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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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Heat map representation of patient antibody profiles in second cohort to the four CMV antigens. The titer values for each serum were log10 transformed and then the titer levels were color-coded as indicated by the log10 scale on the right, in which signal intensities range from green to red indicating low and high titers, respectively. The samples were rank ordered from highest to lowest based on the sum of the antibody titers to the four antigen panel. The samples on the left are from CMV infected sera and the samples in the middle panel represent the uninfected control sera. On the right are samples that were positive by the LIPS assay, but found to be negative according to the ELISA assay.
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Figure 3: Heat map representation of patient antibody profiles in second cohort to the four CMV antigens. The titer values for each serum were log10 transformed and then the titer levels were color-coded as indicated by the log10 scale on the right, in which signal intensities range from green to red indicating low and high titers, respectively. The samples were rank ordered from highest to lowest based on the sum of the antibody titers to the four antigen panel. The samples on the left are from CMV infected sera and the samples in the middle panel represent the uninfected control sera. On the right are samples that were positive by the LIPS assay, but found to be negative according to the ELISA assay.

Mentions: Log 10 transformed antibody titers and color coding were used to create a heatmap to easily visualize the different patient antibody responses toward the panel of antigens and to gain further insight into discordant samples (Figure 3). In this graphic, obvious marked differences in patient antibody responses to the antigen panel were observed, which illustrates the heterogeneity in individual humoral immune responses to the four individual CMV antigens (Figure 3). As shown in Figure 3, many of the ELISA positive/LIPS positive samples showed immunoreactivity to all four of the antigen fragments. One of the ELISA positive samples (Figure 3, bottom of heatmap of CMV positive samples) only showed positive LIPS immunoreactivity with the single anti-pp65-d1 antigen. Analysis of the discordant ELISA negative/LIPS positive samples showed that five of the six CMV ELISA negative samples had highly positive anti-pp150-d1 antibody titers. Furthermore, two of the ELISA negative samples were also positive by LIPS for anti-pp65-d2 and four samples were also positive for anti-pp65-d1 antibodies (Figure 3). It should be noted that many of these ELISA negative/LIPS positive samples showed markedly higher antibody titers than many of the samples that were positive by both ELISA and LIPS. Also, all of the discordant sera had low background binding reactivity with a lysate containing Ruc vector control protein suggesting that the observed immunoreactivity was not due to "sticky sera" or non-specific binding to the Renilla luciferase protein backbone (data not shown). Based on these results, it seems plausible that many of these discordant samples may represent true CMV positive samples that were missed by ELISA.


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)

Heat map representation of patient antibody profiles in second cohort to the four CMV antigens. The titer values for each serum were log10 transformed and then the titer levels were color-coded as indicated by the log10 scale on the right, in which signal intensities range from green to red indicating low and high titers, respectively. The samples were rank ordered from highest to lowest based on the sum of the antibody titers to the four antigen panel. The samples on the left are from CMV infected sera and the samples in the middle panel represent the uninfected control sera. On the right are samples that were positive by the LIPS assay, but found to be negative according to the ELISA assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Heat map representation of patient antibody profiles in second cohort to the four CMV antigens. The titer values for each serum were log10 transformed and then the titer levels were color-coded as indicated by the log10 scale on the right, in which signal intensities range from green to red indicating low and high titers, respectively. The samples were rank ordered from highest to lowest based on the sum of the antibody titers to the four antigen panel. The samples on the left are from CMV infected sera and the samples in the middle panel represent the uninfected control sera. On the right are samples that were positive by the LIPS assay, but found to be negative according to the ELISA assay.
Mentions: Log 10 transformed antibody titers and color coding were used to create a heatmap to easily visualize the different patient antibody responses toward the panel of antigens and to gain further insight into discordant samples (Figure 3). In this graphic, obvious marked differences in patient antibody responses to the antigen panel were observed, which illustrates the heterogeneity in individual humoral immune responses to the four individual CMV antigens (Figure 3). As shown in Figure 3, many of the ELISA positive/LIPS positive samples showed immunoreactivity to all four of the antigen fragments. One of the ELISA positive samples (Figure 3, bottom of heatmap of CMV positive samples) only showed positive LIPS immunoreactivity with the single anti-pp65-d1 antigen. Analysis of the discordant ELISA negative/LIPS positive samples showed that five of the six CMV ELISA negative samples had highly positive anti-pp150-d1 antibody titers. Furthermore, two of the ELISA negative samples were also positive by LIPS for anti-pp65-d2 and four samples were also positive for anti-pp65-d1 antibodies (Figure 3). It should be noted that many of these ELISA negative/LIPS positive samples showed markedly higher antibody titers than many of the samples that were positive by both ELISA and LIPS. Also, all of the discordant sera had low background binding reactivity with a lysate containing Ruc vector control protein suggesting that the observed immunoreactivity was not due to "sticky sera" or non-specific binding to the Renilla luciferase protein backbone (data not shown). Based on these results, it seems plausible that many of these discordant samples may represent true CMV positive samples that were missed by ELISA.

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