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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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.
KeywordsCytochrome c Apoptotic marker ELISA Leucine-rich alpha-2-glycoprotein-1 Leucine-rich repeats
basic local alignment search tool
bovine serum albumin
- Cyt c
enzyme-linked immunosorbent assay
- MALDI-TOF MS
matrix assisted laser desorption ionization time-of-flight mass spectrometry
polyacrylamide gel electrophoresis in sodium dodecylsulfate
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- 19.Urbanski GJ, Margoliash E (1976) The antigenicity of cytochrome c. In: M. Salton, (ed). The immunochemistry of enzymes and their antibodies: J. Wiley and Sons New York pp. 203–226Google Scholar
- 24.Cotter R (1996) Time-of-flight mass spectrometry: Instrumentation and applications in biological research, American Chemical SocietyGoogle Scholar
- 26.Brown JR (1975) Structure of bovine serum albumin. Fed Proc 34:591–591Google Scholar
- 27.O’Donnell LC, Druhan LJ, Avalos BR (2002) Molecular characterization and expression analysis of leucine-rich α2 glycoprotein, a novel marker of granulocytic differentiation. J Leuk Biol 72:478–485Google Scholar