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

Burbelo PD, Issa AT, Ching KH, Exner M, Drew WL, Alter HJ, Iadarola MJ - Virol. J. (2009)

Bottom Line: Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity.Analysis 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).These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA. burbelop@nidcr.nih.gov

ABSTRACT

Background: 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. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity.

Methods: Here 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.

Results: Analysis 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.

Conclusion: These 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.

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Detection of anti-pp150-d1 (A), anti-pp150-d2 (B), anti-pp65-d1 (C), anti-pp65-d2 (D) antibodies by LIPS in the first sera cohort (n = 46). Each symbol represents individual samples from CMV-negative and CMV-positive subjects determined by ELISA. Antibody titers in LU are plotted on a log10 scale. The dashed line, derived from the mean plus five SD of the antibody titer of the 20 uninfected samples, serves as the cut-off level for determining sensitivity and specificity for each individual antigen test. The long solid horizontal lines indicate the GMT of the antibody in each group and the vertical lines show the 95% confidence intervals.
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Figure 1: Detection of anti-pp150-d1 (A), anti-pp150-d2 (B), anti-pp65-d1 (C), anti-pp65-d2 (D) antibodies by LIPS in the first sera cohort (n = 46). Each symbol represents individual samples from CMV-negative and CMV-positive subjects determined by ELISA. Antibody titers in LU are plotted on a log10 scale. The dashed line, derived from the mean plus five SD of the antibody titer of the 20 uninfected samples, serves as the cut-off level for determining sensitivity and specificity for each individual antigen test. The long solid horizontal lines indicate the GMT of the antibody in each group and the vertical lines show the 95% confidence intervals.

Mentions: We generated four different immunodominant fragments of pp150 and pp65 as C-terminal Renilla luciferase (Ruc) fusion proteins using the pREN2 vector [25]. Previously described recombinant CMV protein fragments that were used [32] included two immunodominant fragments of pp150 spanning amino acids 502–692 (pp150-d1), and 859–1048 (pp150-d2) and two immunodominant fragments of pp65 spanning amino acids 2–295 (pp65-d1), and 312–561 (pp65-d2). These four constructs were then 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. Following unmasking of the ELISA data, analysis of the geometric mean titer (GMT) for each of these antibody tests revealed that the CMV-positive sera had 800 to 2000-fold higher antibody titers compared to the CMV-negative sera (Figure 1). For example, in the CMV-negative sera the GMTs for pp150-d1, pp150-d2, pp65-d1 and pp65-d2 were 17; 140; 200; and 7 LU, respectively, while the GMTs in CMV-positive sera were markedly higher with values of 233,715; 297,680; 17,203; and 137,002 LU, respectively. Despite a wide range of titers, the results were highly reproducible. For example, the duplicate interassay LIPS tests for anti-pp150-d1 antibodies had a coefficient of variation (CV) of 14%. These results suggest that one benefit of the dynamic range of the LIPS format is that reproducible antibody titer differences of 100–1000-fold can be detected in the CMV-negative verses CMV-positive sera without the need for serial dilutions.


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

Burbelo PD, Issa AT, Ching KH, Exner M, Drew WL, Alter HJ, Iadarola MJ - Virol. J. (2009)

Detection of anti-pp150-d1 (A), anti-pp150-d2 (B), anti-pp65-d1 (C), anti-pp65-d2 (D) antibodies by LIPS in the first sera cohort (n = 46). Each symbol represents individual samples from CMV-negative and CMV-positive subjects determined by ELISA. Antibody titers in LU are plotted on a log10 scale. The dashed line, derived from the mean plus five SD of the antibody titer of the 20 uninfected samples, serves as the cut-off level for determining sensitivity and specificity for each individual antigen test. The long solid horizontal lines indicate the GMT of the antibody in each group and the vertical lines show the 95% confidence intervals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683803&req=5

Figure 1: Detection of anti-pp150-d1 (A), anti-pp150-d2 (B), anti-pp65-d1 (C), anti-pp65-d2 (D) antibodies by LIPS in the first sera cohort (n = 46). Each symbol represents individual samples from CMV-negative and CMV-positive subjects determined by ELISA. Antibody titers in LU are plotted on a log10 scale. The dashed line, derived from the mean plus five SD of the antibody titer of the 20 uninfected samples, serves as the cut-off level for determining sensitivity and specificity for each individual antigen test. The long solid horizontal lines indicate the GMT of the antibody in each group and the vertical lines show the 95% confidence intervals.
Mentions: We generated four different immunodominant fragments of pp150 and pp65 as C-terminal Renilla luciferase (Ruc) fusion proteins using the pREN2 vector [25]. Previously described recombinant CMV protein fragments that were used [32] included two immunodominant fragments of pp150 spanning amino acids 502–692 (pp150-d1), and 859–1048 (pp150-d2) and two immunodominant fragments of pp65 spanning amino acids 2–295 (pp65-d1), and 312–561 (pp65-d2). These four constructs were then 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. Following unmasking of the ELISA data, analysis of the geometric mean titer (GMT) for each of these antibody tests revealed that the CMV-positive sera had 800 to 2000-fold higher antibody titers compared to the CMV-negative sera (Figure 1). For example, in the CMV-negative sera the GMTs for pp150-d1, pp150-d2, pp65-d1 and pp65-d2 were 17; 140; 200; and 7 LU, respectively, while the GMTs in CMV-positive sera were markedly higher with values of 233,715; 297,680; 17,203; and 137,002 LU, respectively. Despite a wide range of titers, the results were highly reproducible. For example, the duplicate interassay LIPS tests for anti-pp150-d1 antibodies had a coefficient of variation (CV) of 14%. These results suggest that one benefit of the dynamic range of the LIPS format is that reproducible antibody titer differences of 100–1000-fold can be detected in the CMV-negative verses CMV-positive sera without the need for serial dilutions.

Bottom Line: Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity.Analysis 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).These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA. burbelop@nidcr.nih.gov

ABSTRACT

Background: 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. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity.

Methods: Here 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.

Results: Analysis 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.

Conclusion: These 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.

Show MeSH
Related in: MedlinePlus