 patients. It detected low levels of IgG to the AG-4 antigen in 32% of patients' sera known to contain complement fixing antibody to the antigen. Furthermore, the sensitivity of the ELISA system was 2- to 10-fold higher than the complement-fixation system depending on when the sera was taken from the HSV-2 patients. The system also allowed the easy detection and quantitation of AG-4 antigen production when various HSV-1 X HSV-2 intertypic recombinant viruses were used to infect BHK-21 cells.     

Monoclonal antibodies specific for rat IgG1, IgG2a, and IgG2b subclasses, and kappa chain monotypic and allotypic determinants: reagents for use with rat monoclonal antibodies

Mouse monoclonal antibodies to rat IgG were obtained by fusion of immune SJL mouse spleen cells to NSI myeloma cells. Seven monoclonal antibodies have been labeled with 125I and studied as to specificity and avidity by using a panel of rat monoclonal antibodies both as inhibitors and target antigens in soft well plate and indirect cell binding assays. All MAb were selected for high avidity of 4 X 10(7) to greater than or equal to 2 X 10(9) M-1. Four MAb were subclass-specific. RG11/39, RG7/1, and RG7/11 were absolutely specific for the Fc' region of IgG1, IgG2a, and IgG2b, respectively. RG9/6 showed specificity for the Fab' region of IgG2a but crossreacted with lower avidity with IgG2c. Three MAb reacted with rat kappa chains. RG7/9 defined a monotypic (common) kappa chain determinant. RG11/15 and RG7/7 were specific for allelic kappa 1a and kappa 1b determinants, respectively. The monotypic and kappa 1a allotypic determinants are topographically separated. The antibodies can be used as screening reagents in indirect cell binding assays. They have sensitivity similar to affinity-purified rabbit anti-rat IgG and more defined specificity. They do not crossreact with mouse or human IgG, making them particularly suitable companion reagents for rat anti-mouse or anti-human MAb. One Mab, RG7/7, strongly crossreacts with Syrian hamster IgG.     

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

BACKGROUND: Ranking after infections with Chlamydia trachomatis and human papillomavirus, genital herpesvirus is the third most common sexually transmitted disease. The majority of recurrent genital herpes infections are caused by herpes simplex virus type-2 (HSV-2). Seroepidemiological studies on the prevalence of HSV-2 specific IgG antibodies are especially important to study the impact of this infection among risk groups. OBJECTIVE: To evaluate the sensitivity and specificity of the Cobas Core HSV-2 IgG specific assay (available for research use only), that can be run on the Cobas Core fully automated immuno-analyzer. STUDY DESIGN: The Cobas Core HSV-2 specific IgG EIA is based on macro-beads coated with affinity purified glycoprotein G-2 antigen from HSV-2 infected cells. The Cobas Core HSV-2 IgG specific assay was compared with the Chiron rapid immunoblot assay (RIBA), the Gull enzyme-linked immunosorbent assay (EIA) and the Centocor EIA. The respective assays were tested, using 1219 serum samples, from 612 females and 607 males attending the outpatient clinic for sexually transmitted diseases of the Erasmus Medical Center Rotterdam (EMCR). RESULTS: The consensus value, obtained by a concordant result with three out of four assays, demonstrated 350 positive samples (28.7%), 851 negative samples (69.8%) and 18 (1.5%) serum samples with a discordant result. The overall agreement of the Cobas Core HSV-2 EIA against the consensus value was 95.8% and the sensitivity and specificity proved to be 100 and 97.1% respectively. CONCLUSION: The results obtained with the Cobas Core HSV-2 EIA indicate that this is a useful, specific and sensitive assay for the detection of HSV-2 specific IgG antibodies in serum. The advantage of the Cobas Core HVS-2 EIA compared to the other assays, is that this assay can be performed in a fully automated process.     

Serological microarray for detection of HSV-1, HSV-2, VZV, and CMV antibodies

The seroprevalence of human herpesviruses is high and reactivations occur frequently. A microarray was designed and tested for the detection of IgG and IgM antibodies for Puumala hantavirus (PUUV) and IgG antibodies against four herpesviruses. Initially, a microarray platform was set up using an unrelated in-house antigen, PUUV recombinant nucleocapsid protein, to optimize the protocol for the detection of antibodies. Detection of the four herpesviruses was set up in a microarray using the recombinant proteins of herpes simplex virus (HSV) glycoprotein G1 and G2, varicella-zoster virus (VZV) glycoprotein E, and cytomegalovirus (CMV) pp150 phosphoprotein.The results of the PUUV panel were in good agreement with the PUUV IgG immunofluorescent assay and IgM enzyme immunoassay (EIA). Seropositive and negative clinical reference panels were tested for herpesviruses by the serological microarray, and the results were compared to those of individual EIAs used for standard diagnostic purposes.The serologic microarray for HSV, VZV and CMV antibody detection gave good specificities for IgG. However, sensitivities of the assay varied depending on the herpesvirus detected. The serological microarray showed potential for screening purposes. The microarray based analyses were easy to perform, and HSV-1, HSV-2, VZV, and CMV antibodies could be detected on the same microarray.     

