Mimotopes for lupus-derived anti-DNA and nucleosome-specific autoantibodies selected from random peptide phage display libraries: facts and follies.
Autoantibodies against chromatin are the most characteristic serological feature in SLE patients. Anti-dsDNA and nucleosome-specific antibodies are associated with glomerulonephritis, the most serious manifestation of SLE. Identification of peptides mimicking conformational epitopes (so-called mimotopes) on the nucleosome recognized by these antibodies is of considerable interest. Using an approach similar to that used previously to characterize mimotopes for anti-DNA autoantibodies, we have selected and identified a mimotope for a nucleosome-specific autoantibody (#32) by screening a random peptide phage display library. However, the reactivity of monoclonal antibody (mAb) #32 with the selected mimotope (MIMO#0) in ELISA was dependent on the blocking reagents used. Using nonfat dry milk (5%), mAb #32 clearly bound to MIMO#0, but using fetal bovine calf serum (FCS) (5%), there was no binding. Furthermore, again dependent on the blocking reagent used in ELISA, the selected mimotope MIMO#0 was not only recognized by the selecting antibody mAb #32, but also by a large number of other monoclonal anti-DNA, anti-histone and nucleosome-specific autoantibodies (NSA). We could demonstrate that the selected mimotope was able to bind directly to nucleosomal material (DNA/histone complexes) and labeled DNA. This finding was extended to other previously identified mimotopes for anti-DNA autoantibodies. We conclude that nucleosomal material (DNA/histone complexes), derived from reagents used during the mimotope selection procedure, resulted in the selection of DNA-binding peptides from the phage display library, rather than mimotopes. In addition, we could demonstrate that blocking reagents greatly influence the reactivity of anti-DNA, anti-histone and nucleosome-specific autoantibodies in ELISA. Development of blocking reagents devoid of nucleosomal material (DNA/histone complexes) is urgently needed for assay systems in which anti-nuclear autoantibodies are tested.
Dieker
Jürgen W
JW
Nephrology Research Laboratory, Nijmegen Center for Molecular Life Sciences and Division of Nephrology (545), University Medical Center, 6500 HB Nijmegen, The Netherlands.
Sun
Yong-Jiang
YJ
Jacobs
Cor W
CW
Putterman
Chaim
C
Monestier
Marc
M
Muller
Sylviane
S
van der Vlag
Johan
J
Berden
Jo H
JH
D020816:Amino Acid Motifs / Q000276:immunology; D000974:Antibodies, Antinuclear / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology; D004247:DNA / Q000276:immunology*; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D008181:Lupus Nephritis / Q000276:immunology; D018716:Molecular Mimicry / Q000276:immunology*; D009707:Nucleosomes / Q000276:immunology*; D019151:Peptide Library; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification
D000974:Antibodies, Antinuclear; D016056:Immunodominant Epitopes; D009707:Nucleosomes; D019151:Peptide Library; D010455:Peptides; D004247:DNA