Immunohistochemical optimization of a panel of antibodies against amyloid precursor protein for immunodetection of axonal injury in neurologic diseases of sheep

Amyloid precursor protein (APP) is the most sensitive early immunomarker of axonal injury in the brain. Currently, there are several commercially available antibodies to detect axonal spheroids expressing APP. However, immunolabeling of a given molecule can be variable when antibodies are directed to different epitopes. In this study, we optimized a panel of antibodies (22C11, Y188, NAB228, C1/6.1) for labeling APP-immunopositive axonal spheroids in ovine brains and compared the patterns of injury in brains containing spheroids from neurotoxic (clostridial focal symmetrical encephalomalacia [FSE]) and traumatic brain injury. The pattern of axonal spheroid immunolabeling in FSE necrotic foci and trauma-damaged white matter was similar with all 4 antibodies. Only subtle differences in axonal immunolabeling were found probably due to whether the C- or N-terminal fragment of APP was targeted. However, while background labeling with 3 of the antibodies was minimal, there was robust, diffuse labeling of axons due to myelin immunolabeling with the Y188 antibody, which was determined to be specific. Thus, while these antibodies can detect axonal injury with similar efficacy in sheep brains, caution must be taken with Y188 owing to confounding myelin immunolabeling. This study will hopefully serve as a guide for optimal immunodetection of axonal injury in ovine encephalopathies.