Apoptosis

, Volume 11, Issue 7, pp 1121–1129 | Cite as

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

  • Chad Cummings
  • Jennifer Walder
  • Amy Treeful
  • Ronald Jemmerson
Reports

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 

Abbreviations

BLAST

basic local alignment search tool

BSA

bovine serum albumin

Cyt c

cytochrome c

DEAE

diethylaminoethyl

ELISA

enzyme-linked immunosorbent assay

HRP

horseradish peroxidase

mAbs

monoclonal antibodies

LRα2GP1

leucine-rich alpha-2-glycoprotein-1

MALDI-TOF MS

matrix assisted laser desorption ionization time-of-flight mass spectrometry

PBS

phosphate-buffered saline

SDS-PAGE

polyacrylamide gel electrophoresis in sodium dodecylsulfate

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Copyright information

© Springer Science + Business Media, LLC 2006

Authors and Affiliations

  • Chad Cummings
    • 1
  • Jennifer Walder
    • 1
  • Amy Treeful
    • 1
  • Ronald Jemmerson
    • 2
  1. 1.Department of Microbiology and Center for ImmunologyUniversity of MinnesotaMinneapolisUS
  2. 2.Mayo Mail Code 196MinneapolisU.S.A.

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