Plasma proteins in edematous white matter after intracerebral hemorrhage confound immunoblots: an ELISA to quantify contamination.
Intracerebral hemorrhage (ICH) and traumatic brain injury can induce brain tissue edema (i.e., interstitial and/or vasogenic), containing high concentrations of plasma proteins. To understand biochemical processes in edema development following these insults, it would be useful to examine alterations in various proteins (e.g., transcription factors, signaling). However, determining altered protein responses in edematous brain tissue using standard immunoblotting techniques is problematic due to contaminating plasma proteins. To solve this problem, we developed an enzyme-linked immunosorbent assay (ELISA) method to quantify the two major plasma proteins, albumin and immunoglobulin G (IgG), that comprise about 80% of the total plasma proteins. We tested our method on edematous white matter samples from our porcine ICH model. To induce ICH, we infused autologous arterial whole blood (3 mL) into frontal hemispheric white matter of pentobarbital- anesthetized pigs ( approximately 20 kg) over 15 min. We froze brains in situ at various times up to 24 h post- ICH and sampled white matter adjacent and contralateral to hematomas. We prepared cytoplasmic extracts that we subjected to ELISA and immunoblotting analyses. Our results demonstrate that this ELISA method is accurate, reproducible, and enables the concentrations of albumin and IgG in edematous brain tissue samples to be accurately determined. By using this correction method, equal amounts of cellular protein can be loaded onto gels during immunoblotting procedures. This method is applicable to edematous tissue samples in brain injury models in which high plasma protein concentrations result from interstitial or vasogenic edema development.
Loftspring
Matthew C
MC
Department of Neurology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Beiler
Shauna
S
Beiler
Christian
C
Wagner
Kenneth R
KR
D000818:Animals; D001929:Brain Edema / Q000209:etiology / Q000378:metabolism*; D002543:Cerebral Hemorrhage / Q000150:complications / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000378:metabolism*; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000378:metabolism*; D013552:Swine
D007074:Immunoglobulin G; D012709:Serum Albumin