Abstract
Application-specific validation of antibodies is a critical prerequisite for their successful use. Here we introduce an automated framework for characterization and screening of antibodies against synaptic molecules for high-resolution immunofluorescence array tomography (AT). The proposed Synaptic Antibody Characterization Tool (SACT) is designed to provide an automatic, robust, flexible, and efficient tool for antibody characterization at scale. SACT automatically detects puncta of immunofluorescence labeling from candidate antibodies and determines whether a punctum belongs to a synapse. The molecular composition and size of the target synapses expected to contain the antigen is determined by the user, based on biological knowledge. Operationally, the presence of a synapse is defined by the colocalization or adjacency of the candidate antibody punctum to one or more reference antibody puncta. The outputs of SACT are automatically computed measurements such as target synapse density and target specificity ratio that reflect the sensitivity and specificity of immunolabeling with a given candidate antibody. These measurements provide an objective way to characterize and compare the performance of different antibodies against the same target, and can be used to objectively select the antibodies best suited for AT and potentially for other immunolabeling applications.
Highlights
Antibodies are an indispensable tool for the modern biologist
This allows the output of two additional measures: density of target synapses, that is the synapses containing candidate antibody puncta, and target specificity ratio, which is the fraction of candidate antibody puncta that are at the target synapses
In order to evaluate an antibody, a series of measurements are computed; each measurement captures an aspect of the antibody’s performance that would be sought by an expert observer when manually interacting with the data. Each of these measurements provides unbiased quantitative information to help evaluate the intensity, specificity and sensitivity of immunolabeling obtained with a given antibody. These include the density of puncta, and the average punctum volume and standard deviation for each antibody, as well as the target synapse density, and target specificity ratio
Summary
We introduce an automated framework for characterization and screening of antibodies against synaptic molecules for high-resolution immunofluorescence array tomography (AT). SACT automatically detects puncta of immunofluorescence labeling from candidate antibodies and determines whether a punctum belongs to a synapse. The outputs of SACT are automatically computed measurements such as target synapse density and target specificity ratio that reflect the sensitivity and specificity of immunolabeling with a given candidate antibody. These measurements provide an objective way to characterize and compare the performance of different antibodies against the same target, and can be used to objectively select the antibodies best suited for AT and potentially for other immunolabeling applications
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