A competitive inhibition ELISA as a probe for studying the refolding of human serum albumin.
The refolding of iodoacetic acid-blocked human serum albumin (HSA) was studied using a modified competitive inhibition ELISA. A maximum of 89% native activity was detected 24 hours after initiating refolding using an albumin concentration of 600 micrograms/mL. The presence of both monomer and polymer HSA was studied using native polyacrylamide gel electrophoresis of thiol-blocked HSA samples. Monomer HSA was not detected until 2.5 hours after initiating refolding. Fractionated polymer and monomer HSA from a sample trapped at 72 hours after initiating refolding was determined to have 40% and 87% native activity respectively. Both polymer and monomer HSA fractions contribute to the overall immunological activity detected by the ELISA, at various times. The ELISA assay was able to detect the changing HSA conformation associated with refolding of totally reduced HSA.
Clausen
J A
JA
University of Queensland.
Reid
S
S
Randerson
D H
DH
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D011487:Protein Conformation; D012709:Serum Albumin / Q000737:chemistry*
D012709:Serum Albumin