A Reproducible and Quantitative CE-immunoassay for eNOS Protein Expression Measurements Directly in Human Brain Tissue Homogenates

Detailed proteomic characterization of human brain tissue is needed to identify potential novel biomarkers and drug targets for a variety of neurological diseases. The enzyme-linked immunosorbent assay (ELISA) offers quantitative protein expression measurements with high specificity and sensitivity. However, ELISA is challenged by brain tissue samples, which can have significant interference by matrix effects due to the high content of lipids and lipoproteins, as well as sheer protein intricacy that results from the brain’s complexity. As a result, ELISAs for studying protein biomarkers of neurological diseases are limited primarily to peripheral blood and CSF samples. 

 Here, we developed a capillary electrophoresis immunoassay (CE immunoassay) for measuring a biomarker of the cerebrovascular system, eNOS, directly in human brain whole tissue homogenates. We show that the size-based separation provided by the CE immunoassay identified differential expression of eNOS isoforms in brain tissue compared to cultured endothelial and cervical cancer cells which could not be detected by ELISA. Compared to a commercial ELISA kit, the CE immunoassay demonstrated increased sensitivity and dynamic range of detection. Furthermore, differences in tissue homogenization and storage buffer conditions impacted the ability of ELISA to detect eNOS in brain tissue samples but had no observable effect on the CE immunoassay. Finally, the CE immunoassay consumed less brain tissue for analysis, needing only 3 µL of homogenate compared to 50 µL for ELISA. Because the CE immunoassay requires only one target-validated antibody for detection, we anticipate that the CE immunoassay will enable the analysis of additional protein biomarkers in brain tissue samples that historically have been challenging to detect by traditional ELISAs.