A simple dot-immunobinding assay for quantification of synaptophysin-like immunoreactivity in human brain

Neocortical decreases in synaptic density correlate significantly with the cognitive impairment seen in Alzheimer disease. Recently available monoclonal antibodies (MAb) have made possible the highly specific and sensitive detection of synapse-associated proteins in immunocytochemical and immunochemical techniques. We describe a simple yet highly sensitive dot-immunobinding assay for relative quantification of the synapse marker protein synaptophysin in human brain homogenate fractions with the mouse MAb SY38. Fractions prepared from control and Alzheimer specimens were blotted to nitrocellulose membranes and reacted with SY38, rabbit secondary antibody, and iodinated protein A. A relative standard curve was constructed to normalize results from multiple assay runs. We correlated the results with the more complex immunocytochemical synaptic density measurement technique of immunolabeling coupled with laser confocal imaging, showing good correlation at r = 0.821. Results from Alzheimer cases showed a 40% decrease in synaptophysin immunoreactivity in midfrontal cortex compared with normal controls.