Use of densitometric analysis for interpreting HSV serologies based on Western blot
Abstract
Western blot assays have been described for detecting antibodies to herpes simplex virus (HSV). A predominance of antibody binding to either the HSV-1 or the HSV-2-containing blot has been reported to indicate infection with HSV-1 or HSV-2, respectively. By densitometry, differential binding of total antibody on HSV-1 versus HSV-2 strips can be expressed as a ratio. To determine the clinical correlation of these ratios, sera from 81 patients with culture-proven oral or genital herpes were tested by Western blot (WB) and densitometry. Binding ratios accurately identified patients with primary HSV-2 infections. However, ratios on sera with HSV-1 antibody or dual antibody status showed considerable overlap. Densitometry was shown to amplify and clarify the band corresponding to the HSV-2 specific glycoprotein gG-2 and was useful, in this respect, for detecting HSV-2 antibody in the presence of HSV-1 antibody. Sera from 52 patients with asymptomatic HSV-1, HSV-2 or dual infection were also tested by WB. Typing results were confirmed by cross-adsorption of sera (“adsorption blot assay”). Ratios of HSV-2 to HSV-1 binding were higher in asymptomatic versus symptomatic patients with dual antibody (P<0.01). Ratios for those with HSV-1 or HSV-2 antibody types were not affected by disease expression.
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Characterization of polyclonal antibodies against the capsid proteins of Ljungan virus
2008, Journal of Virological MethodsLjungan virus (LV) is a suspected human pathogen isolated from voles in Sweden and North America. To enable virus detection and studies of localization and activity of virion proteins, polyclonal antibodies were produced against bacterially expressed capsid proteins of the LV strain, 87-012G. Specific detection of proteins corresponding to viral antigens in lysates of LV infected cells was demonstrated by immunoblotting using each one of the generated polyclonal antibodies. In addition, native viral antigens present in cell culture infected with LV strains 87-012G or 145SLG were detected in ELISA and by immunofluorescence using the antibodies against the VP0 and VP1 proteins. The anti-VP3 antibody did not react with native proteins of the LV virion, suggesting that the VP3 is less potent in evoking humoral response and may have a less exposed orientation in the virus capsid. No activity of the antibodies was observed against the closely related human parechovirus type 1. The polyclonal antibody against the VP1 protein was further used for detection of LV infected myocytes in a mouse model of LV-induced myocarditis. Thus, polyclonal antibodies against recombinant viral capsid proteins enabled detection of natural LV virions by several different immunological methods.
A population-based study of squamous cell vaginal cancer: HPV and cofactors
2002, Gynecologic OncologyBackground. Little is known about the etiology of in situ or invasive squamous cell cancer of the vagina. It is thought that some vaginal cancers may have the same etiology as cervical cancer. It is also not known whether in situ and invasive vaginal cancer share the same etiologic factors. We conducted a study to evaluate risk factors for in situ and invasive vaginal cancer and their potential relationship to prior exposure to human papillomaviruses (HPV).
Methods. A population-based case–control study included 156 women with squamous cell in situ or invasive vaginal cancer diagnosed between January 1981 and June 1998 and 2041 control women identified through random-digit dialing in western Washington state. Cases and controls were interviewed in person and provided blood samples; archival tumor tissue was retrieved for cases. Blood samples were tested for antibodies to HPV, and tumor tissue was tested for HPV DNA.
Results. Women with vaginal cancer were more likely to have five or more lifetime sexual partners (OR = 3.1, 95% CI 1.9 to 4.9), to have an early age at first intercourse (<17 years OR = 2.0, 95% CI 1.2 to 3.5), and to be current smokers at diagnosis (OR = 2.1, 95% CI 1.4 to 3.1) than control women. Approximately 30% of all cases had been treated for a prior anogenital tumor, most often of the cervix. Prior hysterectomy was a risk factor only among women who had no history of prior anogenital cancer (OR = 3.9 95% CI 2.5 to 6.1). Antibodies to HPV16 L1 were strongly related to risk of vaginal cancer (OR = 4.3, 95% CI 3.0 to 6.2). We detected HPV DNA in tumor blocks from over 80% of the patients with in situ and 60% of the patients with invasive cancers.
Conclusions. In situ and invasive vaginal neoplasia have many of the same risk factors as cervical cancer, including a strong relationship to HPV infection. Women who have been treated for a prior anogenital cancer, particularly of the cervix, have a high relative risk, although low absolute risk, of being diagnosed with vaginal cancer.