The use of cellular reagents as an adjunct to typing for HLA-DRw10

Two human T cell lines recognizing DRw10 were generated. One line was directly alloreactive, while a second cloned line recognized mumps virus in the context of a DRw10-like determinant. In panel studies, the lines responded only to DRw10 cells, and not to DR1 (DRw10-) cells. When compared to 5 anti-DRw10 sera, the cellular reagents gave virtually identical results. The generation and use of such cellular reagents may be of particular value for the identification of HLA-D-region specificites that are rare, poorly defined, or for which serologic typing reagents are limited.     

Humoral immune response to HSV-1 and HSV-2 viral proteins in patients with primary genital herpes

The humoral immune response to HSV-1 and HSV-2 proteins was examined in patients with primary first-episode genital herpes. Ten patients had culture-proven HSV-1 infections, 37 had HSV-2 infections, and all were seronegative to HSV proteins before developing their infections. Development of serum antibodies to individual HSV proteins and glycoproteins was determined by immunoprecipitation of radiolabeled HSV-1- and HSV-2-infected cell proteins and subsequent gel electrophoresis. In HSV-1 patients, a sequential development of antibodies to HSV-1 proteins was observed with early appearance of antibodies to the nucleocapsid protein p148 and to glycoproteins gB and gC. Seroconversion to gD and to a polypeptide of 88,000 molecular weight (p88) occurred next, and, finally, seroconversion to gE and to a nonglycosylated 66,000 dalton protein p66. In HSV-2 patients, antibodies to HSV-2 proteins p148, gB, and p88 appeared within 1 week of onset of symptoms. Seroconversion to p66, gD, and to a complex of glycoproteins gC and gE ("g80") occurred later, at a mean time of approximately 3 weeks. Seroconversion to HSV-1 gB, p88, and p66 occurred significantly later than seroconversion to the homologous counterparts. Seroconversion within 21 days of onset to HSV-2 gD, g80, and p66 was associated with a longer time to the first recurrence in HSV-2 patients, suggesting a possible role of these antibodies, alone or in combination, in the maintenance of HSV-2 latency in humans.     

The pH-dependent binding of goat IgG1 and IgG2 to protein A-Sepharose

Hjelm et al. (1972) introduced protein A-Sepharose to separate protein A-reactive from nonreactive immunoglubulins. In the conventional use of this procedure, all bound immunoglobulins are eluted in a single step with 1 M acetic acid. We used instead a pH gradient to elute column-bound goat immunoglobulins and resolved two major IgG components; one eluting with a peak at pH 6.7 and the other at pH 5.8. We investigated the relationship between these components and the known IgG subclasses which, in the goat, are separable by DEAE-cellulose chromatography. Protein A-Sepharose chromatography of DEAE fractions indicates that the second DEAE peak, in which IgG1 is known to predominate, corresponds to the IgG eluting at pH 6.7 and that the first DEAE peak, in which IgG2 predominates, corresponds to the IgG eluting at pH 5.8. Commercial anti-bovine IgG1 reacted strongly upon double immunodiffusion with the pH 6.7 IgG and only slightly with the pH 5.8 IgG; anti-bovine IgG2, however, failed to react with either sub-population of goat IgG.     

Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG-2).

The glycoprotein G (gG-2) purified from HSV-2 infected cells has been reported to be useful for determination of HSV-2 type-specific antibodies using conventional ELISA formats. This study further confirmed the specificity of gG-2 and demonstrated the feasibility of a specific IgM assay. The gG-2 ELISA was developed to detect HSV-2 specific IgG and IgM antibodies in human sera with high levels of sensitivity and specificity. Of 45 patients with culture-proven recurrent HSV-2 genital infection 44 were reactive for gG-2 IgG. Of 30 sera from patients with culture-proven recent initial HSV-2 genital infection 29 were positive for gG-2 IgM. Three patients with primary HSV-2 genital infection showed gG-2 IgM in the convalescent but not in the acute sera. The IgG- and IgM-gG-2 ELISA showed high specificity. None of 40 sera from children were reactive by either assay. Only one of 94 sera from patients with antibody to herpesviruses other than HSV reacted in the IgG assay but none reacted in the IgM assay. There was no cross-reaction with sera from patients with proven HSV-1 infection with the gG-2 antigen. The results suggest that the IgG assay can be used for demonstration of past HSV-2 infection and the IgM assay for the diagnosis of HSV-2 in neonatal herpes and primary genital herpes, when cultures or rapid diagnostic techniques are unavailable.