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

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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.

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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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Authors and Affiliations

  1. Department of Microbiology and Center for Immunology, University of Minnesota, Minneapolis, MN, US

    Chad Cummings, Jennifer Walder & Amy Treeful

  2. Mayo Mail Code 196, 420 Delaware St. S.E., Minneapolis, MN, 55455, U.S.A.

    Ronald Jemmerson

Authors
  1. Chad Cummings
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  2. Jennifer Walder
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  3. Amy Treeful
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  4. Ronald Jemmerson
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Corresponding author

Correspondence to Ronald Jemmerson.

Additional information

Supported by: NIH NINDS 5R21-NS45589

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Cummings, C., Walder, J., Treeful, A. et al. Serum leucine-rich alpha-2-glycoprotein-1 binds cytochrome c and inhibits antibody detection of this apoptotic marker in enzyme-linked immunosorbent assay. Apoptosis 11, 1121–1129 (2006). https://doi.org/10.1007/s10495-006-8159-3

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