Evaluation of a fully automated glycoprotein G-2 based assay for the detection of HSV-2 specific IgG antibodies in serum and plasma
1999, Journal of Clinical VirologyBackground: 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 HSV-2 EIA compared to the other assays, is that this assay can be performed in a fully automated process.
Detection of Epstein-Barr virus-specific antibodies by an automated enzyme immunoassay
1998, Diagnostic Microbiology and Infectious DiseaseDetection of antibodies to specific antigens of Epstein-Barr virus (EBV) has been conventionally performed by an immunofluorescence assay (IFA). The procedure is labor intensive and expensive, and interpretation of results is subjective. We evaluated an automated enzyme immunoassay (EIA) (INCSTAR® Corp., Stillwater, MN, USA) using 290 serum specimens submitted for the diagnosis of acute infection with EBV. Antibodies (IgG, IgM) to EBV capsid antigen and IgG class antibodies to the nuclear antigen of the virus were obtained using the LABOTECH™ Automated Microplate Analyzer (BioChem ImmunoSystems Inc., Allentown, PA, USA) and were compared to the antibody profile results obtained by IFA and Western blot as the “gold standard.” For detection of acute infection with EBV (presence of IgM and IgG antibodies to the capsid antigen; absence of antibodies to the nuclear antigen), the EIA had 100% sensitivity (11 of 11) and 99% specificity (275 of 279) compared to IFA and Western blot results. A cost analysis of IFA and EIA procedures, based on an estimated annual volume of 12,000 procedures, indicated that $236,000 direct cost and 1,400 h of technologist time could be saved with the automated compared with immunofluorescence procedure. The automated EIA for determination of antibodies provides cost-effective, accurate diagnosis of EBV infections in laboratories processing high numbers of specimens now processed by IFA.
Evaluation of new reagents for typing IgG to HSV-1 and HSV-2
1997, Opportunistic PathogensUntil recently, the lack of suitable type specific assays has hampered the serological diagnosis of herpes simplex virus (HSV) infections, due to the high crossreactivity between types 1 and 2. The aim of the present paper is the evaluation of new commercial methods for the detection of HSV-1 and HSV-2-specific IgG using glycoprotein G as antigen (BioElisa HSV-1 and BioElisa HSV-2), in their application to viral diagnosis and seroepidemiological studies. Eighty two serum samples from HSV recent infections (30 samples from 13 cases), normal children (28 samples), and patients attended in clinics for sexually transmitted diseases (STD) (24 samples from 20 patients) were studied by such methods and the results compared with those obtained by a conventional indirect ELISA, and by the complement fixation test. The methods gave a type specific identification of the antibody response in nine of the 13 HSV patients. Positive results for anti-HSV-2 IgG were obtained in four cases among the STD patients but in none among the normal children. Nineteen of the former and seven of the latter were positive for anti-HSV-1. Only one sample was reactive in the HSV-1 assay, and negative by the indirect ELISA. Type specific HSV-1 and HSV-2 assays may help the serological diagnosis of HSV infections, since they allow the correct characterization of the antibody response. Bearing in mind the high specificity of the method for HSV-2 IgG, it might be useful in screening of populations for anti-HSV-2 and especially in prevention of the neonatal HSV-2 infection.
Asymptomatic maternal shedding of herpes simplex virus at the onset of labor: Relationship to preterm labor
1996, Obstetrics and GynecologyTo determine if fetal growth restriction and prematurity are observed with subclinical shedding of herpes simplex virus (HSV) at the onset of labor.
Within 48 hours of delivery, cultures were taken from the cervix and external genitalia of 15,923 asymptomatic pregnant women without symptoms or signs of genital HSV infection; results were positive for HSV in 57. Each of these 57 women were compared with a control group composed of the three culture-negative women delivering immediately before and the three delivering immediately after each woman shedding HSV.
The median birth weight for infants born to the 57 women with asymptomatic shedding was 3050 g, compared with 3360 g among the 342 women without asymptomatic shedding, a statistically significant difference (P < .002). These differences were due to very low birth weight (LBW) among the five infants of women with subclinical viral shedding secondary to recently acquired primary genital herpes; these five infants had a median gestational age of 33 weeks, compared with 37 weeks for the 14 infants of mothers with nonprimary, first-episode disease and 39 weeks for the 33 infants of women with reactivation disease, also a significant difference (P=.018).
Asymptomatic genital shedding of HSV at the onset of labor because of subclinical primary genital HSV infection is associated with preterm delivery. Women who acquire genital HSV-2 before pregnancy and are shedding subclinically at the onset of labor experience no increase in adverse outcome. Thus, prevention of the prematurity and LBW associated with genital herpes means that acquisition of the infection in late pregnancy must be prevented.