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Large scale immune profiling of infected humans and goats reveals differential recognition of Brucella melitensis antigens.

Liang L, Leng D, Burk C, Nakajima-Sasaki R, Kayala MA, Atluri VL, Pablo J, Unal B, Ficht TA, Gotuzzo E, Saito M, Morrow WJ, Liang X, Baldi P, Gilman RH, Vinetz JM, Tsolis RM, Felgner PL - PLoS Negl Trop Dis (2010)

Bottom Line: To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins.Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans.These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, University of California Irvine, Irvine, California, USA.

ABSTRACT
Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

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Related in: MedlinePlus

Immunostrips probing.(a)Thirteen serodiagnostic antigens were printed onto nitrocellulose paper in adjacent stripes using a BioDot jet dispenser as described in Materials and Methods. Strips were probed with Culture Positive or Peruvian naive sera diluted 1/200 followed by alkaline phosphatase conjugated secondary antibody and enzyme substrate. Weak reactivity in the naïve healthy controls can be distinguished from the strong reactivity in infected group. (b). The LOOCV ROC curve was generated and sensitivity and specificity of immunostrips probing test is 94% and 89%, respectively.
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pntd-0000673-g005: Immunostrips probing.(a)Thirteen serodiagnostic antigens were printed onto nitrocellulose paper in adjacent stripes using a BioDot jet dispenser as described in Materials and Methods. Strips were probed with Culture Positive or Peruvian naive sera diluted 1/200 followed by alkaline phosphatase conjugated secondary antibody and enzyme substrate. Weak reactivity in the naïve healthy controls can be distinguished from the strong reactivity in infected group. (b). The LOOCV ROC curve was generated and sensitivity and specificity of immunostrips probing test is 94% and 89%, respectively.

Mentions: To test the feasibility of using the serodiagnostic antigens in an alternative analytical assay, thirteen serodiagnostic proteins were printed onto Nitrocellulose membranes using a BioDot jet dispenser. The paper was then cut into 3 mm strips (Fig. 5a). The individual strips were probed with 42 different culture positive sera and 44 Peruvian naive sera. Brucellosis patients reacted strongly against the serodiagnostic antigens with variable signal intensity among the patients. Naïve samples showed much lower reactivity against these serodiagnostic antigens. To assess the ability of antigens to separate disease and healthy controls, the LOOCV ROC curve was also generated (Fig. 5b). The ROC curve shows that this probing test yielded a high AUC of 0.962 with sensitivity rate of 94% and specificity rate of 89%. Thus, thirteen differentially reactive serodiagnostic antigens identified by microarray analysis in immunostrip format validated the list of serodiagnostic antigens to correctly classify B. melitensis positive sera.


Large scale immune profiling of infected humans and goats reveals differential recognition of Brucella melitensis antigens.

Liang L, Leng D, Burk C, Nakajima-Sasaki R, Kayala MA, Atluri VL, Pablo J, Unal B, Ficht TA, Gotuzzo E, Saito M, Morrow WJ, Liang X, Baldi P, Gilman RH, Vinetz JM, Tsolis RM, Felgner PL - PLoS Negl Trop Dis (2010)

Immunostrips probing.(a)Thirteen serodiagnostic antigens were printed onto nitrocellulose paper in adjacent stripes using a BioDot jet dispenser as described in Materials and Methods. Strips were probed with Culture Positive or Peruvian naive sera diluted 1/200 followed by alkaline phosphatase conjugated secondary antibody and enzyme substrate. Weak reactivity in the naïve healthy controls can be distinguished from the strong reactivity in infected group. (b). The LOOCV ROC curve was generated and sensitivity and specificity of immunostrips probing test is 94% and 89%, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864264&req=5

pntd-0000673-g005: Immunostrips probing.(a)Thirteen serodiagnostic antigens were printed onto nitrocellulose paper in adjacent stripes using a BioDot jet dispenser as described in Materials and Methods. Strips were probed with Culture Positive or Peruvian naive sera diluted 1/200 followed by alkaline phosphatase conjugated secondary antibody and enzyme substrate. Weak reactivity in the naïve healthy controls can be distinguished from the strong reactivity in infected group. (b). The LOOCV ROC curve was generated and sensitivity and specificity of immunostrips probing test is 94% and 89%, respectively.
Mentions: To test the feasibility of using the serodiagnostic antigens in an alternative analytical assay, thirteen serodiagnostic proteins were printed onto Nitrocellulose membranes using a BioDot jet dispenser. The paper was then cut into 3 mm strips (Fig. 5a). The individual strips were probed with 42 different culture positive sera and 44 Peruvian naive sera. Brucellosis patients reacted strongly against the serodiagnostic antigens with variable signal intensity among the patients. Naïve samples showed much lower reactivity against these serodiagnostic antigens. To assess the ability of antigens to separate disease and healthy controls, the LOOCV ROC curve was also generated (Fig. 5b). The ROC curve shows that this probing test yielded a high AUC of 0.962 with sensitivity rate of 94% and specificity rate of 89%. Thus, thirteen differentially reactive serodiagnostic antigens identified by microarray analysis in immunostrip format validated the list of serodiagnostic antigens to correctly classify B. melitensis positive sera.

Bottom Line: To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins.Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans.These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, University of California Irvine, Irvine, California, USA.

ABSTRACT
Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

Show MeSH
Related in: MedlinePlus