KSHV/HHV-8 IgG Antibody ELISA Kit

Catalog Number: 
15-501-000
Status: 
Current product--usually ships within 2 business days
Size: 
Kit
Use: 
For research use only. Not for use in diagnostic procedures.
Storage Temperature: 
2 to 8˚C

Intended Use

Advanced Biotechnologies Inc’s Indirect Human Herpesvirus-8 (HHV-8) IgG antibody Enzyme Linked ImmunoSorbent Assay (ELISA) is intended for the detection of HHV-8 IgG specific antibodies in human serum or plasma.

Introduction

Chang et al. first recognized Human Herpesvirus-8 (HHV-8), also known as Kaposi’s Sarcoma Herpesvirus (KSHV),1 in Kaposi’s Sarcoma (KS) lesions by representational difference analysis. HHV-8 is a gamma 2 herpesvirus and the only member of human rhadinovirus.2,3,4,5 HHV-8 has a genome size of 165 kb2-5 and shares a 51% homology to ORF-26 encoding for the capsid protein of VP23 of Herpesvirus saimiri (HVS) found in squirrel monkeys, a gamma herpesvirus of nonhuman new world primates.1,5 Similarly, the KS 330 Bam fragment of HHV-8 is also 39% homologous to BDLF-1 ORF (Tegument) of EBV, another human gamma herpesvirus.1,2-5 Using KS 330 Bam of KSHV (HHV-8), Chang et al.,1 reported that 90% of KS tissues from AIDS patients contained HHV-8 DNA sequences by PCR. Later HHV-8 DNA sequences were also reported from classical KS lesions (a neoplasm most prevalent in elderly Jewish men of Mediterranean origin), and from KS lesions, which occur in men and women from Central Africa.2-7 Localization of HHV-8 DNA was reported in vascular endothelial cells and from perivascular, spindleshaped cells of KS patients by in situ hybridization.7 HHV-8 has also been detected in cell lines established from body cavity lymphomas, from both AIDS and non-AIDS patients.2,3 While most of these established KSHV cell lines are co-infected with EBV2, several cell lines are free of EBV and HIV-1.8-13 More recently, HHV-8 was isolated from a KS lesion culture by co-cultivation with an SV40 immortalized Simian endothelial cell line (293).14

HHV-8, in addition to being implicated in human KS and body cavity lymphomas,1-6 has also been detected in HIV-1 negative patients with Bowen’s disease,15 in malignant squamous cell carcinomas, in multicentric Castleman’s disease, in HIV negative immunoblastic adenopathy, in the KS lesions in transplant patients, and in normal gastrointestinal mucosa from HIV seropositives.2-4,6 HHV-8 DNA sequences were detected in semen from AIDS patients, indicating possible sexual transmission.16 The presence of HHV-8 was also recently reported in a normal, healthy blood donor, suggesting that the virus had persisted over a long period of time in the host.17

The seroprevalence rate of HHV-8 in the general population has been reported in the scientific literature to vary from 0-57% depending on the geographic region36 and the type of immunologic assay employed. Low HHV-8 seroprevalance rates (0-5%) were found in northern Europe, Asia and North America; intemediate HHV-8 seroprevalance rates (5%-35%) were reported in the Mediterranean, Eastern European and Caribbean countries; and high HHV-8 seroprevalance rates (>50%) were detected in Central and Southern Africa and Peru.2-4,18,24,37 Some of the assays used to date include the immunofluorescence assay (IFA) to latent and lytic antigens, using the body cavity lymphoma cell lines (BC-3 or BCBL-1) (with and without TPA induction), immunoblots, and ELISA to a single HHV-8 peptide,20 or to a recombinant HHV-8 minor capsid protein.20,21,25 Because the sensitivity and specificity of antibody detection varies for the different assay formats, there is a need to compare the data from the various assays on known coded human sera panels. These sera panels will make it possible to define reliable assay systems that can accurately predict the HHV-8 seroprevalence rates in the general population.24,26-28

It may soon be possible to differentiate between lytic and latent HHV-8 infections in patients.34,35 Published data suggests that HHV-8 infections are limited in the general population and that the virus is not ubiquitous in the general population.2-4,26-31 In a number of these reported studies, highly elevated IgG antibody titers were observed in KS patients, but not in normal blood donors.18-29 The seroprevalence of HHV-8 is 80 - 95% in classic KS patients, and 40 - 50% in HIV-1 patients without KS.35 Thus the seroprevalence rate of HHV-8 is different from other human herpesviruses (i.e., EBV, HHV-6, HHV-7, CMV, or HSV-1) where >80% of the population is antibody-positive for these viruses.2,3

HHV-8 whole virus extract was prepared from sucrose gradient purified virus that was isolated from the KS-1 cell line. The KS-1 cell line was derived from a body cavity lymphoma of an EBV/HIV-negative patient.9 Virus antigen from the KS-1 cell line was then used for the development of an ELISA kit which measures antibody to the majority of HHV-8 structural proteins. These KS-1 cell cultures demonstrate ?40% antigen expressing cells by IFA using sera from KS (classical) or HIV-KS patients, but do not react with numerous human sera obtained from normal blood donors. KS-1 cells are not contaminated with EBV, CMV, HHV-6, HHV-7, or HIV-1 and are also Mycoplasma-free.

The diagnosis for HHV-8 infection in a patient can be confirmed by PCR analysis and by immunological assays; e.g., IFA and ELISA. PCR detection of HHV-8 DNA in peripheral blood identifies only 50% of patients with Kaposi’s sarcoma25, 33, 34, while serology tests are more accurate: 80-90% of patients with Kaposi’s sarcoma are seropositive for antibody to HHV-8. PCR is expensive and less practical for a clinical setting because there are more chances for contamination in handling the samples used for evaluation. It is also more practical to use ELISA and IFA immunoassay kits in evaluating the patient sera for HHV-8 infection, as well as to follow the course of KS disease. ELISA has certain advantages over IFA, since it can be automated, and a large number of serologic samples can be tested at one time. Clinical research studies are still needed utilizing serial bleeds to define the rise and fall of antibody levels to HHV-8 and to correlate that with HHV-8 infection and disease states such as KS and other malignancies where HHV-8 may be involved in the pathogenesis.

Principle of the ELISA Assay

Advanced Biotechnologies Inc’s indirect HHV-8 IgG Antibody ELISA Kit is intended for the detection of HHV-8 IgG antibodies in human serum or plasma. 8-well strips are coated with optimal amounts of solubilized HHV-8 whole virus extract. The test procedure involves three incubation steps:

  1. Tested human samples are added to the microtiter wells and any specific antibodies present to HHV- 8 will bind to the antigen-coated plates, formulating immunological complexes. After the incubation, the strips are washed to remove unbound sample components.
  2. Anti-human IgG conjugated to the enzyme horseradish peroxidase (HRP) is added to the wells and during incubation, will bind to the antibody-antigen complexes already present in the wells. After incubation, the wells are washed to remove unbound conjugate.
  3. HRP substrate tetramethylbenzidine (TMB) is added to the wells. During incubation, enzyme mediated cleavage of the substrate results in a color change. The reaction is stopped by adding Stop Solution, and the wells with positive samples (i.e., contain antibodies to HHV-8) will change color from blue to yellow. The wells with negative samples (i.e., do not contain antibodies to HHV-8) will remain almost colorless. The color intensity is proportional to the level of HHV-8 IgG antibodies in a sample, and is measured photometrically.

References

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  20. Simpson, G.R., et al., Lancet 348:1133-1138, 1996
  21. Miller, G., et al., New Eng. J. Med. 334:1292-1297, 1996
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