Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies

Peter D Burbelo,Alexandra T Issa,Maurice Exner,Kathryn H Ching,Harvey J Alter,W Lawrence Drew,Michael J Iadarola

doi:10.1186/1743-422x-6-45

Abstract

BackgroundHuman cytomegalovirus infection is associated with a variety of pathological conditions including retinitis, pneumonia, hepatitis and encephalitis that may be transmitted congenitally, horizontally and parenterally and occurs both as a primary infection and as reactivation in immunocompromised individuals. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity.MethodsHere we investigated whether luciferase immunoprecipitation systems (LIPS) would provide a more quantitative and sensitive method for detecting anti-CMV antibodies. Four protein fragments of immunodominant regions of CMV antigens pp150 and pp65 were generated as Renilla luciferase (Ruc) fusion proteins and used in LIPS with two cohorts of CMV positive and negative sera samples previously tested by ELISA.ResultsAnalysis of the antibody responses to two of these antigen fragments, pp150-d1 and pp150-d2, revealed geometric mean antibody titers in the first cohort that were 100–1000 fold higher in the CMV positive sera compared to the CMV negative samples (p < 0.0001) and infection status exactly matched the ELISA results for the 46 samples of the first cohort (100% sensitivity and 100% specificity). Two additional antigen fragments, pp65-d1 and pp65-d2 also showed robust antibody titers in some CMV-infected sera and yielded 50% and 96% sensitivity, respectively. Analysis of a second cohort of 70 samples using a mixture of the 4 antigens, which simplifies data collection and analysis, yielded values which correlated well with the sum of the values from the 4 separate tests (rs = 0.93, p < 0.00001). While comparison of the LIPS results from this second cohort with ELISA showed 100% sensitivity, LIPS detected six additional CMV positive samples that were not detected by ELISA. Heat map analysis revealed that several of the LIPS positive/ELISA negative samples had positive LIPS immunoreactivity with 3–4 of the CMV antigens.ConclusionThese results suggest that LIPS provides a highly robust and quantitative method for studying anti-CMV antibodies and has the potential to more accurately document CMV infection than standard ELISA.

Highlights

Cytomegalovirus (CMV) is the largest member of the herpesvirus family, with a genome of approximately 230 kb encoding 160 genes [1]
In order to circumvent some of the problems with solid phase ELISAs, we developed a liquid phase luciferase immunoprecipitation systems (LIPS)
Described recombinant CMV protein fragments that were used [32] included two immunodominant fragments of pp150 spanning amino acids 502–692, and 859–1048 and two immunodominant fragments of pp65 spanning amino acids 2–295, and 312–561. These four constructs were expressed in Cos1 cells and the lysates were used in the LIPS assay to evaluate a blinded sera cohort containing CMV seronegative and seropositive samples previously tested by ELISA

Summary

Introduction

Cytomegalovirus (CMV) is the largest member of the herpesvirus family, with a genome of approximately 230 kb encoding 160 genes [1]. In immunocompromised individuals, including HIV-infected patients, CMV infection and reactivation can lead to ocular infections, encephalitis, and hepatitis [4]. CMV infection is a common cause of febrile illnesses and graft rejection in transplant patients [5] and transfusion can lead to primary infection or reactivation of the virus [6]. CMV infection likely plays a role in vascular injury [7] and a variety of neurological problems including Guillain Barré syndrome [4,8]. Human cytomegalovirus infection is associated with a variety of pathological conditions including retinitis, pneumonia, hepatitis and encephalitis that may be transmitted congenitally, horizontally and parenterally and occurs both as a primary infection and as reactivation in immunocompromised individuals. There is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity

Methods

Results

Conclusion