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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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CMV antibody titers to the sum of the four individual tests and using a mixture format. Antibody titers to the sum of the 4 antigens in the first cohort (A), second cohort (B) or using a 4 antigen mixture format in the second cohort (C). Each symbol represents individual samples from CMV-negative and CMV-positive samples determined by ELISA. In the case of the first cohort, the sum of the titer values from the four individual tests and a cut-off value of 15,000, showed 100% sensitivity and 100% specificity. In the case of the second cohort, the LIPS tests from the sum of the 4 individual tests (B) or tested simultaneously as a mixture of four antigens (C) showed almost identical results; in each case, LIPS detected 6 samples that were ELSA negative. The solid horizontal lines indicate the GMT of the antibodies in each group and the vertical lines show the 95% confidence intervals.
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Figure 2: CMV antibody titers to the sum of the four individual tests and using a mixture format. Antibody titers to the sum of the 4 antigens in the first cohort (A), second cohort (B) or using a 4 antigen mixture format in the second cohort (C). Each symbol represents individual samples from CMV-negative and CMV-positive samples determined by ELISA. In the case of the first cohort, the sum of the titer values from the four individual tests and a cut-off value of 15,000, showed 100% sensitivity and 100% specificity. In the case of the second cohort, the LIPS tests from the sum of the 4 individual tests (B) or tested simultaneously as a mixture of four antigens (C) showed almost identical results; in each case, LIPS detected 6 samples that were ELSA negative. The solid horizontal lines indicate the GMT of the antibodies in each group and the vertical lines show the 95% confidence intervals.

Mentions: In addition to analyzing the data from the four antigens separately and to compare to the mixture of antigens analyzed in a second, independent cohort (see below), we summed the antibody titers from the 4 individual tests and used a 15,000 LU cutoff, which was derived from the mean plus five standard deviations of the CMV negative samples. In this analysis, the 26 CMV positive samples detected in the individual tests were also detected in this combined approach and the 20 CMV negative samples again tested negative (Figure 2A). Together these results suggest that the single antigen tests (especially the pp150-d1 and pp150-d2 tests) and the combined results from the four individual tests provide an extraordinarily sensitive and specific method for profiling anti-CMV antibodies to diagnose infection.


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)

CMV antibody titers to the sum of the four individual tests and using a mixture format. Antibody titers to the sum of the 4 antigens in the first cohort (A), second cohort (B) or using a 4 antigen mixture format in the second cohort (C). Each symbol represents individual samples from CMV-negative and CMV-positive samples determined by ELISA. In the case of the first cohort, the sum of the titer values from the four individual tests and a cut-off value of 15,000, showed 100% sensitivity and 100% specificity. In the case of the second cohort, the LIPS tests from the sum of the 4 individual tests (B) or tested simultaneously as a mixture of four antigens (C) showed almost identical results; in each case, LIPS detected 6 samples that were ELSA negative. The solid horizontal lines indicate the GMT of the antibodies 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 2: CMV antibody titers to the sum of the four individual tests and using a mixture format. Antibody titers to the sum of the 4 antigens in the first cohort (A), second cohort (B) or using a 4 antigen mixture format in the second cohort (C). Each symbol represents individual samples from CMV-negative and CMV-positive samples determined by ELISA. In the case of the first cohort, the sum of the titer values from the four individual tests and a cut-off value of 15,000, showed 100% sensitivity and 100% specificity. In the case of the second cohort, the LIPS tests from the sum of the 4 individual tests (B) or tested simultaneously as a mixture of four antigens (C) showed almost identical results; in each case, LIPS detected 6 samples that were ELSA negative. The solid horizontal lines indicate the GMT of the antibodies in each group and the vertical lines show the 95% confidence intervals.
Mentions: In addition to analyzing the data from the four antigens separately and to compare to the mixture of antigens analyzed in a second, independent cohort (see below), we summed the antibody titers from the 4 individual tests and used a 15,000 LU cutoff, which was derived from the mean plus five standard deviations of the CMV negative samples. In this analysis, the 26 CMV positive samples detected in the individual tests were also detected in this combined approach and the 20 CMV negative samples again tested negative (Figure 2A). Together these results suggest that the single antigen tests (especially the pp150-d1 and pp150-d2 tests) and the combined results from the four individual tests provide an extraordinarily sensitive and specific method for profiling anti-CMV antibodies to diagnose infection.

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