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Evaluation and validation of commercial antibodies for the detection of Shb.

ABSTRACT: Antibodies are among the most important tools for protein detection but, prior to their usage, proper validation of their appropriateness for given applications is required. The utility of an antibody depends on its sensitivity and specificity. We studied these two aspects in a panel of commercial antibodies against Shb, a platform protein involved in receptor tyrosine kinase signalling, but the function of which is still incompletely understood. Several of the antibodies showed shortcomings or were not acceptable for detection of the endogenous protein. The few that could detect Shb were doing so in either western blotting or immunoprecipitation experiments but a given antibody could not work in both applications. This article provides a resource for the available molecular tools that can be used in future research on Shb.

SUBMITTER: Vanli G

PROVIDER: S-EPMC5711028 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Evaluation and validation of commercial antibodies for the detection of Shb.

Vanli Güliz G Cuesta-Marban Alvaro A Widmann Christian C

PloS one 20171201 12

Antibodies are among the most important tools for protein detection but, prior to their usage, proper validation of their appropriateness for given applications is required. The utility of an antibody depends on its sensitivity and specificity. We studied these two aspects in a panel of commercial antibodies against Shb, a platform protein involved in receptor tyrosine kinase signalling, but the function of which is still incompletely understood. Several of the antibodies showed shortcomings or ...[more]

PMID: 29194461

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Project description:BackgroundTransfusion with Plasmodium-infected blood represents a risk for malaria transmission, a rare but severe event. Several non-endemic countries implement a strategy for the screening of candidate blood donors including questionnaire for the identification of at-risk subjects and laboratory testing of blood samples, often serology-based, with temporary deferral from donation for individuals with a positive result. In Italy, the most recent legislation, issued in November 2015, introduced the use of serological tests for the detection of anti-Plasmodium antibodies.MethodsIn the absence of a gold standard for malaria serology, the aim of this work was to evaluate five commercial ELISA kits, and to determine their accuracy (sensitivity and specificity) in comparison to immuno-fluorescence antibody test (IFAT), and their agreement (concordance of results). Serum samples from malaria patients or from subjects with malaria history (N = 64), malaria naïve patients with other parasitic infections (N = 15), malaria naïve blood donors (N = 8) and malaria exposed candidate blood donors (N = 36) were tested.ResultsThe specificity of all ELISA kits was 100%, while sensitivity ranged between 53 and 64% when compared to IFAT on malaria patients samples. When tested on candidate blood donors' samples, ELISA kits showed highly variable agreement (42-94%) raising the possibility that the same individual could be included or excluded from donation depending on the test in use by the transfusion centre.ConclusionsThese preliminary results indicate how the lack of a gold standard for malaria serology must be taken into account in the application and future revision of current legislation. There is need of developing more sensitive serological assays. Moreover, the adoption of a unique serological test at national level is recommended, as well as the development of screening algorithms based on multiple laboratory tests, including molecular assays.

Dataset Information

Evaluation and validation of commercial antibodies for the detection of Shb.

Publications

Evaluation and validation of commercial antibodies for the detection of Shb.

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