Reports

Apoptosis

, Volume 11, Issue 7, pp 1121-1129

First online:

Serum leucine-rich alpha-2-glycoprotein-1 binds cytochrome c and inhibits antibody detection of this apoptotic marker in enzyme-linked immunosorbent assay

  • Chad CummingsAffiliated withDepartment of Microbiology and Center for Immunology, University of Minnesota
  • , Jennifer WalderAffiliated withDepartment of Microbiology and Center for Immunology, University of Minnesota
  • , Amy TreefulAffiliated withDepartment of Microbiology and Center for Immunology, University of Minnesota
  • , Ronald JemmersonAffiliated withMayo Mail Code 196 Email author 

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Abstract

Cytochrome c (Cyt c) has been implicated as a serum marker for aberrant apoptosis and, thus, has considerable clinical potential. Using a sandwich enzyme-linked immunosorbent assay (ELISA) we found that the sensitivity of Cyt c detection is reduced in the presence of serum. The inhibitory factor responsible was purified from both fetal bovine serum and human serum employing standard chromatography procedures followed by affinity chromatography on Affi-Gel 10-bound Cyt c. In SDS-PAGE, bands at 44 kD and 50 kD were observed for the bovine and human proteins, respectively. Mass spectrometry analysis identified the serum inhibitory factor as leucine-rich alpha-2-glycoprotein-1 (LRα2GP1). This identification may lead to a modified ELISA to quantify total Cyt c in patients’ sera. LRα2GP1 is the first extracellular ligand for Cyt c that has been identified. A physiological function associated with binding is suggested.

Keywords

Cytochrome c Apoptotic marker ELISA Leucine-rich alpha-2-glycoprotein-1 Leucine-rich repeats