Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells

Proc Natl Acad Sci U S A. 2007 Sep 11;104(37):14610-5. doi: 10.1073/pnas.0706806104. Epub 2007 Sep 5.

Abstract

The autofluorescent lipofuscin pigment A2E accumulates in retinal pigment epithelial cells with age and is particularly abundant in some retinal disorders. To generate a polyclonal antibody that recognizes this pyridinium bisretinoid molecule, we immunized rabbits with bovine serum albumin (BSA) conjugates in which the protein was linked to the A2E molecule via its pyridinium ethanolamine moiety. Analysis by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) of the A2E-BSA conjugate indicated the presence of five intact A2E molecules covalently linked to BSA, thus deeming it a suitable antigen for immunization. By immunocytochemical staining, the rabbit polyclonal antibody recognized A2E that had accumulated in cultured cells, whereas dot-blot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but no cross-reactivity with various retinoids. Preimmune serum was nonreactive. In fluorescence spectroscopy studies, antibody-A2E binding was evidenced by a fluorescence increase and by a blue-shift in the emission maximum consistent with a change in A2E milieu upon antibody binding. The changes in fluorescence emission upon antibody binding could reflect several processes including restrictions on trans-cis isomerization and intersystem crossing of photo-excited A2E.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cattle
  • Cells, Cultured
  • Circular Dichroism
  • Fluorescent Antibody Technique, Indirect
  • Humans
  • Immunohistochemistry
  • Lipofuscin / chemistry
  • Lipofuscin / metabolism*
  • Models, Chemical
  • Molecular Structure
  • Pigment Epithelium of Eye / cytology
  • Pigment Epithelium of Eye / metabolism*
  • Pyridinium Compounds / chemistry
  • Pyridinium Compounds / metabolism*
  • Rabbits
  • Retina / cytology*
  • Retinoids / biosynthesis
  • Retinoids / chemistry
  • Retinoids / metabolism*
  • Serum Albumin, Bovine / immunology
  • Serum Albumin, Bovine / metabolism
  • Spectrometry, Fluorescence
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Spectrophotometry, Ultraviolet

Substances

  • A2-E (N-retinylidene-N-retinylethanolamine)
  • Lipofuscin
  • Pyridinium Compounds
  • Retinoids
  • Serum Albumin, Bovine